RNA CONSTRUCTS AND USES THEREOF BACKGROUND Use of RNA polynucleotides as therapeutics is a new and emerging field. SUMMARY The present disclosure identifies certain challenges that can be associated with in vitro production of RNA, for example of RNA therapeutics. For example, in some embodiments, the present disclosure identifies the source of certain problems that can be encountered with expression of polypeptides encoded by RNA therapeutics. Among other things, the present disclosure provides technologies for improving capping efficiency (e.g., percentage of capped transcripts in an in vitro transcription reaction), quality of an RNA preparation (e.g., of an in vitro transcribed RNA, such as, e.g., the amount of short polynucleotide byproducts produced), translation efficiency of an RNA encoding a payload, and/or expression of a polypeptide payload encoded by an RNA. In some embodiments, translation efficiency and/or expression of an RNA-encoded payload can be improved with an RNA polynucleotide comprising: a 5’ cap as defined and described herein; a 5’ UTR comprising a cap proximal sequence as defined and described herein, and a sequence encoding a payload. Without wishing to be bound by a particular theory, the present disclosure proposes that improved RNA transcription, capping efficiency, translation efficiency, and/or polypeptide payload expression and/or reduced transcription byproduct formation can be achieved through use of a 5’ cap structure as decribed herein in combination with certain transcription start site sequences of a template DNA. In some embodiments, the present disclosure recognizes that certain caps provide improved RNA transcription, capping efficiency, translation efficiency, and/or polypeptide payload expression and/or reduced byproduct formation. In some embodiments, the present disclosure recognizes that certain caps when utilized with particular transcription start sites provide improved RNA transcription, capping efficiency, translation efficiency, and/or polypeptide payload expression and/or reduced byproduct formation. T7 RNA polymerase most commonly utilizes a GGG transcriptional start site (e.g., generating an RNA whose first three residues are each “G”), and, moreover, has been reported to prefer “G” as an initiating residue (e.g., generating an RNA whose first residue is “G”). Conrad, et al. (2020) Communications Biology 3:439. Studies comparing T7 transcription of templates with different initiating residues report levels of transcripts beginning with “A” are only 25% of those observed for transcripts beginning with “G”. Milligan, et al. (1987) Nucleic Acids Research 15:8783-8798. The 3’ end of commonly used dinucleotide cap analogs also employ “G” (e.g., m ₂ ^{7,2’- O} GppSpG “β-S-ARCA” or “D1”). Grudzien-Nogalska, et al. RNA 13:1745-1755. Indeed, certain such caps, e.g., β-S-ARCA, provide advantages including, e.g., being more resistant to human decapping enzymes (Kowalska et al. (2008) RNA 14:1119-1131) and interferon-induced proteins with tetratricopeptide repeats (IFITs), which inhibit Cap0-dependent translation (Diamond et al. (2014) Cytokine & Growth Factor Reviews 25:543-550; and Miedziak et al. (2019) RNA 25:58-68). However, poor capping efficiency is sometimes observed. Without wishing to be bound by any particular theory, the present disclosure proposes that competition with GTP in the transcription reaction may contribute to such poor capping efficiency. In some embodiments, cap1 analogs (including, e.g., ones that are commercially available) can be incorporated into synthetic RNAs (e.g., RNAs produced by in vitro transcription (IVT)) in the correct orientation to produce cap1 RNA with a high capping efficiency, e.g., all in a rapid co-transcriptional reaction. For example, a cap analog for a synthetic self-amplifying RNA (saRNA) may be or comprise CleanCap AU, TriLink (#N7-114). For example, a cap analog for a synthetic mRNA may be or comprise CleanCap AG, Trilink, #N7-413. See Henderson, J.M., et al. (2021) Current protocols, 1, e39. An appealing feature of these trinucleotide cap1 analogs is that they require an A initiator, which may avoid potential slippage of RNA polymerases on the DNA template strand as opposed to those containing a G triplet as a transcriptional start site. See Imburgio, et al. (2000) Biochemistry, 39, 10419–10430. Furthermore, anti reverse cap analog (ARCA)-capped mRNA may possess higher translation efficiency compared to conventional cap analogs. See Stepinski, J., et al. (2001) RNA (New York, N.Y.), 7, 1486–1495; and Kuhn, A.N., et al. (2010) Gene therapy, 17, 961–971. For example, a variant of the CleanCap AG with a modification at the C3’ position of 7- methylguanosine (CleanCap AG 3’ OMe) may play an important role in the progress of immunotherapeutic vaccination strategy against SARS-CoV-2. See Sahin, U. et al. (2021) Nature, 595, 572–577. In some embodiments, without wishing to be bound to a particular theory, the present disclosure provides the recognition that an ARCA cap1 analog may exhibit better translational efficiency and/or biological activity as compared to those capped with its non-ARCA version (See Figure 1). Additionally or alternatively, cap analogs paired with particular start sequences have been described to attempt to address one or more of these problems, e.g., WO 2021/214204A1. Additionally or alternatively, incorporation of nucleoside modifications (e.g., modified uridines (including, e.g., N1-methylpseudouridine (m1Ψ)) and/or modified adenosines (including, e.g., N6-methyladenine (m6A)) into synthetic RNA (e.g., in some embodiments IVTmRNAs) may increase biological stability and thereby enhance the durability of the encoded protein compared to unmodified RNAs. See Karikó, K., et al. (2008) Molecular therapy: the journal of the American Society of Gene Therapy, 16, 1833–1840; and Gao, Y., et al. (2020) Immunity, 52, 1007-1021.e8. However, without wishing to be bound to a particular theory, because certain saRNA cannot contain modified nucleosides, use of such modified nucleosides has primarily been limited to preventive vaccines against infectious diseases in contrast to non- replicating mRNAs. See Bloom, K., et al. (2021) Gene therapy, 28, 117–129. Additionally or alternatively, non-replicating mRNAs also have great potential in research areas such as gene editing, protein replacement therapy, where the reduction and elimination of immunomodulation is important for reaching the appropriate therapeutic goal. While potential advantages of using various modified nucleosides have been contemplated, the effects of cap analogs containing modified nucleosides on quality, translational efficiency, and biological activity or immunogenicity of mRNA that encodes a potentially therapeutic protein are not understood. In some embodiments, the present disclosure also provides the recognition that 5’ caps comprising modified nucleoside(s) may be a promising alternative to current capping strategies in mRNA vaccines and especially in RNA-based therapeutics. In some embodiments, the present disclosure recognizes that certain 5’ cap structures (e.g., trinucleotide caps comprising N1pN2, wherein N1 is A or an analog thereof, and N2 is U or an analog thereof), e.g., when paired with certain transcription start sites (e.g., AUN, such as AUA), provide improved RNA transcription, improved translation efficiency, and/or improved and/or prolonged polypeptide payload expression as compared to transcripts comprising other 5’ cap structures (such as, e.g., CC114 or CC413 caps used). Additionally or alternatively, in some embodiments, the present disclosure recognizes that certain 5’ cap structures (e.g., trinucleotide caps comprising N1pN2, wherein N1 is A or an analog thereof, and N2 is U or an analog thereof), e.g., when paired with certain transcription start sites (e.g., AUN, such as AUA), result in higher capping efficiency, reduced amounts of short contaminants, and reduced toxicity due to cytokine/chemokine secretion as compared to transcripts comprising other 5’ cap structures (such as, e.g., CC114 or CC413 caps used). Additionally or alternatively, in some embodiments, the present disclosure recognizes that the demonstrated effects of certain 5’ cap structures (e.g., trinucleotide caps comprising N1pN2, wherein N1 is A or an analog thereof, and N2 is U or an analog thereof), e.g., when paired with certain transcription start sites (e.g., AUN, such as AUA), can be adapted not only to replicative mRNA, but also to non-replicative mRNA. Additionally or alternatively, in some embodiments, the present disclosure recognizes that disclosed 5’ cap structures where N2 is a modified U (e.g., pseudouridine, i.e., Ψ, and analogs thereof, such as 1-methylpseudouridine ((m ¹ )Ψ)) display improved RNA transcription, improved translation efficiency, and/or improved and/or prolonged polypeptide payload expression as compared to transcripts comprising other 5’ cap structures (such as, e.g., CC114 or CC413 caps, or caps comprising unmodified U). Additionally or alternatively, in some embodiments, the present disclosure recognizes that disclosed 5’ cap structures where N ₂ is a modified U (e.g., pseudouridine, i.e., Ψ, and analogs thereof, such as (m ¹ )Ψ) result in higher capping efficiency, lesser amounts of short contaminants, and reduced toxicity due to cytokine/chemokine secretion as compared to transcripts comprising other 5’ cap structures (such as, e.g., CC114 or CC413 caps, or caps comprising unmodified U). Additionally or alternatively, in some embodiments, the present disclosure recognizes that the demonstrated effects of disclosed 5’ cap structures where N ₂ is a modified U (e.g., pseudouridine, i.e., Ψ, and analogs thereof, such as (m ¹ )Ψ) display can be adapted not only to replicative mRNA, but also to non-replicative mRNA. Accordingly, in some embodiments, the present disclosure provides, inter alia, a composition or medical preparation comprising an RNA polynucleotide, comprising: (i) a 5’ cap, e.g., as disclosed herein; (ii) a cap proximal sequence, e.g., as disclosed herein; and (iii) a sequence encoding a payload. Also disclosed herein are methods of making and using the same to, e.g., induce an immune response in a subject. In some embodiments, the present disclosure also provides trinucleotide caps G*N1pN2, or a salt thereof, wherein: G* comprises a structure of formula I′: Iʹ wherein: each R ² and R ³ is independently -OH or -OCH ₃ ; and X is OH or SH; N1 is A or an analog thereof; N ₂ is U or an analog thereof; and p is a group selected from phosphate (e.g., -P(=O)(OH)- or -P(=O)(O-)-) or thiophosphate (e.g., -P(=S)(OH)- or -P(=S)(O-)-). In some embodiments, it will be appreciated that trinucleotide caps having the structure of formula I′ (e.g., trinucleotide caps comprising a N ₂ nucleotide of formula II′′ or formula II′′′) demonstrate surprising advantages such as, for example, improved translation efficiencies, as discussed in greater detail herein. BRIEF DESCRIPTION OF THE DRAWING Figure 1 shows a comparison of levels of murine EPO and hematocrit %. CC113 corresponds to ; CC413 corresponds to (m ₂ ^7,3'-O )Gppp(m ^2'-O )ApG. Translational efficiency as well as biological activity of EPO mRNA capped with CC413 is significantly better than CC113. Figure 2A shows a comparison of RNA quality after in vitro transcription using (m ₂ ^{7,3’- O} )Gppp(m ^2’-O )ApU cap (i.e., compound I′-1) and various start sites. The highest yield was observed with AUAGU start site. Figure 2B shows a comparison of capping efficiency by 21% Urea-PAGE. A high yield was observed when (m2 ^7,3’-O )Gppp(m ^2’-O )ApU cap (i.e., compound I′- 1) was used in the range of 3-6mM concentration. Capping efficiency is close to 100% regardless of the concentration used Figure 3 shows a comparison of capping efficiency by 21% Urea-PAGE. Cap 1 corresponds to compound I′-1 ((m2 ^7,3’-O )Gppp(m ^2’-O )ApU); Cap 2 corresponds to compound I′-6 ((m ₂ ^7,3’-O )Gppp(m ^2’-O )Ap(m ¹ )Ψ); CC114 corresponds to (m ⁷ )Gppp(m ^2'-O )ApU; CC413 corresponds to (m2 ^7,3'-O )Gppp(m ^2'-O )ApG. The capping efficiency of compound I′-1 and compound I′-6 is close to 100% and is comparable to CC114 and CC413. Figure 4 shows a comparison of amounts of short contaminants for certain caps and start sites. Cap 1 corresponds to compound I′-1 ((m ₂ ^7,3’-O )Gppp(m ^2’-O )ApU); Cap 2 corresponds to compound I′-6 ((m2 ^7,3’-O )Gppp(m ^2’-O )Ap(m ¹ )Ψ); CC114 corresponds to (m ⁷ )Gppp(m ^2'-O )ApU; CC413 corresponds to (m ₂ ^7,3'-O )Gppp(m ^2'-O )ApG. A minimial amount of short contaminants was observed for compound I′-6 and CC413 mRNA, while significant amount was observed for other unmodified mRNAs tested, independent of the cap. Figure 5 shows a comparison of XTT assay of viable PMBCs at 24 hours. Cap 1 corresponds to compound I′-1 ((m ₂ ^7,3’-O )Gppp(m ^2’-O )ApU); Cap 2 corresponds to compound I′-6 ((m2 ^7,3’-O )Gppp(m ^2’-O )Ap(m ¹ )Ψ); CC114 corresponds to (m ⁷ )Gppp(m ^2'-O )ApU; CC413 corresponds to (m2 ^7,3'-O )Gppp(m ^2'-O )ApG. No toxic effect on cell viability of PMBCs up to 1 µg/well mRNA originating from compound I′-1 or compound I′-6 is observed. Transfection of unmodified mRNAs leads to decrease in cellular viability starting at dose 0.333 µg/well, however this effect does not depend on the cap used, but on the mRNA modification. Figures 6A, 6B, 6C, 6D, 6E, 6F, and 6G show a comparison of cytokine/chemokine secrection in human PBMCs. Cap 1 corresponds to compound I′-1 ((m ₂ ^7,3’-O )Gppp(m ^2’-O )ApU); Cap 2 corresponds to compound I′-6 ((m2 ^7,3’-O )Gppp(m ^2’-O )Ap(m ¹ )Ψ); CC114 coresponds to (m ⁷ )Gppp(m ^2'-O )ApU; CC413 corresponds to (m ₂ ^7,3'-O )Gppp(m ^2'-O )ApG. Compound I′-6 is comparable to CC413 in regard to the amount of cytokines/chemokines secreted by human PBMCs after transfection of m1Ψ-modified mRNA. In regard to cytokines/chemokines of unmodified mRNA, compound I′-1 is comparable to CC114 and leads to significantly higher cytokines/chemokines in comparison to CC413. Figure 7 shows a comparison of EPO secrection in human hepatocytes at day 1 after transfection of 0.1 µg/well TransIT-EPO mRNA (IV187). Cap 1 corresponds to compound I′-1 ((m ₂ ^7,3’-O )Gppp(m ^2’-O )ApU); Cap 2 corresponds to compound I′-6 ((m ₂ ^7,3’-O )Gppp(m ^{2’- O} )Ap(m ¹ )Ψ); CC114 is (m ⁷ )Gppp(m ^2'-O )ApU; CC413 is (m ₂ ^7,3'-O )Gppp(m ^2'-O )ApG. Compound I′-1 and compound I′-6 show higher translation compared to CC413 in human hepatocytes at 24 hrs. Figure 8A shows a comparison of plasma EPO mice IV invjected with 3 µg TransIT- formulated somEPO mRNA (JR81). Cap 1 corresponds to compound I′-1 ((m ₂ ^7,3’-O )Gppp(m ^{2’- O} )ApU); Cap 2 corresponds to compound I′-6 ((m2 ^7,3’-O )Gppp(m ^2’-O )Ap(m ¹ )Ψ); CC114 corresponds to (m ⁷ )Gppp(m ^2'-O )ApU; CC413 corresponds to (m2 ^7,3'-O )Gppp(m ^2'-O )ApG. EPO mRNA capped with compound I′-6 translated 2- to 3-fold greater at later time points compared to those capped with CC413, demonstrating that compound I′-6 has a strong beneficial effect on translational capacity and biological activity of mRNA. Figure 8B shows hematocrit level in mice IV injected with 3 µg TransIT-complexed somEPO mRNA (hAg) capped with certain caps. Cap 1 corresponds to compound I′-1 ((m ₂ ^7,3’-O )Gppp(m ^2’-O )ApU); Cap 2 corresponds to compound I′-6 ((m2 ^7,3’-O )Gppp(m ^2’-O )Ap(m ¹ )Ψ); CC114 corresponds to (m ⁷ )Gppp(m ^2'-O )ApU; CC413 corresponds to (m2 ^7,3'-O )Gppp(m ^2'-O )ApG. Hemoatocrit values in mice injected with EPO mRNA capped with compound I′-6 are very high and further increased after day 14 after injection. Figure 9 depicts a comparison of plasma EPO mice IV injected with 3 µg TransIT- formulated somEPO mRNA capped with cap analogs of formula I′. CC114 corresponds to (m ⁷ )Gppp(m ^2'-O )ApU; CC413 corresponds to (m ₂ ^7,3'-O )Gppp(m ^2'-O )ApG. I′-1 corresponds to (m2 ^7,3’-O )Gppp(m ^2’-O )ApU. I′-2 corresponds to (m2 ^7,2’-O )Gppp(m ^2’-O )ApU. I′-13 corresponds to m7Gppp(m ^2’-O )Ap(m1)Ѱ. I′-5 corresponds to (m ₂ ^7,2’-O )Gppp(m ^2’-O )Ap(m1)Ѱ. I′-6 corresponds to (m ₂ ^7,3’-O )Gppp(m ^2’-O )Ap(m ¹ )Ψ. ARCA analogs I′-1 and I′-6 translated significantly better compared to non-ARCA caps CC114 and I′-13 regardless of the RNA modification. m1Ѱ- mRNA capped with non-ARCA cap I′-13 containing m1Ѱ-modified RNA translated 8 and 25- fold more than U-mRNA capped with non-ARCA CC114 without nucleoside modification at 6 and 24 h after injection, respectively. Figure 10 shows a comparison of EPO levels in mice injected with 3 µg TransIT- formulated with U-containing mRNA capped with I′-1 and m1Ѱ-modified mRNA capped with I′-6. I′-6 (i.e., mRNA with the combination of m1Ѱ-m1Ѱ) performed the best and translated 2-3- fold more than I′-1 at each time points after administration. U-containing mRNA capped with I′- 1 showed translational capacity which is significantly lower in each time point than that observed for m1Ѱ modification is present both in the cap analog (I′-6) and in the mRNA. Figure 11 shows a comparison of EPO levels in mice injected with 3 µg TransIT- formulated m1Ѱ-modified mRNA with caps comprising unmodified uridine (I′-1) and unmodified pseudouridine (I′-3) and modified uridine (U) or pseudouridine (Ѱ) (N5- methyluridine (I′-9), N5-methoxyuridine (I′-12), N1-methylpseudouridine (I′-6), and N1- propargylpseudouridine (I′-16). At 48 and 72 hours after injection. EPO level in mice injected with mRNA capped with Ѱ-containing cap analog (I′-3 - (m2 ^7,3'-O )G(5′)ppp(5′)(m ^2'-O )ApѰ) is equal or slightly less to those bearing m1Ѱ (I′-6 - (m ₂ ^7,3'-O )G(5′)ppp(5′)(m ^2'-O )Apm1Ѱ) (Figure 11). Neither uridine (U) and its derivatives (5-methylU, 5-methoxyU) nor pseudouridine derivative 1-propargylѰ could improve the potency of N1-methylpseudouridine (1-methylѰ)- containing cap (I′-6). Figure 12 depicts the effect on levels of cytokines and chemokines after application of Lipoplex (LPX)-formulated EPO mRNAs. Figure 12A depicts the effect CC413 and I′-6 on IL- 6 levels. Figure 12B depicts the effect CC413 and I′-6 on TNF-α levels. Figure 12C depicts the effect CC413 and I′-6 on IL-1β levels. Figure 12D depicts the effect CC413 and I′-6 on IFN-γ levels. Figure 12E depicts the effect CC413 and I′-6 on MIP-1β levels. I′-6 demonstrated less of an increase in the levels of proinflammatory cytokines and chemokines (i.e., I′-6 showed less immunogenicity) as compared to CC413 across the concentrations tested. Figure 13 shows a comparison of EPO levels in primary human hepatocytes transfected with 0.1 µg/well TransIT-formulated somEPO mRNA. EPO level was measured from supernatants transfected using I′-6 or CC114- capped uRNA. Increased secretion of EPO in human primary cells was detected at all three tested time points: 24h, 48h and 144h. These results suggested that cap1 analogs such as I′-6 are suitable for translation of the encoded protein and can be used for synthesizing non-replicating functional mRNAs. Figure 14 shows a comparison of EPO uRNA capped with I′-6 and I′-1. In this case both mRNAs had the same TAGT 5´end. I′-6 showed benefit leading to significantly lower cytokines (IL-6 (Fig.14A), TNF-α (Fig.14B), IL-1β (Fig.14C) and IFN-γ (Fig.14D)) 24h after application to human PBMCs. Thus, I′-6 results in lower immunogenicity. Figure 15 shows EPO secretion after application of EPO-encoding Ѱ-mRNA capped with uridine (U) or pseudouridine (Ѱ) derivatives CC413 and I′-3, respectively. The level of EPO was higher at 24h and 48h when I′-3 was used compared to CC413. Figure 16 shows a comparison of EPO-encoding mRNA capped with cap1 analogs bearing N5-methyluridine (I′-9), N5-methoxyuridine (I′-12), N1-methylpseudouridine (I′-6) and N1-propargylpseudouridine (I′-16). I′-6 showed increased level of secreted EPO at 24h as compared to other caps. In addition, mRNAs with modified caps led to increase in EPO secretion at 24h and 48h when compared to the unmodified I′-1. CERTAIN DEFINITIONS Although the present disclosure is described in detail below, it is to be understood that this disclosure is not limited to the particular methodologies, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. Preferably, the terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", H.G.W. Leuenberger, B. Nagel, and H. Kölbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995). The practice of the present disclosure will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA techniques which are explained in the literature in the field (cf., e.g., Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989). Compounds of the present disclosure include those described generally above, and are further illustrated by the classes, subclasses, and species disclosed herein. As used herein, the following definitions shall apply unless otherwise indicated. For purposes of this disclosure, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed. Additionally, general principles of organic chemistry are described in “Organic Chemistry”, Thomas Sorrell, University Science Books, Sausalito: 1999, and “March’s Advanced Organic Chemistry”, 5th Ed., Ed.: Smith, M.B. and March, J., John Wiley & Sons, New York: 2001, the entire contents of which are hereby incorporated by reference. Combinations of substituents envisioned by this disclosure are preferably those that result in the formation of stable or chemically feasible compounds. The term “stable”, as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein. The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof. As used herein, the term “pharmaceutically acceptable salt” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1–19, incorporated herein by reference. Pharmaceutically acceptable salts include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxyl-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2–naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3–phenylpropionate, phosphate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p–toluenesulfonate, undecanoate, valerate salts, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N ⁺ (C _1–4 alkyl) ₄ salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate and aryl sulfonate. Unless otherwise stated, structures depicted herein are also meant to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) forms of the structure; for example, the R and S configurations for each asymmetric center, Z and E double bond isomers, and Z and E conformational isomers. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present compounds are within the scope of the present disclosure. Unless otherwise stated, all tautomeric forms are within the scope of the disclosure. Additionally, unless otherwise stated, the present disclosure also includes compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures including the replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a ¹³ C- or ¹⁴ C-enriched carbon are within the scope of this disclosure. Such compounds are useful, for example, as analytical tools, as probes in biological assays, or as therapeutic agents in accordance with the present disclosure. In some embodiments, compounds of this disclosure comprise one or more deuterium atoms. In the following, the elements of the present disclosure will be described. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and embodiments should not be construed to limit the present disclosure to only the explicitly described embodiments. This description should be understood to disclose and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed elements. Furthermore, any permutations and combinations of all described elements should be considered disclosed by this description unless the context indicates otherwise. The term "about" means approximately or nearly, and in the context of a numerical value or range set forth herein in some embodiments means ± 20%, ± 10%, ± 5%, or ± 3% of the numerical value or range recited or claimed. The terms "a" and "an" and "the" and similar reference used in the context of describing the disclosure (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it was individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as"), provided herein is intended merely to better illustrate the disclosure and does not pose a limitation on the scope of the claims. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the disclosure. Unless expressly specified otherwise, the term "comprising" is used in the context of the present document to indicate that further members may optionally be present in addition to the members of the list introduced by "comprising". It is, however, contemplated as a specific embodiment of the present disclosure that the term "comprising" encompasses the possibility of no further members being present, i.e., for the purpose of this embodiment "comprising" is to be understood as having the meaning of "consisting of" or "consisting essentially of". Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the present disclosure was not entitled to antedate such disclosure. In the following, definitions will be provided which apply to all aspects of the present disclosure. The following terms have the following meanings unless otherwise indicated. Any undefined terms have their art recognized meanings. Agent: As used herein, the term “agent”, may refer to a physical entity or phenomenon. In some embodiments, an agent may be characterized by a particular feature and/or effect. In some embodiments, an agent may be a compound, molecule, or entity of any chemical class including, for example, a small molecule, polypeptide, nucleic acid, saccharide, lipid, metal, or a combination or complex thereof. In some embodiments, the term “agent” may refer to a compound, molecule, or entity that comprises a polymer. In some embodiments, the term may refer to a compound or entity that comprises one or more polymeric moieties. In some embodiments, the term “agent” may refer to a compound, molecule, or entity that is substantially free of a particular polymer or polymeric moiety. In some embodiments, the term may refer to a compound, molecule, or entity that lacks or is substantially free of any polymer or polymeric moiety. Aliphatic or aliphatic group: as used herein, means a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is completely saturated or that contains one or more units of unsaturation, or a monocyclic hydrocarbon or bicyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic (also referred to herein as “carbocycle”, “carbocyclic”, “cycloaliphatic” or “cycloalkyl”), that has a single point of attachment to the rest of the molecule. Unless otherwise specified, aliphatic groups contain 1-6 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-5 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-4 aliphatic carbon atoms. In still other embodiments, aliphatic groups contain 1-3 aliphatic carbon atoms, and in yet other embodiments, aliphatic groups contain 1-2 aliphatic carbon atoms. In some embodiments, “cycloaliphatic” (or “carbocycle” or “cycloalkyl”) refers to a monocyclic C3-C6 hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic, that has a single point of attachment to the rest of the molecule. Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl groups and hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl. Unsaturated: as used herein, means that a moiety has one or more units of unsaturation. Partially unsaturated: as used herein, refers to a ring moiety that includes at least one double or triple bond. The term “partially unsaturated”, as used herein, is intended to encompass rings having multiple sites of unsaturation, but is not intended to include aryl or heteroaryl moieties, as herein defined. Amino acid: in its broadest sense, as used herein, the term “amino acid” refers to a compound and/or substance that can be, is, or has been incorporated into a polypeptide chain, e.g., through formation of one or more peptide bonds. In some embodiments, an amino acid has the general structure H2N–C(H)(R)–COOH. In some embodiments, an amino acid is a naturally- occurring amino acid. In some embodiments, an amino acid is a non-natural amino acid; in some embodiments, an amino acid is a D-amino acid; in some embodiments, an amino acid is an L- amino acid. “Standard amino acid” refers to any of the twenty standard L-amino acids commonly found in naturally occurring peptides. “Nonstandard amino acid” refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source. In some embodiments, an amino acid, including a carboxy- and/or amino-terminal amino acid in a polypeptide, can contain a structural modification as compared with the general structure above. For example, in some embodiments, an amino acid may be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and/or substitution (e.g., of the amino group, the carboxylic acid group, one or more protons, and/or the hydroxyl group) as compared with the general structure. In some embodiments, such modification may, for example, alter the circulating half-life of a polypeptide containing the modified amino acid as compared with one containing an otherwise identical unmodified amino acid. In some embodiments, such modification does not significantly alter a relevant activity of a polypeptide containing the modified amino acid, as compared with one containing an otherwise identical unmodified amino acid. As will be clear from context, in some embodiments, the term “amino acid” may be used to refer to a free amino acid; in some embodiments it may be used to refer to an amino acid residue of a polypeptide. Analog: As used herein, the term “analog” refers to a substance that shares one or more particular structural features, elements, components, or moieties with a reference substance. Typically, an “analog” shows significant structural similarity with the reference substance, for example sharing a core or consensus structure, but also differs in certain discrete ways. In some embodiments, an analog is a substance that can be generated from the reference substance, e.g., by chemical manipulation of the reference substance. In some embodiments, an analog is a substance that can be generated through performance of a synthetic process substantially similar to (e.g., sharing a plurality of steps with) one that generates the reference substance. In some embodiments, an analog is or can be generated through performance of a synthetic process different from that used to generate the reference substance. Antibody agent: As used herein, the term “antibody agent” refers to an agent that specifically binds to a particular antigen. In some embodiments, the term encompasses a polypeptide or polypeptide complex that includes immunoglobulin structural elements sufficient to confer specific binding. For example, in some embodiments, an antibody agent is or comprises a polypeptide whose amino acid sequence includes one or more structural elements recognized by those skilled in the art as a complementarity determining region (CDR); in some embodiments an antibody agent is or comprises a polypeptide whose amino acid sequence includes at least one CDR (e.g., at least one heavy chain CDR and/or at least one light chain CDR) that is substantially identical to one found in a reference antibody. In some embodiments an included CDR is substantially identical to a reference CDR in that it is either identical in sequence or contains between 1-5 amino acid substitutions as compared with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that it shows at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that it shows at least 96%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that at least one amino acid within the included CDR is deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical with that of the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that 1-5 amino acids within the included CDR are deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical to the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that at least one amino acid within the included CDR is substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical with that of the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that 1-5 amino acids within the included CDR are deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical to the reference CDR. In some embodiments, an antibody agent is or comprises a polypeptide whose amino acid sequence includes structural elements recognized by those skilled in the art as an immunoglobulin variable domain. In some embodiments, an antibody agent in or comprises a polypeptide whose amino acid sequence includes structural elements recognized by those skilled in the art to correspond to CDRs1, 2, and 3 of an antibody variable domain; in some such embodiments, an antibody agent in or comprises a polypeptide or set of polypeptides whose amino acid sequence(s) together include structural elements recognized by those skilled in the art to correspond to both heavy chain and light chain variable region CDRs, e.g., heavy chain CDRs 1, 2, and/or 3 and light chain CDRs 1, 2, and/or 3. In some embodiments, an antibody agent is a polypeptide protein having a binding domain which is homologous or largely homologous to an immunoglobulin-binding domain. In some embodiments, an antibody agent may be or comprise a polyclonal antibody preparation. In some embodiments, an antibody agent may be or comprise a monoclonal antibody preparation. In some embodiments, an antibody agent may include one or more constant region sequences that are characteristic of a particular organism, such as a camel, human, mouse, primate, rabbit, rat; in many embodiments, an antibody agent may include one or more constant region sequences that are characteristic of a human. In some embodiments, an antibody agent may include one or more sequence elements that would be recognized by one skilled in the art as a humanized sequence, a primatized sequence, a chimeric sequence, etc. In some embodiments, an antibody agent may be a canonical antibody (e.g., may comprise two heavy chains and two light chains). In some embodiments, an antibody agent may be in a format selected from, but not limited to, intact IgA, IgG, IgE or IgM antibodies; bi- or multi- specific antibodies (e.g., Zybodies®, etc); antibody fragments such as Fab fragments, Fab’ fragments, F(ab’)2 fragments, Fd’ fragments, Fd fragments, and isolated CDRs or sets thereof; single chain Fvs; polypeptide- Fc fusions; single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof); cameloid antibodies; masked antibodies (e.g., Probodies®); Small Modular ImmunoPharmaceuticals (“SMIPs ^TM” ); single chain or Tandem diabodies (TandAb®); VHHs; Anticalins®; Nanobodies® minibodies; BiTE®s; ankyrin repeat proteins or DARPINs®; Avimers®; DARTs; TCR-like antibodies;, Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®; MicroProteins; Fynomers®, Centyrins®; and KALBITOR®s. In some embodiments, an antibody may lack a covalent modification (e.g., attachment of a glycan) that it would have if produced naturally. In some embodiments, an antibody may contain a covalent modification (e.g., attachment of a glycan, a payload [e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc], or other pendant group [e.g., poly-ethylene glycol, etc.]. Associated: Two events or entities are “associated” with one another, as that term is used herein, if the presence, level, degree, type and/or form of one is correlated with that of the other. For example, a particular entity (e.g., polypeptide, genetic signature, metabolite, microbe, etc) is considered to be associated with a particular disease, disorder, or condition, if its presence, level and/or form correlates with incidence of, susceptibility to, severity of, stage of, etc the disease, disorder, or condition (e.g., across a relevant population). In some embodiments, two or more entities are physically “associated” with one another if they interact, directly or indirectly, so that they are and/or remain in physical proximity with one another. In some embodiments, two or more entities that are physically associated with one another are covalently linked to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another but are non-covalently associated, for example by means of hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetism, and combinations thereof. Binding: It will be understood that the term “binding”, as used herein, typically refers to a non-covalent association between or among two or more entities. “Direct” binding involves physical contact between entities or moieties; indirect binding involves physical interaction by way of physical contact with one or more intermediate entities. Binding between two or more entities can typically be assessed in any of a variety of contexts – including where interacting entities or moieties are studied in isolation or in the context of more complex systems (e.g., while covalently or otherwise associated with a carrier entity and/or in a biological system or cell). Binding between two entities may be considered “specific” if, under the conditions assessed, the relevant entities are more likely to associate with one another than with other available binding partners. Biological Sample: As used herein, the term “biological sample” typically refers to a sample obtained or derived from a biological source (e.g., a tissue or organism or cell culture) of interest, as described herein. In some embodiments, a source of interest comprises an organism, such as an animal or human. In some embodiments, a biological sample is or comprises biological tissue or fluid. In some embodiments, a biological sample may be or comprise bone marrow; blood; blood cells; ascites; tissue or fine needle biopsy samples; cell-containing body fluids; free floating nucleic acids; sputum; saliva; urine; cerebrospinal fluid, peritoneal fluid; pleural fluid; feces; lymph; gynecological fluids; skin swabs; vaginal swabs; oral swabs; nasal swabs; washings or lavages such as a ductal lavages or broncheoalveolar lavages; aspirates; scrapings; bone marrow specimens; tissue biopsy specimens; surgical specimens; feces, other body fluids, secretions, and/or excretions; and/or cells therefrom, etc. In some embodiments, a biological sample is or comprises cells obtained from an individual. In some embodiments, obtained cells are or include cells from an individual from whom the sample is obtained. In some embodiments, a sample is a “primary sample” obtained directly from a source of interest by any appropriate means. For example, in some embodiments, a primary biological sample is obtained by methods selected from the group consisting of biopsy (e.g., fine needle aspiration or tissue biopsy), surgery, collection of body fluid (e.g., blood, lymph, feces etc.), etc. In some embodiments, as will be clear from context, the term “sample” refers to a preparation that is obtained by processing (e.g., by removing one or more components of and/or by adding one or more agents to) a primary sample. For example, filtering using a semi-permeable membrane. Such a “processed sample” may comprise, for example nucleic acids or proteins extracted from a sample or obtained by subjecting a primary sample to techniques such as amplification or reverse transcription of mRNA, isolation and/or purification of certain components, etc. Combination therapy: As used herein, the term “combination therapy” refers to those situations in which a subject is simultaneously exposed to two or more therapeutic regimens (e.g., two or more therapeutic agents). In some embodiments, the two or more regimens may be administered simultaneously; in some embodiments, such regimens may be administered sequentially (e.g., all “doses” of a first regimen are administered prior to administration of any doses of a second regimen); in some embodiments, such agents are administered in overlapping dosing regimens. In some embodiments, “administration” of combination therapy may involve administration of one or more agent(s) or modality(ies) to a subject receiving the other agent(s) or modality(ies) in the combination. For clarity, combination therapy does not require that individual agents be administered together in a single composition (or even necessarily at the same time), although in some embodiments, two or more agents, or active moieties thereof, may be administered together in a combination composition, or even in a combination compound (e.g., as part of a single chemical complex or covalent entity). Complementary: As used herein, the term “complementary” is used in reference to oligonucleotide hybridization related by base-pairing rules. For example, the sequence “C-A-G- T” is complementary to the sequence “G-T-C-A.” Complementarity can be partial or total. Thus, any degree of partial complementarity is intended to be included within the scope of the term “complementary” provided that the partial complementarity permits oligonucleotide hybridization. Partial complementarity is where one or more nucleic acid bases is not matched according to the base pairing rules. Total or complete complementarity between nucleic acids is where each and every nucleic acid base is matched with another base under the base pairing rules. Comparable: As used herein, the term “comparable” refers to two or more agents, entities, situations, sets of conditions, etc., that may not be identical to one another but that are sufficiently similar to permit comparison there between so that one skilled in the art will appreciate that conclusions may reasonably be drawn based on differences or similarities observed. In some embodiments, comparable sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantially identical features and one or a small number of varied features. Those of ordinary skill in the art will understand, in context, what degree of identity is required in any given circumstance for two or more such agents, entities, situations, sets of conditions, etc to be considered comparable. For example, those of ordinary skill in the art will appreciate that sets of circumstances, individuals, or populations are comparable to one another when characterized by a sufficient number and type of substantially identical features to warrant a reasonable conclusion that differences in results obtained or phenomena observed under or with different sets of circumstances, individuals, or populations are caused by or indicative of the variation in those features that are varied. Corresponding to: As used herein, the term “corresponding to” refers to a relationship between two or more entities. For example, the term “corresponding to” may be used to designate the position/identity of a structural element in a compound or composition relative to another compound or composition (e.g., to an appropriate reference compound or composition). For example, in some embodiments, a monomeric residue in a polymer (e.g., an amino acid residue in a polypeptide or a nucleic acid residue in a polynucleotide) may be identified as “corresponding to” a residue in an appropriate reference polymer. For example, those of ordinary skill will appreciate that, for purposes of simplicity, residues in a polypeptide are often designated using a canonical numbering system based on a reference related polypeptide, so that an amino acid "corresponding to" a residue at position 190, for example, need not actually be the 190 ^th amino acid in a particular amino acid chain but rather corresponds to the residue found at 190 in the reference polypeptide; those of ordinary skill in the art readily appreciate how to identify "corresponding" amino acids. For example, those skilled in the art will be aware of various sequence alignment strategies, including software programs such as, for example, BLAST, CS-BLAST, CUSASW++, DIAMOND, FASTA, GGSEARCH/GLSEARCH, Genoogle, HMMER, HHpred/HHsearch, IDF, Infernal, KLAST, USEARCH, parasail, PSI- BLAST, PSI-Search, ScalaBLAST, Sequilab, SAM, SSEARCH, SWAPHI, SWAPHI-LS, SWIMM, or SWIPE that can be utilized, for example, to identify “corresponding” residues in polypeptides and/or nucleic acids in accordance with the present disclosure. Those of skill in the art will also appreciate that, in some instances, the term “corresponding to” may be used to describe an event or entity that shares a relevant similarity with another event or entity (e.g., an appropriate reference event or entity). To give but one example, a gene or protein in one organism may be described as “corresponding to” a gene or protein from another organism in order to indicate, in some embodiments, that it plays an analogous role or performs an analogous function and/or that it shows a particular degree of sequence identity or homology, or shares a particular characteristic sequence element. Designed: As used herein, the term “designed” refers to an agent (i) whose structure is or was selected by the hand of man; (ii) that is produced by a process requiring the hand of man; and/or (iii) that is distinct from natural substances and other known agents. Dosing regimen: Those skilled in the art will appreciate that the term “dosing regimen” may be used to refer to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses. In some embodiments, a dosing regimen comprises a plurality of doses each of which is separated in time from other doses. In some embodiments, individual doses are separated from one another by a time period of the same length; in some embodiments, a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses. In some embodiments, all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount. In some embodiments, a dosing regimen is correlated with a desired or beneficial outcome when administered across a relevant population (i.e., is a therapeutic dosing regimen). Encode: As used herein, the term “encode” or “encoding” refers to sequence information of a first molecule that guides production of a second molecule having a defined sequence of nucleotides (e.g., mRNA) or a defined sequence of amino acids. For example, a DNA molecule can encode an RNA molecule (e.g., by a transcription process that includes a DNA-dependent RNA polymerase enzyme). An RNA molecule can encode a polypeptide (e.g., by a translation process). Thus, a gene, a cDNA, or a single-stranded RNA (e.g., an mRNA) encodes a polypeptide if transcription and translation of mRNA corresponding to that gene produces the polypeptide in a cell or other biological system. In some embodiments, a coding region of a single-stranded RNA encoding a target polypeptide agent refers to a coding strand, the nucleotide sequence of which is identical to the mRNA sequence of such a target polypeptide agent. In some embodiments, a coding region of a single-stranded RNA encoding a target polypeptide agent refers to a non-coding strand of such a target polypeptide agent, which may be used as a template for transcription of a gene or cDNA. Engineered: In general, the term “engineered” refers to the aspect of having been manipulated by the hand of man. For example, a polynucleotide is considered to be “engineered” when two or more sequences that are not linked together in that order in nature are manipulated by the hand of man to be directly linked to one another in the engineered polynucleotide and/or when a particular residue in a polynucleotide is non-naturally occurring and/or is caused through action of the hand of man to be linked with an entity or moiety with which it is not linked in nature. Epitope: as used herein, the term “epitope” refers to a moiety that is specifically recognized by an immunoglobulin (e.g., antibody or receptor) binding component. In some embodiments, an epitope is comprised of a plurality of chemical atoms or groups on an antigen. In some embodiments, such chemical atoms or groups are surface-exposed when the antigen adopts a relevant three-dimensional conformation. In some embodiments, such chemical atoms or groups are physically near to each other in space when the antigen adopts such a conformation. In some embodiments, at least some such chemical atoms are groups are physically separated from one another when the antigen adopts an alternative conformation (e.g., is linearized). Expression: As used herein, the term “expression” of a nucleic acid sequence refers to the generation of any gene product from the nucleic acid sequence. In some embodiments, a gene product can be a transcript. In some embodiments, a gene product can be a polypeptide. In some embodiments, expression of a nucleic acid sequence involves one or more of the following: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, etc); (3) translation of an RNA into a polypeptide or protein; and/or (4) post-translational modification of a polypeptide or protein. Improved, increased or reduced: As used herein, these terms, or grammatically comparable comparative terms, indicate values that are relative to a comparable reference measurement. For example, in some embodiments, an assessed value achieved with an agent of interest may be “improved” relative to that obtained with a comparable reference agent. Alternatively or additionally, in some embodiments, an assessed value achieved in a subject or system of interest may be “improved” relative to that obtained in the same subject or system under different conditions (e.g., prior to or after an event such as administration of an agent of interest), or in a different, comparable subject (e.g., in a comparable subject or system that differs from the subject or system of interest in presence of one or more indicators of a particular disease, disorder or condition of interest, or in prior exposure to a condition or agent, etc.). In some embodiments, comparative terms refer to statistically relevant differences (e.g., that are of a prevalence and/or magnitude sufficient to achieve statistical relevance). Those skilled in the art will be aware, or will readily be able to determine, in a given context, a degree and/or prevalence of difference that is required or sufficient to achieve such statistical significance. In vitro: The term “in vitro” as used herein refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel (e.g., a bioreactor), in cell culture, etc., rather than within a multi-cellular organism. In vitro transcription: As used herein, the term "in vitro transcription" or "IVT" refers to the process whereby transcription occurs in vitro in a non-cellular system to produce a synthetic RNA product for use in various applications, including, e.g., production of protein or polypeptides. Such synthetic RNA products can be translated in vitro or introduced directly into cells, where they can be translated. Such synthetic RNA products include, e.g., but not limited to mRNAs, antisense RNA molecules, shRNA molecules, long non-coding RNA molecules, ribozymes, aptamers, guide RNAs (e.g., for CRISPR), ribosomal RNAs, small nuclear RNAs, small nucleolar RNAs, and the like. An IVT reaction typically utilizes a DNA template (e.g., a linear DNA template) as described and/or utilized herein, ribonucleotides (e.g., non-modified ribonucleotide triphosphates or modified ribonucleotide triphosphates), and an appropriate RNA polymerase. Pharmaceutical composition: As used herein, the term “pharmaceutical composition” refers to an active agent, formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, pharmaceutical compositions may be specially formulated for parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation. Polypeptide: As used herein refers to a polymeric chain of amino acids. In some embodiments, a polypeptide has an amino acid sequence that occurs in nature. In some embodiments, a polypeptide has an amino acid sequence that does not occur in nature. In some embodiments, a polypeptide has an amino acid sequence that is engineered in that it is designed and/or produced through action of the hand of man. In some embodiments, a polypeptide may comprise or consist of natural amino acids, non-natural amino acids, or both. In some embodiments, a polypeptide may comprise or consist of only natural amino acids or only non- natural amino acids. In some embodiments, a polypeptide may comprise D-amino acids, L- amino acids, or both. In some embodiments, a polypeptide may comprise only D-amino acids. In some embodiments, a polypeptide may comprise only L-amino acids. In some embodiments, a polypeptide may include one or more pendant groups or other modifications, e.g., modifying or attached to one or more amino acid side chains, at the polypeptide’s N-terminus, at the polypeptide’s C-terminus, or any combination thereof. In some embodiments, such pendant groups or modifications may be selected from the group consisting of acetylation, amidation, lipidation, methylation, pegylation, etc., including combinations thereof. In some embodiments, a polypeptide may be cyclic, and/or may comprise a cyclic portion. In some embodiments, a polypeptide is not cyclic and/or does not comprise any cyclic portion. In some embodiments, a polypeptide is linear. In some embodiments, a polypeptide may be or comprise a stapled polypeptide. In some embodiments, the term “polypeptide” may be appended to a name of a reference polypeptide, activity, or structure; in such instances it is used herein to refer to polypeptides that share the relevant activity or structure and thus can be considered to be members of the same class or family of polypeptides. For each such class, the present specification provides and/or those skilled in the art will be aware of exemplary polypeptides within the class whose amino acid sequences and/or functions are known; in some embodiments, such exemplary polypeptides are reference polypeptides for the polypeptide class or family. In some embodiments, a member of a polypeptide class or family shows significant sequence homology or identity with, shares a common sequence motif (e.g., a characteristic sequence element) with, and/or shares a common activity (in some embodiments at a comparable level or within a designated range) with a reference polypeptide of the class; in some embodiments with all polypeptides within the class). For example, in some embodiments, a member polypeptide shows an overall degree of sequence homology or identity with a reference polypeptide that is at least about 30-40%, and is often greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more and/or includes at least one region (e.g., a conserved region that may in some embodiments be or comprise a characteristic sequence element) that shows very high sequence identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99%. Such a conserved region usually encompasses at least 3-4 and often up to 20 or more amino acids; in some embodiments, a conserved region encompasses at least one stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids. In some embodiments, a relevant polypeptide may comprise or consist of a fragment of a parent polypeptide. Prevent or prevention: as used herein when used in connection with the occurrence of a disease, disorder, and/or condition, refers to reducing the risk of developing the disease, disorder and/or condition and/or to delaying onset of one or more characteristics or symptoms of the disease, disorder or condition. Prevention may be considered complete when onset of a disease, disorder or condition has been delayed for a predefined period of time. Reference: As used herein describes a standard or control relative to which a comparison is performed. For example, in some embodiments, an agent, animal, individual, population, sample, sequence or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence or value. In some embodiments, a reference or control is tested and/or determined substantially simultaneously with the testing or determination of interest. In some embodiments, a reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as would be understood by those skilled in the art, a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment. Those skilled in the art will appreciate when sufficient similarities are present to justify reliance on and/or comparison to a particular possible reference or control. Ribonucleotide: As used herein, the term “ribonucleotide” encompasses unmodified ribonucleotides and modified ribonucleotides. For example, unmodified ribonucleotides include the purine bases adenine (A) and guanine (G), and the pyrimidine bases cytosine (C) and uracil (U). Modified ribonucleotides may include one or more modifications including, but not limited to, for example, (a) end modifications, e.g., 5' end modifications (e.g., phosphorylation, dephosphorylation, conjugation, inverted linkages, etc.), 3' end modifications (e.g., conjugation, inverted linkages, etc.), (b) base modifications, e.g. , replacement with modified bases, stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, or conjugated bases, (c) sugar modifications (e.g., at the 2' position or 4' position) or replacement of the sugar, and (d) internucleoside linkage modifications, including modification or replacement of the phosphodiester linkages. The term “ribonucleotide” also encompasses ribonucleotide triphosphates including modified and non-modified ribonucleotide triphosphates. Risk: as will be understood from context, “risk” of a disease, disorder, and/or condition refers to a likelihood that a particular individual will develop the disease, disorder, and/or condition. In some embodiments, risk is expressed as a percentage. In some embodiments, risk is from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 up to 100%. In some embodiments risk is expressed as a risk relative to a risk associated with a reference sample or group of reference samples. In some embodiments, a reference sample or group of reference samples have a known risk of a disease, disorder, condition and/or event. In some embodiments a reference sample or group of reference samples are from individuals comparable to a particular individual. In some embodiments, relative risk is 0,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. In some embodiments, risk may reflect one or more genetic attributes, e.g., which may predispose an individual toward development (or not) of a particular disease, disorder and/or condition. In some embodiments, risk may reflect one or more epigenetic events or attributes and/or one or more lifestyle or environmental events or attributes. Susceptible to: An individual who is “susceptible to” a disease, disorder, and/or condition is one who has a higher risk of developing the disease, disorder, and/or condition than does a member of the general public. In some embodiments, an individual who is susceptible to a disease, disorder and/or condition may not have been diagnosed with the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition may exhibit symptoms of the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition may not exhibit symptoms of the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will develop the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will not develop the disease, disorder, and/or condition. Vaccination: As used herein, the term “vaccination” refers to the administration of a composition intended to generate an immune response, for example to a disease-associated (e.g., disease-causing) agent. In some embodiments, vaccination can be administered before, during, and/or after exposure to a disease-associated agent, and in certain embodiments, before, during, and/or shortly after exposure to the agent. In some embodiments, vaccination includes multiple administrations, appropriately spaced in time, of a vaccine composition. In some embodiments, vaccination generates an immune response to an infectious agent. In some embodiments, vaccination generates an immune response to a tumor; in some such embodiments, vaccination is “personalized” in that it is partly or wholly directed to epitope(s) (e.g., which may be or include one or more neoepitopes) determined to be present in a particular individual’s tumors. Variant: As used herein in the context of molecules, e.g., nucleic acids, proteins, or small molecules, the term “variant” refers to a molecule that shows significant structural identity with a reference molecule but differs structurally from the reference molecule, e.g., in the presence or absence or in the level of one or more chemical moieties as compared to the reference entity. In some embodiments, a variant also differs functionally from its reference molecule. In general, whether a particular molecule is properly considered to be a “variant” of a reference molecule is based on its degree of structural identity with the reference molecule. As will be appreciated by those skilled in the art, any biological or chemical reference molecule has certain characteristic structural elements. A variant, by definition, is a distinct molecule that shares one or more such characteristic structural elements but differs in at least one aspect from the reference molecule. In some embodiments, a variant polypeptide or nucleic acid may differ from a reference polypeptide or nucleic acid as a result of one or more differences in amino acid or nucleotide sequence and/or one or more differences in chemical moieties (e.g., carbohydrates, lipids, phosphate groups) that are covalently components of the polypeptide or nucleic acid (e.g., that are attached to the polypeptide or nucleic acid backbone). In some embodiments, a variant polypeptide or nucleic acid shows an overall sequence identity with a reference polypeptide or nucleic acid that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%. In some embodiments, a variant polypeptide or nucleic acid does not share at least one characteristic sequence element with a reference polypeptide or nucleic acid. In some embodiments, a reference polypeptide or nucleic acid has one or more biological activities. In some embodiments, a variant polypeptide or nucleic acid shares one or more of the biological activities of the reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid lacks one or more of the biological activities of the reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid shows a reduced level of one or more biological activities as compared to the reference polypeptide or nucleic acid. In some embodiments, a polypeptide or nucleic acid of interest is considered to be a “variant” of a reference polypeptide or nucleic acid if it has an amino acid or nucleotide sequence that is identical to that of the reference but for a small number of sequence alterations at particular positions. Typically, fewer than about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, or about 2% of the residues in a variant are substituted, inserted, or deleted, as compared to the reference. In some embodiments, a variant polypeptide or nucleic acid comprises about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 substituted residues as compared to a reference. Often, a variant polypeptide or nucleic acid comprises a very small number (e.g., fewer than about 5, about 4, about 3, about 2, or about 1) number of substituted, inserted, or deleted, functional residues (i.e., residues that participate in a particular biological activity) relative to the reference. In some embodiments, a variant polypeptide or nucleic acid comprises not more than about 5, about 4, about 3, about 2, or about 1 addition or deletion, and, in some embodiments, comprises no additions or deletions, as compared to the reference. In some embodiments, a variant polypeptide or nucleic acid comprises fewer than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6, and commonly fewer than about 5, about 4, about 3, or about 2 additions or deletions as compared to the reference. In some embodiments, a reference polypeptide or nucleic acid is one found in nature. DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS The present disclosure provides, among other things, an RNA polynucleotide comprising (i) a 5’ cap; (ii) a 5’ UTR sequence comprising a cap proximal sequence, e.g., as disclosed herein; and (iii) a sequence encoding a payload. Also provided herein are compositions and medical preparations comprising the same, as well as methods of making and using the same. In some embodiments, translation efficiency of an RNA encoding a payload, and/or expression of a payload encoded by an RNA, can be improved with an RNA polynucleotide comprising a 5’ cap having a structure disclosed herein; a 5’ UTR comprising a cap proximal sequence disclosed herein, and a sequence encoding a payload. In some embodiments, absence of a self-hybridizing sequence in an RNA polynucleotide encoding a payload can further improve translation efficiency of an RNA encoding a payload, and/or expression of a payload encoded by an RNA payload. RNA polynucleotides The term "polynucleotide" or "nucleic acid", as used herein, refers to DNA and RNA such as genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules. A nucleic acid may be single-stranded or double-stranded. RNA includes synthetic RNA. In some embodiments, synthetic RNA is or comprises in vitro transcribed RNA (IVT RNA). According to the invention, a polynucleotide is preferably isolated. In some embodiments, nucleic acids may be comprised in a vector. The term "vector" as used herein includes any vectors known to the skilled person including plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as retroviral, adenoviral or baculoviral vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or P1 artificial chromosomes (PAC). In some embodiments, a vector may be an expression vector; alternatively or additionally, in some embodiments, a vector may be a cloning vector. Those skilled in the art will appreciate that, in some embodiments, an expression vector may be, for example, a plasmid; alternatively or additionally, in some embodiments, an expression vector may be a viral vector. Typically, an expression vector will contain a desired coding sequence and appropriate other sequences necessary for the expression of the operably linked coding sequence in a particular host organism (e.g., bacteria, yeast, plant, insect, or mammal) or in in vitro expression systems. Cloning vectors are generally used to engineer and amplify a certain desired fragment (typically a DNA fragment), and may lack functional sequences needed for expression of the desired fragment(s). In some embodiments, a nucleic acid as described and/or utilized herein may be or comprise recombinant and/or isolated molecules. Those skilled in the art, reading the present disclosure, will understand that the term "RNA" typically refers to a nucleic acid molecule which includes ribonucleotide residues. In some embodiments, an RNA contains all or a majority of ribonucleotide residues. As used herein, "ribonucleotide" refers to a nucleotide with a hydroxyl group at the 2'-position of a β-D- ribofuranosyl group. In some embodiments, an RNA may be partly or fully double stranded RNA; in some embodiments, an RNA may comprise two or more distinct nucleic acid strands (e.g., separate molecules) that are partly or fully hybridized with one another. In many embodiments, an RNA is a single strand, which may in some embodiments, self-hybridize or otherwise fold into secondary and/or tertiary structures. In some embodiments, an RNA as described and/or utilized herein does not self-hybridize, at least with respect to certain sequences as described herein. In some embodiments, an RNA may be an isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, and/or a modified RNA (where the term “modified” is understood to indicate that one or more residues or other structural elements of the RNA differs from naturally occurring RNA; for example, in some embodiments, a modified RNA differs by the addition, deletion, substitution and/or alteration of one or more nucleotides and/or by one or more moieties or characteristics of a nucleotide- e.g., of a nucleoside or of a backbone structure or linkage). In some embodiments, a modification may be or comprise addition of non-nucleotide material to internal RNA nucleotides or to the end(s) of RNA. It is also contemplated herein that nucleotides in RNA (e.g., in a modified RNA) may be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. For the present disclosure, these altered RNAs are considered analogs of naturally-occurring RNA. As appreciated by a person skilled in the art, the RNA polynucleotides disclosed herein can comprise or consist of naturally occurring ribonucleotides and/or modified ribonucleotides. Therefore, a person skilled in the art will understand references to A, U, G, or C throughout the specification described herein can refer to a naturally occurring ribonucleotide and/or a modified ribonucleotide described herein. For example, in some embodiments, a U is uridine. In some embodiments, a U is modified uridine (e.g., pseudouridine, 1-methyl pseudouridine). In some embodiments of the present disclosure, an RNA is or comprises messenger RNA (mRNA) that relates to an RNA transcript which encodes a polypeptide. In some embodiments, an RNA disclosed herein comprises: a 5’ cap disclosed herein; a 5' untranslated region comprising a cap proximal sequence (5'-UTR), a sequence encoding a payload (e.g., a polypeptide); a 3' untranslated region (3'-UTR); and/or a polyadenylate (PolyA) sequence. In some embodiments, an RNA disclosed herein comprises the following components in 5’ to 3’ orientation: a 5’ cap disclosed herein; a 5' untranslated region comprising a cap proximal sequence (5'-UTR), a sequence encoding a payload (e.g., a polypeptide); a 3' untranslated region (3'-UTR); and a PolyA sequence. In some embodiments, an RNA is produced by in vitro transcription or chemical synthesis. In some embodiments, an mRNA is produced by in vitro transcription using a DNA template where DNA refers to a nucleic acid that contains deoxyribonucleotides. In some embodiments, an RNA disclosed herein is in vitro transcribed RNA (IVT-RNA) and may be obtained by in vitro transcription of an appropriate DNA template. The promoter for controlling transcription can be any promoter for any RNA polymerase. A DNA template for in vitro transcription may be obtained by cloning of a nucleic acid, in particular cDNA, and introducing it into an appropriate vector for in vitro transcription. The cDNA may be obtained by reverse transcription of RNA. In some embodiments, an RNA is "replicon RNA" or simply a "replicon", in particular "self-replicating RNA" or "self-amplifying RNA". In some embodiments, a replicon or self- replicating RNA is derived from or comprises elements derived from a ssRNA virus, in particular a positive-stranded ssRNA virus such as an alphavirus. Alphaviruses are typical representatives of positive-stranded RNA viruses. Alphaviruses replicate in the cytoplasm of infected cells (for review of the alphaviral life cycle see José et al., Future Microbiol., 2009, vol. 4, pp.837–856). The total genome length of many alphaviruses typically ranges between 11,000 and 12,000 nucleotides, and the genomic RNA typically has a 5’-cap and a 3’ poly(A) tail. The genome of alphaviruses encodes non-structural proteins (involved in transcription, modification and replication of viral RNA and in protein modification) and structural proteins (forming the virus particle). There are typically two open reading frames (ORFs) in the genome. The four non-structural proteins (nsP1–nsP4) are typically encoded together by a first ORF beginning near the 5′ terminus of the genome, while alphavirus structural proteins are encoded together by a second ORF which is found downstream of the first ORF and extends near the 3’ terminus of the genome. Typically, the first ORF is larger than the second ORF, the ratio being roughly 2:1. In cells infected by an alphavirus, only the nucleic acid sequence encoding non-structural proteins is translated from the genomic RNA, while the genetic information encoding structural proteins is translatable from a subgenomic transcript, which is an RNA polynucleotide that resembles eukaryotic messenger RNA (mRNA; Gould et al., 2010, Antiviral Res., vol.87 pp.111–124). Following infection, i.e. at early stages of the viral life cycle, the (+) stranded genomic RNA directly acts like a messenger RNA for the translation of the open reading frame encoding the non-structural poly-protein (nsP1234). Alphavirus-derived vectors have been proposed for delivery of foreign genetic information into target cells or target organisms. In simple approaches, the open reading frame encoding alphaviral structural proteins is replaced by an open reading frame encoding a protein of interest. Alphavirus-based trans-replication systems rely on alphavirus nucleotide sequence elements on two separate nucleic acid molecules: one nucleic acid molecule encodes a viral replicase, and the other nucleic acid molecule is capable of being replicated by said replicase in trans (hence the designation trans-replication system). Trans-replication requires the presence of both these nucleic acid molecules in a given host cell. The nucleic acid molecule capable of being replicated by the replicase in trans must comprise certain alphaviral sequence elements to allow recognition and RNA synthesis by the alphaviral replicase. In some embodiments, an RNA described herein may have modified nucleosides. In some embodiments, an RNA comprises a modified nucleoside in place of at least one (e.g., every) uridine.

The term "uracil," as used herein, describes one of the nucleobases that can occur in the nucleic acid of RNA. The structure of uracil is: . The term "uridine," as used herein, describes one of the nucleosides that can occur in RNA. The structure of uridine is: . UTP (uridine 5’-triphosphate) has the following structure: . Pseudo-UTP (pseudouridine-5’-triphosphate) has the following structure: . "Pseudouridine" is one example of a modified nucleoside that is an isomer of uridine, where the uracil is attached to the pentose ring via a carbon-carbon bond instead of a nitrogen-carbon glycosidic bond. Another exemplary modified nucleoside is N1-methylpseudouridine (m1Ψ), which has the structure: . N1-methylpseudouridine-5’-triphosphate (m1ΨTP) has the following structure: . Another exemplary modified nucleoside is 5-methyluridine (m5U), which has the structure: . In some embodiments, one or more uridines in an RNA described herein is replaced by a modified nucleoside. In some embodiments, a modified nucleoside is a modified uridine. In some embodiments, an RNA comprises a modified nucleoside in place of at least one uridine. In some embodiments, an RNA comprises a modified nucleoside in place of each uridine. In some embodiments, a modified nucleoside is independently selected from pseudouridine (Ψ), N1-methylpseudouridine (m1Ψ), and 5-methyluridine (m5U). In some embodiments, a modified nucleoside comprises pseudouridine (Ψ). In some embodiments, a modified nucleoside comprises N1-methyl-pseudouridine (m1Ψ). In some embodiments, a modified nucleoside comprises 5-methyluridine (m5U). In some embodiments, an RNA may comprise more than one type of modified nucleoside, and the modified nucleosides are independently selected from pseudouridine (Ψ), N1-methylpseudouridine (m1Ψ), and 5- methyluridine (m5U). In some embodiments, the modified nucleosides comprise pseudouridine (Ψ) and N1-methylpseudouridine (m1Ψ). In some embodiments, the modified nucleosides comprise pseudouridine (Ψ) and 5-methyluridine (m5U). In some embodiments, the modified nucleosides comprise N1-methylpseudouridine (m1Ψ) and 5-methyluridine (m5U). In some embodiments, the modified nucleosides comprise pseudouridine (Ψ), N1-methylpseudouridine (m1Ψ), and 5-methyluridine (m5U). In some embodiments, a modified nucleoside replacing one or more, e.g., all, uridines in the RNA may be any one or more of 3-methyl-uridine (m ³ U), 5-methoxy-uridine (mo ⁵ U), 5-aza- uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s ² U), 4-thio-uridine (s ⁴ U), 4-thio- pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho ⁵ U), 5-aminoallyl-uridine, 5-halo- uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5-oxyacetic acid (cmo ⁵ U), uridine 5- oxyacetic acid methyl ester (mcmo ⁵ U), 5-carboxymethyl-uridine (cm ⁵ U), 1-carboxymethyl- pseudouridine, 5-carboxyhydroxymethyl-uridine (chm ⁵ U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm ⁵ U), 5-methoxycarbonylmethyl-uridine (mcm ⁵ U), 5- methoxycarbonylmethyl-2-thio-uridine (mcm ⁵ s ² U), 5-aminomethyl-2-thio-uridine (nm ⁵ s ² U), 5- methylaminomethyl-uridine (mnm ⁵ U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio- uridine (mnm ⁵ s ² U), 5-methylaminomethyl-2-seleno-uridine (mnm ⁵ se ² U), 5-carbamoylmethyl- uridine (ncm ⁵ U), 5-carboxymethylaminomethyl-uridine (cmnm ⁵ U), 5- carboxymethylaminomethyl-2-thio-uridine (cmnm ⁵ s ² U), 5-propynyl-uridine, 1-propynyl- pseudouridine, 5-taurinomethyl-uridine (τm ⁵ U), 1-taurinomethyl-pseudouridine, 5- taurinomethyl-2-thio-uridine(τm5s2U), 1-taurinomethyl-4-thio-pseudouridine), 5-methyl-2-thio- uridine (m ⁵ s ² U), 1-methyl-4-thio-pseudouridine (m ¹ s ⁴ ψ), 4-thio-1-methyl-pseudouridine, 3- methyl-pseudouridine (m ³ ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6- dihydrouridine, 5-methyl-dihydrouridine (m ⁵ D), 2-thio-dihydrouridine, 2-thio- dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3- carboxypropyl)uridine (acp ³ U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp ³ ψ), 5-(isopentenylaminomethyl)uridine (inm ⁵ U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm ⁵ s ² U), α-thio-uridine, 2′-O-methyl-uridine (Um), 5,2′-O-dimethyl-uridine (m ⁵ Um), 2′-O- methyl-pseudouridine (ψm), 2-thio-2′-O-methyl-uridine (s ² Um), 5-methoxycarbonylmethyl-2′- O-methyl-uridine (mcm ⁵ Um), 5-carbamoylmethyl-2′-O-methyl-uridine (ncm ⁵ Um), 5- carboxymethylaminomethyl-2′-O-methyl-uridine (cmnm ⁵ Um), 3,2′-O-dimethyl-uridine (m ³ Um), 5-(isopentenylaminomethyl)-2′-O-methyl-uridine (inm ⁵ Um), 1-thio-uridine, deoxythymidine, 2′- F-ara-uridine, 2′-F-uridine, 2′-OH-ara-uridine, 5-(2-carbomethoxyvinyl) uridine, 5-[3-(1-E- propenylamino)uridine, or any other modified uridine known in the art. In some embodiments, an RNA comprises other modified nucleosides or comprises further modified nucleosides, e.g., modified cytidine. For example, in some embodiments of an RNA, 5- methylcytidine is substituted partially or completely, preferably completely, for cytidine. In some embodiments, an RNA comprises 5-methylcytidine and one or more nucleosides selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U). In some embodiments, an RNA comprises 5-methylcytidine and N1-methyl-pseudouridine (m1ψ). In some embodiments, the RNA comprises 5-methylcytidine in place of each cytidine and N1-methyl- pseudouridine (m1ψ) in place of each uridine. In some embodiments, an RNA encoding a payload, e.g., a vaccine antigen, is expressed in cells of a subject treated to provide a payload, e.g., vaccine antigen. In some embodiments, the RNA is transiently expressed in cells of the subject. In some embodiments, the RNA is in vitro transcribed RNA. In some embodiments, expression of a payload, e.g., a vaccine antigen is at the cell surface. In some embodiments, a payload, e.g., a vaccine antigen is expressed and presented in the context of MHC. In some embodiments, expression of a payload, e.g., a vaccine antigen is into the extracellular space, i.e., the vaccine antigen is secreted. In the context of the present disclosure, the term "transcription" relates to a process, wherein the genetic code in a DNA sequence is transcribed into RNA. Subsequently, the RNA may be translated into peptide or protein. According to the present invention, the term "transcription" comprises "in vitro transcription", wherein the term "in vitro transcription" relates to a process wherein RNA, in particular mRNA, is in vitro synthesized in a cell-free system, preferably using appropriate cell extracts. Preferably, cloning vectors are applied for the generation of transcripts. These cloning vectors are generally designated as transcription vectors and are according to the present invention encompassed by the term "vector". According to the present invention, the RNA used in the present invention preferably is in vitro transcribed RNA (IVT-RNA) and may be obtained by in vitro transcription of an appropriate DNA template. The promoter for controlling transcription can be any promoter for any RNA polymerase. Particular examples of RNA polymerases are the T7, T3, and SP6 RNA polymerases. Preferably, the in vitro transcription according to the invention is controlled by a T7 or SP6 promoter. A DNA template for in vitro transcription may be obtained by cloning of a nucleic acid, in particular cDNA, and introducing it into an appropriate vector for in vitro transcription. The cDNA may be obtained by reverse transcription of RNA. With respect to RNA, the term "expression" or "translation" relates to the process in the ribosomes of a cell by which a strand of mRNA directs the assembly of a sequence of amino acids to make a peptide or protein. In some embodiments, after administration of an RNA described herein, e.g., formulated as RNA lipid particles, at least a portion of the RNA is delivered to a target cell. In some embodiments, at least a portion of the RNA is delivered to the cytosol of the target cell. In some embodiments, the RNA is translated by the target cell to produce the peptide or protein it encodes. In some embodiments, the target cell is a spleen cell. In some embodiments, the target cell is an antigen presenting cell such as a professional antigen presenting cell in the spleen. In some embodiments, the target cell is a dendritic cell or macrophage. RNA particles such as RNA lipid particles described herein may be used for delivering RNA to such target cell. Accordingly, the present disclosure also relates to a method for delivering RNA to a target cell in a subject comprising the administration of the RNA particles described herein to the subject. In some embodiments, the RNA is delivered to the cytosol of the target cell. In some embodiments, the RNA is translated by the target cell to produce the peptide or protein encoded by the RNA. "Encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA. In some embodiments, nucleic acid compositions described herein, e.g., compositions comprising a lipid nanoparticle encapsulated mRNA are characterized by (e.g., when administered to a subject) sustained expression of an encoded polypeptide. For example, in some embodiments, such compositions are characterized in that, when administered to a human, they achieve detectable polypeptide expression in a biological sample (e.g., serum) from such human and, in some embodiments, such expression persists for a period of time that is at least 36 hours or longer, including, e.g., at least 48 hours, at least 60 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 148 hours, or longer. In some embodiments, an RNA encoding a payload to be administered according to the present disclosure is non-immunogenic. RNA encoding immunostimulant may be administered according to the invention to provide an adjuvant effect. The RNA encoding immunostimulant may be standard RNA or non-immunogenic RNA. The term "non-immunogenic RNA" as used herein refers to RNA that does not induce a response by the immune system upon administration, e.g., to a mammal, or induces a weaker response than would have been induced by the same RNA that differs only in that it has not been subjected to the modifications and treatments that render the immunogenic RNA non- immunogenic, i.e., than would have been induced by standard RNA (stdRNA). In one preferred embodiment, non-immunogenic RNA, which is also termed modified RNA (modRNA) herein, is rendered non-immunogenic by incorporating modified nucleosides suppressing RNA-mediated activation of innate immune receptors into the RNA and removing double-stranded RNA (dsRNA). For rendering the immunogenic RNA non-immunogenic by the incorporation of modified nucleosides, any modified nucleoside may be used as long as it lowers or suppresses immunogenicity of the RNA. Particularly preferred are modified nucleosides that suppress RNA- mediated activation of innate immune receptors. In some embodiments, the modified nucleosides comprise a replacement of one or more uridines with a nucleoside comprising a modified nucleobase. In some embodiments, the modified nucleobase is a modified uracil. In some embodiments, the nucleoside comprising a modified nucleobase is selected from the group consisting of 3-methyl-uridine (m ³ U), 5-methoxy-uridine (mo ⁵ U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s ² U), 4-thio-uridine (s ⁴ U), 4-thio-pseudouridine, 2-thio- pseudouridine, 5-hydroxy-uridine (ho ⁵ U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo- uridine or 5-bromo-uridine), uridine 5-oxyacetic acid (cmo ⁵ U), uridine 5-oxyacetic acid methyl ester (mcmo ⁵ U), 5-carboxymethyl-uridine (cm ⁵ U), 1-carboxymethyl-pseudouridine, 5- carboxyhydroxymethyl-uridine (chm ⁵ U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm ⁵ U), 5-methoxycarbonylmethyl-uridine (mcm ⁵ U), 5-methoxycarbonylmethyl-2-thio- uridine (mcm ⁵ s ² U), 5-aminomethyl-2-thio-uridine (nm ⁵ s ² U), 5-methylaminomethyl-uridine (mnm ⁵ U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine (mnm ⁵ s ² U), 5- methylaminomethyl-2-seleno-uridine (mnm ⁵ se ² U), 5-carbamoylmethyl-uridine (ncm ⁵ U), 5- carboxymethylaminomethyl-uridine (cmnm ⁵ U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm ⁵ s ² U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (τm ⁵ U), 1- taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine(τm5s2U), 1-taurinomethyl-4-thio- pseudouridine), 5-methyl-2-thio-uridine (m ⁵ s ² U), 1-methyl-4-thio-pseudouridine (m ¹ s ⁴ ψ), 4-thio- 1-methyl-pseudouridine, 3-methyl-pseudouridine (m ³ ψ), 2-thio-1-methyl-pseudouridine, 1- methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m ⁵ D), 2-thio- dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4- methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3- amino-3-carboxypropyl)uridine (acp ³ U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp ³ ψ), 5-(isopentenylaminomethyl)uridine (inm ⁵ U), 5-(isopentenylaminomethyl)-2-thio- uridine (inm ⁵ s ² U), α-thio-uridine, 2′-O-methyl-uridine (Um), 5,2′-O-dimethyl-uridine (m ⁵ Um), 2′-O-methyl-pseudouridine (ψm), 2-thio-2′-O-methyl-uridine (s ² Um), 5- methoxycarbonylmethyl-2′-O-methyl-uridine (mcm ⁵ Um), 5-carbamoylmethyl-2′-O-methyl- uridine (ncm ⁵ Um), 5-carboxymethylaminomethyl-2′-O-methyl-uridine (cmnm ⁵ Um), 3,2′-O- dimethyl-uridine (m ³ Um), 5-(isopentenylaminomethyl)-2′-O-methyl-uridine (inm ⁵ Um), 1-thio- uridine, deoxythymidine, 2′-F-ara-uridine, 2′-F-uridine, 2′-OH-ara-uridine, 5-(2- carbomethoxyvinyl)uridine, and 5-[3-(1-E-propenylamino)uridine. In one particularly preferred embodiment, the nucleoside comprising a modified nucleobase is pseudouridine (ψ), N1-methyl- pseudouridine (m1ψ) or 5-methyl-uridine (m5U), in particular N1-methyl-pseudouridine. In some embodiments, the replacement of one or more uridines with a nucleoside comprising a modified nucleobase comprises a replacement of at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 25%, at least 50%, at least 75%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of the uridines. During synthesis of mRNA by in vitro transcription (IVT) using T7 RNA polymerase significant amounts of aberrant products, including double-stranded RNA (dsRNA) are produced due to unconventional activity of the enzyme. dsRNA induces inflammatory cytokines and activates effector enzymes leading to protein synthesis inhibition. dsRNA can be removed from RNA such as IVT RNA, for example, by ion-pair reversed phase HPLC using a non-porous or porous C-18 polystyrene-divinylbenzene (PS-DVB) matrix. Alternatively, an enzymatic based method using E. coli RNaseIII that specifically hydrolyzes dsRNA but not ssRNA, thereby eliminating dsRNA contaminants from IVT RNA preparations can be used. Furthermore, dsRNA can be separated from ssRNA by using a cellulose material. In some embodiments, an RNA preparation is contacted with a cellulose material and the ssRNA is separated from the cellulose material under conditions which allow binding of dsRNA to the cellulose material and do not allow binding of ssRNA to the cellulose material. As the term is used herein, "remove" or "removal" refers to the characteristic of a population of first substances, such as non-immunogenic RNA, being separated from the proximity of a population of second substances, such as dsRNA, wherein the population of first substances is not necessarily devoid of the second substance, and the population of second substances is not necessarily devoid of the first substance. However, a population of first substances characterized by the removal of a population of second substances has a measurably lower content of second substances as compared to the non-separated mixture of first and second substances. In some embodiments, the removal of dsRNA from non-immunogenic RNA comprises a removal of dsRNA such that less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.3%, or less than 0.1% of the RNA in the non- immunogenic RNA composition is dsRNA. In some embodiments, the non-immunogenic RNA is free or essentially free of dsRNA. In some embodiments, the non-immunogenic RNA composition comprises a purified preparation of single-stranded nucleoside modified RNA. For example, in some embodiments, the purified preparation of single-stranded nucleoside modified RNA is substantially free of double stranded RNA (dsRNA). In some embodiments, the purified preparation is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% single stranded nucleoside modified RNA, relative to all other nucleic acid molecules (DNA, dsRNA, etc.). In some embodiments, the non-immunogenic RNA is translated in a cell more efficiently than standard RNA with the same sequence. In some embodiments, translation is enhanced by a factor of 2-fold relative to its unmodified counterpart. In some embodiments, translation is enhanced by a 3-fold factor. In some embodiments, translation is enhanced by a 4-fold factor. In some embodiments, translation is enhanced by a 5-fold factor. In some embodiments, translation is enhanced by a 6-fold factor. In some embodiments, translation is enhanced by a 7-fold factor. In some embodiments, translation is enhanced by an 8-fold factor. In some embodiments, translation is enhanced by a 9-fold factor. In some embodiments, translation is enhanced by a 10- fold factor. In some embodiments, translation is enhanced by a 15-fold factor. In some embodiments, translation is enhanced by a 20-fold factor. In some embodiments, translation is enhanced by a 50-fold factor. In some embodiments, translation is enhanced by a 100-fold factor. In some embodiments, translation is enhanced by a 200-fold factor. In some embodiments, translation is enhanced by a 500-fold factor. In some embodiments, translation is enhanced by a 1000-fold factor. In some embodiments, translation is enhanced by a 2000-fold factor. In some embodiments, the factor is 10-1000-fold. In some embodiments, the factor is 10-100-fold. In some embodiments, the factor is 10-200-fold. In some embodiments, the factor is 10-300-fold. In some embodiments, the factor is 10-500-fold. In some embodiments, the factor is 20-1000-fold. In some embodiments, the factor is 30-1000-fold. In some embodiments, the factor is 50-1000- fold. In some embodiments, the factor is 100-1000-fold. In some embodiments, the factor is 200- 1000-fold. In some embodiments, translation is enhanced by any other significant amount or range of amounts. In some embodiments, the non-immunogenic RNA exhibits significantly less innate immunogenicity than standard RNA with the same sequence. In some embodiments, the non- immunogenic RNA exhibits an innate immune response that is 2-fold less than its unmodified counterpart. In some embodiments, innate immunogenicity is reduced by a 3-fold factor. In some embodiments, innate immunogenicity is reduced by a 4-fold factor. In some embodiments, innate immunogenicity is reduced by a 5-fold factor. In some embodiments, innate immunogenicity is reduced by a 6-fold factor. In some embodiments, innate immunogenicity is reduced by a 7-fold factor. In some embodiments, innate immunogenicity is reduced by a 8-fold factor. In some embodiments, innate immunogenicity is reduced by a 9-fold factor. In some embodiments, innate immunogenicity is reduced by a 10-fold factor. In some embodiments, innate immunogenicity is reduced by a 15-fold factor. In some embodiments, innate immunogenicity is reduced by a 20- fold factor. In some embodiments, innate immunogenicity is reduced by a 50-fold factor. In some embodiments, innate immunogenicity is reduced by a 100-fold factor. In some embodiments, innate immunogenicity is reduced by a 200-fold factor. In some embodiments, innate immunogenicity is reduced by a 500-fold factor. In some embodiments, innate immunogenicity is reduced by a 1000-fold factor. In some embodiments, innate immunogenicity is reduced by a 2000-fold factor. The term "exhibits significantly less innate immunogenicity" refers to a detectable decrease in innate immunogenicity. In some embodiments, the term refers to a decrease such that an effective amount of the non-immunogenic RNA can be administered without triggering a detectable innate immune response. In some embodiments, the term refers to a decrease such that the non-immunogenic RNA can be repeatedly administered without eliciting an innate immune response sufficient to detectably reduce production of the protein encoded by the non- immunogenic RNA. In some embodiments, the decrease is such that the non-immunogenic RNA can be repeatedly administered without eliciting an innate immune response sufficient to eliminate detectable production of the protein encoded by the non-immunogenic RNA. "Immunogenicity" is the ability of a foreign substance, such as RNA, to provoke an immune response in the body of a human or other animal. The innate immune system is the component of the immune system that is relatively unspecific and immediate. It is one of two main components of the vertebrate immune system, along with the adaptive immune system. As used herein "endogenous" refers to any material from or produced inside an organism, cell, tissue or system. As used herein, the term "exogenous" refers to any material introduced from or produced outside an organism, cell, tissue or system. The term "expression" as used herein is defined as the transcription and/or translation of a particular nucleotide sequence. As used herein, the terms "linked," "fused", or "fusion" are used interchangeably. These terms refer to the joining together of two or more elements or components or domains. In some embodiments, the present disclosure provides an RNA polynucleotide comprising: a 5’ cap; a cap proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; and a sequence encoding a payload, wherein: (i) the 5’ cap is a trinucleotide cap structure comprises N1pN2, wherein N1 is position +1 of the RNA polynucleotide and N ₂ is position +2 of the RNA polynucleotide, and wherein N1 is A or an analog thereof; and N ₂ is U or an analog thereof; and (ii) the cap proximal sequence comprises: N1 and N2 of the trinucleotide cap structure and a sequence comprising N3N4N5 at positions +3, +4, and +5 respectively of the RNA polynucleotide, wherein N3, N4, and N5 are each independently selected from: A, C, G, and U. Codon optimization In some embodiments, a payload (e.g., a polypeptide) described herein is encoded by a coding sequence which is codon-optimized and/or the G/C content of which is increased compared to wild type coding sequence. In some embodiments, one or more sequence regions of the coding sequence are codon-optimized and/or increased in the G/C content compared to the corresponding sequence regions of the wild type coding sequence. In some embodiments, codon- optimization and/or increased the G/C content does not change the sequence of the encoded amino acid sequence. The term "codon-optimized" is understood by those in the art to refer to alteration of codons in the coding region of a nucleic acid molecule to reflect the typical codon usage of a host organism without preferably altering the amino acid sequence encoded by the nucleic acid molecule. Within the context of the present disclosure, coding regions are preferably codon- optimized for optimal expression in a subject to be treated using an RNA polynucleotide described herein. Codon-optimization is based on the finding that the translation efficiency is also determined by a different frequency in the occurrence of tRNAs in cells. Thus, the sequence of RNA may be modified such that codons for which frequently occurring tRNAs are available are inserted in place of "rare codons". In some embodiments, guanosine/cytidine (G/C) content of a coding region (e.g., of a payload sequence) of an RNA is increased compared to the G/C content of the corresponding coding sequence of a wild type RNA encoding the payload, wherein the amino acid sequence encoded by the RNA is preferably not modified compared to the amino acid sequence encoded by the wild type RNA. This modification of the RNA sequence is based on the fact that the sequence of any RNA region to be translated is important for efficient translation of that mRNA. Sequences having an increased G (guanosine)/C (cytidine) content are more stable than sequences having an increased A (adenosine)/U (uridine) content. In respect to the fact that several codons code for one and the same amino acid (so-called degeneration of the genetic code), the most favourable codons for the stability can be determined (so-called alternative codon usage). Depending on the amino acid to be encoded by the RNA, there are various possibilities for modification of the RNA sequence, compared to its wild type sequence. In particular, codons which contain A and/or U nucleosides can be modified by substituting these codons by other codons, which code for the same amino acids but contain no A and/or U or contain a lower content of A and/or U nucleosides. In some embodiments, G/C content of a coding region of an RNA described herein is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, or even more compared to the G/C content of a coding region of a wild type RNA. 5’ cap A structural feature of mRNAs is a cap structure at the five-prime (5’) terminus. Natural eukaryotic mRNA comprises a 7-methylguanosine cap linked to the mRNA via a 5´ to 5´- triphosphate bridge resulting in a cap0 structure (m7GpppN). In most eukaryotic mRNA and some viral mRNA, further modifications can occur at the 2'-hydroxy-group (2’-OH) (e.g., the 2'- hydroxyl group may be methylated to form 2'-O-Me) of the first and subsequent nucleotides producing “cap1” and “cap2” five-prime ends, respectively. Diamond, et al., (2014) Cytokine & growth Factor Reviews, 25:543–550 reported that cap0-mRNA cannot be translated as efficiently as cap1-mRNA in which the role of 2'-O-Me in the penultimate position at the mRNA 5’ end is determinant. Lack of the 2'-O-Me has been shown to trigger innate immunity and activate IFN response. Daffis, et al. (2010) Nature, 468:452-456; and Züst et al. (2011) Nature Immunology, 12:137-143. RNA capping is well researched and is described, e.g., in Decroly E et al. (2012) Nature Reviews 10: 51-65; and in Ramanathan A. et al., (2016) Nucleic Acids Res; 44(16): 7511–7526, the entire contents of each of which is hereby incorporated by reference. In some embodiments, to imitate the 5’ cap structure of natural mRNA, in vitro-transcribed mRNA (IVT mRNA) can be capped either post-transcriptionally using recombinant Vaccinia virus-derived enzymes (see., e.g., Kyrieleis, et al. (1993) Structure 22:452-465; and Corbett, et al. (2020) The New England Journal of Medicine 383:1544-1555) or co-transcriptionally by adding cap analogs immediately into the in vitro transcription reaction (see, e.g., Jemielity, et al. (2003) RNA 9:1108-1122; and Kocmik, et al. (2018) Cell Cycle 17:1624-1636). In some embodiments, enzymatic capping can yield cap1-mRNA, but can be time-consuming since it requires an extra purification step and demands a heating step to improve the accessibility of structured 5’ends, thereby further increasing the risk of RNA degradation. Among other things, cotranscriptional capping can be highly reproducible and less expensive than enzymatic capping. mRNA generated in the presence of cap analogs can be resistant to the human decapping enzymes ( see, e.g., Kowalska et al. (2008) RNA 14:1119-1131) and/or interferon-induced proteins with tetratricopeptide repeats (IFITs) which inhibits cap0-dependent translation (see, e.g., Diamond et al. (2014) Cytokine & Growth Factor Reviews 25:543-550; and Miedziak, et al. (2019) RNA 26:58-68). However, in cotranscriptional capping, GTP is typically competing with cap analogs during transcription, which can lead to poor capping efficiency and result inweak translational capacity. Certain cap1 structures can be incorporated into IVT mRNA in the right orientation for producing cap1-mRNA with high capping efficiency in a rapid co-transcriptional reaction. See, e.g., Henderson et al., (2021) Current Protocols 1:e39. For example, a trinucleotide cap1 structure comprising an AG initiator can reduce the slippage of RNA polymerases on a DNA template strand (e.g., as compared to a DNA template containing a G triplet as a transcriptional start site). See, e.g., Imburgio, et al. (2000) Biochemistry 39:10419-10430. In some embodiments, a 5’ cap includes a Cap-0 structure (also referred herein as “Cap0”), a Cap-1 structure (also referred herein as “Cap1”), or a Cap-2 structure (also referred herein as “Cap2”). See, e.g., Figure 1 of Ramanathan A et al., and Figure 1 of Decroly E et al. The term "5'-cap" as used herein refers to a structure found on the 5'-end of an RNA, e.g., mRNA, and generally includes a guanosine nucleotide connected to an RNA, e.g., mRNA, via a 5'- to 5'-triphosphate linkage (also referred to as Gppp or G(5')ppp(5')). In some embodiments, a guanosine nucleoside included in a 5’ cap may be modified, for example, by methylation at one or more positions (e.g., at the 7-position) on a base (guanine), and/or by methylation at one or more positions of a ribose. In some embodiments, a guanosine nucleoside included in a 5’ cap comprises a 3’O methylation at a ribose (denoted as “(m ^3’-O )G” or “3’OMeG”). In some embodiments, a guanosine nucleoside included in a 5’ cap comprises methylation at the 7-position of guanine (denoted as “(m ⁷ )G” or “m7G”). In some embodiments, a guanosine nucleoside included in a 5’ cap comprises methylation at the 7-position of guanine and a 3’ O methylation at a ribose (denoted as “(m2 ^7,3’-O )G” or “m7(3’OMeG)”). In some embodiments, a guanosine nucleoside included in a 5’ cap comprises a 2’ O methylation at a ribose (denoted as “(m ^2’-O )G” or “2’OMeG”). In some embodiments, a guanosine nucleoside included in a 5’ cap comprises methylation at the 7-position of guanine and a 2’ O methylation at a ribose (denoated as “(m2 ^7,2’-O )G” or “m7(2’OMeG)”). It will be understood that the notation used in the above paragraph, e.g., “(m ₂ ^7,3’-O )G” or “m7(3’OMeG)”, applies to other structures described herein. In some embodiments, providing an RNA with a 5'-cap disclosed herein or a 5'-cap analog may be achieved by in vitro transcription, in which a 5'-cap is co-transcriptionally incorporated into an RNA strand. In some embodiments, a 5’ cap may be attached to an RNA post-transcriptionally using capping enzymes. In some embodiments, co-transcriptional capping with a cap disclosed herein, e.g., a cap0, cap1, or cap2 structure, improves the capping efficiency of an RNA compared to co-transcriptional capping with an appropriate reference comparator. In some embodiments, improving capping efficiency can increase a translation efficiency and/or translation rate of an RNA, and/or increase expression of an encoded polypeptide. In some embodiments, an RNA described herein comprises a 5’-cap or a 5’ cap analog, e.g., a 5’-cap comprising a Cap0, a Cap1 or a Cap2 structure. In some embodiments, a provided RNA does not have uncapped 5'-triphosphates. In some embodiments, an RNA may be capped with a 5'-cap analog. In some embodiments, an RNA described herein comprises a Cap0 structure. In some embodiments, an RNA described herein comprises a Cap1 structure, e.g., as described herein. In some embodiments, an RNA described herein comprises a Cap2 structure. In some embodiments, a Cap0 structure comprises a guanosine nucleoside methylated at the 7-position of guanine (m7G). In some embodiments, a Cap0 structure is connected to an RNA via a 5'- to 5'-triphosphate linkage and is also referred to herein as m7Gppp or m7G(5')ppp(5'). In some embodiments, a Cap1 structure comprises a guanosine nucleoside methylated at the 7-position of guanine (m7G) and a 2’O methylated first nucleotide in an RNA (2'OMeN ₁ ). In some embodiments, a Cap1 structure is connected to an RNA via a 5'- to 5'-triphosphate linkage and is also referred to herein as m7Gppp(2'OMeN1) or m7G(5')ppp(5')(2'OMeN1), wherein N ₁ is as defined and described herein. In some embodiments, a m7G(5')ppp(5')(2'OMeN ₁ ) Cap1 structure comprises a second nucleotide, N ₂ which is a cap proximal nucleotide at position 2 (m7G(5')ppp(5')(2'OMeN1)N2) wherein each of N1 and N2 is as defined and described herein. In some embodiments, the 5’ cap is a trinucleotide cap structure. In some embodiments, the 5’ cap is a trinucleotide cap structure comprising N1pN2, wherein N1 and N2 are as defined and described herein. In some embodiments, the 5’ cap is a trinucleotide cap G*N ₁ pN ₂ , wherein N ₁ and N ₂ are as defined above and herein, and: G* comprises a structure of formula (I): (I) or a salt thereof, wherein each R ² and R ³ is -OH or -OCH ₃ ; and X is OH or SH. It will be understood that each nucleotide, e.g., N1 and N2 are linked via a phosphate group “p” (e.g., -P(=O)(OH)-, or a salt thereof such as -P(=O)(O-)-. In some embodiments, R ² is -OH. In some embodiments, R ² is -OCH ₃ . In some embodiments, R ³ is -OH. In some embodiments, R ³ is -OCH3. In some embodiments, R ² is -OH and R ³ is -OH. In some embodiments, R ² is -OH and R ³ is -CH3. In some embodiments, R ² is - CH ₃ and R ³ is -OH. In some embodiments, R ² is -CH ₃ and R ³ is -CH ₃ . In some embodiments, R ² is -OH and R ³ is -OCH3 In some embodiments R ² is -OCH3 and R ³ is -OH In some embodiments, R ² is -OCH3 and R ³ is -OCH3. It will be understood, that X being OH or SH includes salts thereof, e.g., O- or S-. In some embodiments, X is OH. In some embodiments, X is SH. In some embodiments, X is O-. In some embodiments, X is S-. In some embodiments, the 5’ cap is a trinucleotide Cap0 structure (e.g. (m ⁷ )GpppN1pN2, (m ₂ ^7,2’-O )GpppN ₁ pN ₂ , or (m ₂ ^7,3’-O )GpppN ₁ pN ₂ , wherein N ₁ and N ₂ are as defined and described herein). In some embodiments, the 5’ cap is a trinucleotide Cap1 structure (e.g., (m ⁷ )Gppp(m ^{2’- O} )N1pN2, (m2 ^7,2’-O )Gppp(m ^2’-O )N1pN2, (m2 ^7,3’-O )Gppp(m ^2’-O )N1pN2, wherein N1 and N2 are as defined and described herein. In some mebodiments, the 5’ cap is a trinucleotide Cap2 structure (e.g., (m ⁷ )Gppp(m ^2’-O )N ₁ p(m ^2’-O )N ₂ , (m ₂ ^7,2’-O )Gppp(m ^2’-O )N ₁ p(m ^2’-O )N ₂ , (m ₂ ^7,3’-O )Gppp(m ^{2’- O} )N1p(m ^2’-O )N2, wherein N1 and N2 are as defined and described herein. In some embodiments, N1 is A or an analog thereof. In some embodiments, N1 is adenosine. In some embodiments, N ₁ is 6-methyladenosine. In some embodiments, N ₁ is: , , , or , wherein % represents the point of attachment to G*. In some embodiments, N ₂ is U or an analog thereof. In some embodiments, N ₂ is a modified U. In some embodiments, N2 is 3-methyl-uridine (m ³ U), 5-methoxy-uridine (mo ⁵ U), 5- aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s ² U), 4-thio-uridine (s ⁴ U), 4-thio- pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho ⁵ U), 5-aminoallyl-uridine, 5-halo- uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5-oxyacetic acid (cmo ⁵ U), uridine 5- oxyacetic acid methyl ester (mcmo ⁵ U), 5-carboxymethyl-uridine (cm ⁵ U), 1-carboxymethyl- pseudouridine, 5-carboxyhydroxymethyl-uridine (chm ⁵ U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm ⁵ U), 5-methoxycarbonylmethyl-uridine (mcm ⁵ U), 5- methoxycarbonylmethyl-2-thio-uridine (mcm ⁵ s ² U), 5-aminomethyl-2-thio-uridine (nm ⁵ s ² U), 5- methylaminomethyl-uridine (mnm ⁵ U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio- uridine (mnm ⁵ s ² U), 5-methylaminomethyl-2-seleno-uridine (mnm ⁵ se ² U), 5-carbamoylmethyl- uridine (ncm ⁵ U) 5-carboxymethylaminomethyl-uridine (cmnm ⁵ U) 5- carboxymethylaminomethyl-2-thio-uridine (cmnm ⁵ s ² U), 5-propynyl-uridine, 1-propynyl- pseudouridine, 5-taurinomethyl-uridine (τm ⁵ U), 1-taurinomethyl-pseudouridine, 5- taurinomethyl-2-thio-uridine(τm5s2U), 1-taurinomethyl-4-thio-pseudouridine), 5-methyl-2-thio- uridine (m ⁵ s ² U), 1-methyl-4-thio-pseudouridine (m ¹ s ⁴ ψ), 4-thio-1-methyl-pseudouridine, 3- methyl-pseudouridine (m ³ ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6- dihydrouridine, 5-methyl-dihydrouridine (m ⁵ D), 2-thio-dihydrouridine, 2-thio- dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3- carboxypropyl)uridine (acp ³ U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp ³ ψ), 5-(isopentenylaminomethyl)uridine (inm ⁵ U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm ⁵ s ² U), α-thio-uridine, 2′-O-methyl-uridine (Um), 5,2′-O-dimethyl-uridine (m ⁵ Um), 2′-O- methyl-pseudouridine (ψm), 2-thio-2′-O-methyl-uridine (s ² Um), 5-methoxycarbonylmethyl-2′- O-methyl-uridine (mcm ⁵ Um), 5-carbamoylmethyl-2′-O-methyl-uridine (ncm ⁵ Um), 5- carboxymethylaminomethyl-2′-O-methyl-uridine (cmnm ⁵ Um), 3,2′-O-dimethyl-uridine (m ³ Um), 5-(isopentenylaminomethyl)-2′-O-methyl-uridine (inm ⁵ Um), 1-thio-uridine, deoxythymidine, 2′- F-ara-uridine, 2′-F-uridine, 2′-OH-ara-uridine, 5-(2-carbomethoxyvinyl) uridine, 5-[3-(1-E- propenylamino)uridine, or any other modified uridine known in the art. In some embodiments, N ₂ is 5-methyluridine (m ⁵ U). In some embodiments, N ₂ is 1-methyl-pseudouridine (m ¹ ψ). In some embodiments, N ₂ is pseudouridine (ψ). In some embodiments, N ₂ is 1-(2,2,2- trifluoroethyl)pseudouridine (tfet ¹ ψ). In some embodiments, N2 is 1-propargylpseudouridine (ppg) ¹ ψ). In some embodiments, N ₂ is 1-benzylpseudouridine (bn ¹ ψ). In some embodiments, N ₂ is 1-(cyclopropylmethyl)pseudouridine (cpm ¹ ψ). In some embodiments, N ₂ is 1-(pyridin-4- ylmethyl)pseudouridine ((4-pm) ¹ ψ).

In some embodiments, N2 is of formula II: II or a salt thereof, wherein: each is independently a single or double bond, as allowed by valency; Y ¹ is O or S; Y ² is N, C, or CH; Y ³ is N, NR ^a1 , CR ^a1 , or CHR ^a1 ; Y ⁴ is NR ^a2 or CHR ^a2 ; each of R ^a1 or R ^a2 is independently hydrogen or C1-6 aliphatic; R ⁴ is -OH or -OMe; and # represents the point of attachment to p of N ₁ p. In some embodiments, Y ¹ is O. In some embodiments, Y ¹ is S. In some embodiments, Y ² is N. In some embodiments, Y ² is C or CH. In some embodiments, Y ² is C. In some embodiments, Y ² is CH. In some embodiments, Y ³ is N or CR ^a1 . In some embodiments, Y ³ is N. In some embodiments, Y ³ is CR ^a1 . In some embodiments, Y ³ is CH or C(CH3). In some embodiments, Y ³ is CH. In some embodiments, Y ³ is C(CH ₃ ). In some embodiments, Y ³ is NR ^a1 or CHR ^a1 . In some embodiments, Y ³ is NH or N(CH ₃ ). In some embodiments, Y ³ is NH. In some embodiments, Y ³ is N(CH3). In some embodiments, Y ³ is CH2 or CH(CH3). In some embodiments, Y ³ is CH2. In some embodiments, Y ³ is CH(CH3). In some embodiments, Y ⁴ is NR ^a2 . In some embodiments, Y ⁴ is NH or NCH ₃ . In some embodiments, Y ⁴ is NH. In some embodiments, Y ⁴ is NCH3. In some embodiments, Y ⁴ is CHR ^a2 . In some embodiments, Y ⁴ is CH2 or CH(CH3). In some embodiments, Y ⁴ is CH2. In some embodiments, Y ⁴ is CH(CH ₃ ). In some embodiments, R ^a1 is hydrogen. In some embodiments, R ^a1 is C _1-6 aliphatic. In some embodiments, R ^a1 is methyl, ethyl, n-propyl, or isopropyl. In some embodiments, R ^a1 is methyl. In some embodiments, R ^a2 is hydrogen. In some embodiments, R ^a2 is C1-6 aliphatic. In some embodiments, R ^a2 is methyl, ethyl, n-propyl, or isopropyl. In some embodiments, R ^a2 is methyl. In some embodiments, R ⁴ is -OH. In some embodiments, R ⁴ is -OMe. In some embodiments, N ₂ is of formula IIa: IIa or a salt thereof, wherein each of Y ¹ , Y ³ , R ⁴ , and # is as defined above and described herein. In some embodiments of formula IIa, Y ¹ is O. In some embodiments of formula IIa, Y ³ is CR ^a1 . In some such embodiments, R ^a1 is hydrogen, C _1-6 aliphatic or –O(C _1-4 alkyl). In some embodiments of formula IIa, R ^a1 is hydrogen, C1-3 aliphatic or –O(C1-2 alkyl). In some embodiments of formula IIa, R ^a1 is –CH3 or –OCH3. In some embodiments, N2 is of formula IIb: IIb or a salt thereof, wherein each of Y ¹ , Y ³ , R ⁴ , and # is as defined above and described herein. In some embodiments of formula IIb, Y ³ is CR ^a1 . In some embodiments of formula IIb, R ^a1 is hydrogen. In some embodiments, R ^a1 is C1-6 aliphatic. In some embodiments of formula IIb, R ^a1 is C1-3 aliphatic. In some embodiments of formula IIb, R ^a1 is –CH3. In some embodiments of formula IIb, R ^a1 is -CH2R. In some such embodiments, R is C1-4 aliphatic substituted with halogen. In some embodiments of formula IIb, R ^a1 is -CH ₂ R, wherein R is C _1-2 aliphatic substituted with halogen. In some embodiments of formula IIb, R ^a1 is -CH2R, wherein R is –CF3. In some embodiments of formula IIb, R ^a1 is -CH2R, wherein R is phenyl. In some embodiments of formula IIb, R ^a1 is -CH ₂ R, wherein R is a 3- to 6-membered saturated carbocyclic ring. In some embodiments of formula IIb, R ^a1 is -CH ₂ R, wherein R is a 3- to 4- membered saturated carbocyclic ring. In some embodiments of formula IIb, R ^a1 is -CH2R, wherein R is a 3-membered saturated carbocyclic ring. In some embodiments of formula IIb, R ^a1 is -CH ₂ R, wherein R is a 5- to 6-membered heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments of formula IIb, R ^a1 is -CH2R, wherein R is a 6-membered heteroaryl ring having 1-3 nitrogen atoms. In some embodiments of formula IIb, R ^a1 is -CH ₂ R, wherein R is a 6-membered heteroaryl ring having 1 nitrogen atom. In some embodiments, N2 is of formula II′′: II′′ or a salt thereof, wherein: each is independently a single or double bond, as allowed by valency; Y ¹ is O or S; Y ² is N, C, or CH; Y ³ is N, NR ^a1 , CR ^a1 , or CHR ^a1 ; Y ⁴ is NR ^a2 or CHR ^a2 ; each of R ^a1 or R ^a2 is independently hydrogen, C1-6 aliphatic, -CH2R, or –O(C1-4 alkyl); R is C _1-4 aliphatic substituted with halogen, phenyl, a 3- to 6-membered saturated carbocyclic ring, or a 5- to 6-membered heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; R ⁴ is -OH or -OMe; and # represents the point of attachment to p of N1p. In some embodiments of formula II′′, Y ¹ is O. In some embodiments of formula II′′, Y ¹ is S. In some embodiments of formula II′′, Y ² is N. In some embodiments of formula II′′, Y ² is C or CH. In some embodiments of formula II′′, Y ² is C. In some embodiments of formula II′′, Y ² is CH. In some embodiments of formula II′′, Y ³ is N or CR ^a1 . In some embodiments of formula II′′, Y ³ is N. In some embodiments of formula II′′, Y ³ is CR ^a1 . In some embodiments of formula II′′, Y ³ is NR ^a1 or CHR ^a1 . In some embodiments of formula II′′, Y ³ is NR ^a1 . In some embodiments of formula II′′, Y ³ is CHR ^a1 . In some embodiments of formula II′′, Y ⁴ is NR ^a2 . In some embodiments of formula II′′, Y ⁴ is CHR ^a2 . In some embodiments of formula II′′, R ^a1 is hydrogen. In some embodiments of formula II′′, R ^a1 is C _1-6 aliphatic. In some embodiments of formula II′′, R ^a1 is C _1-3 aliphatic. In some embodiments of formula II′′, R ^a1 is methyl, ethyl, n-propyl, or isopropyl. In some embodiments of formula II′′, R ^a1 is methyl. In some embodiments of formula II′′, R ^a1 is ethyl. In some embodiments of formula II′′, R ^a1 is –CH=CH ₂ . In some embodiments of formula II′′, R ^a1 is n- propyl. In some embodiments of formula II′′, R ^a1 is isopropyl. In some embodiments of formula II′′, R ^a1 is –CH2C≡CH. In some embodiments of formula II′′, R ^a1 is –CH2CH=CH2. In some embodiments of formula II′′, R ^a1 is -CH ₂ R. In some embodiments of formula II′′, R ^a1 is – O(C _1-4 alkyl). In some embodiments of formula II′′, R ^a1 is –OMe. In some embodiments of formula II′′, R ^a2 is hydrogen. In some embodiments of formula II′′, R ^a2 is C1-6 aliphatic. In some embodiments of formula II′′, R ^a2 is C1-3 aliphatic. In some embodiments of formula II′′, R ^a2 is methyl, ethyl, n-propyl, or isopropyl. In some embodiments of formula II′′, R ^a2 is methyl. In some embodiments of formula II′′, R ^a2 is ethyl. In some embodiments of formula II′′, R ^a2 is –CH=CH2. In some embodiments of formula II′′, R ^a2 is n- propyl. In some embodiments of formula II′′, R ^a2 is isopropyl. In some embodiments of formula II′′, R ^a2 is –CH ₂ C≡CH. In some embodiments of formula II′′, R ^a2 is –CH ₂ CH=CH ₂ . In some embodiments of formula II′′, R ^a2 is -CH2R. In some embodiments of formula II′′, R ^a2 is – O(C1-4 alkyl). In some embodiments of formula II′′, R ^a2 is –OMe. In some embodiments of formula II′′, R is C1-4 aliphatic substituted with halogen. In some embodiments of formula II′′, R is C _1-2 aliphatic substituted with halogen. In some embodiments of formula II′′, R is –CF3. Accordingly, in some embodiments of formula II′′, R ^a1 or R ^a2 is –CH2CF3. In some embodiments of formula II′′, R is phenyl. Accordingly, in some embodiments of formula II′′, R ^a1 or R ^a2 is benzyl (i.e., ). In some embodiments of formula II′′, R is a 3- to 6-membered saturated carbocyclic ring. In some embodiments of formula II′′, R is a 3- to 4-membered saturated carbocyclic ring. In some embodiments of formula II′′, R is a 3-membered saturated carbocyclic ring. Accordingly, in some embodiments of formula II′′, R ^a1 or R ^a2 is . In some embodiments of formula II′′, R is a 5- to 6-membered heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments of formula II′′, R is a 6-membered heteroaryl ring having 1-3 nitrogen atoms. In some embodiments of formula II′′, R is a 6-membered heteroaryl ring having 1-2 nitrogen atoms. In some embodiments of formula II′′, R is a 6-membered heteroaryl ring having 1 nitrogen atom. In some embodiments of formula II′′, R is 4-pyridyl. Accordingly, in some embodiments of formula II′′, R ^a1 or R ^a2 is . In some embodiments of formula II′′, R ⁴ is -OH. In some embodiments of formula II′′, R ⁴ is -OMe. In some embodiments, N ₂ is of formula IIa′′ IIa′′ or a salt thereof, wherein each of Y ¹ , Y ³ , R ⁴ , and # is as defined above and described herein for formula II′′. In some embodiments, N2 is of formula IIb′′: IIb′′ or a salt thereof, wherein each of Y ¹ , Y ³ , R ⁴ , and # is as defined above and described herein for formula II′′. In some embodiments, N ₂ is of formula II′′′: II′′′ or a salt thereof, wherein: each is independently a single or double bond, as allowed by valency; Y ¹ is O or S; Y ² is N, C, or CH; Y ³ is N, NR ^a1 , CR ^a1 , or CHR ^a1 ; Y ⁴ is NR ^a2 or CHR ^a2 ; Y ⁵ is CR ^a3 ; each of R ^a1 , R ^a2 or R ^a3 is independently hydrogen, C _1-6 aliphatic, -CH ₂ R, or –O(C _1-4 alkyl); R is C1-4 aliphatic substituted with halogen, phenyl, a 3- to 6-membered saturated carbocyclic ring, or a 5- to 6-membered heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; R ⁴ is -OH or -OMe; and # represents the point of attachment to p of N1p. In some embodiments of formula II′′′, Y ¹ is O. In some embodiments of formula II′′′, Y ¹ is S. In some embodiments of formula II′′′, Y ² is N. In some embodiments of formula II′′′, Y ² is C or CH. In some embodiments of formula II′′′, Y ² is C. In some embodiments of formula II′′′, Y ² is CH. In some embodiments of formula II′′′, Y ³ is N or CR ^a1 . In some embodiments of formula II′′′, Y ³ is N. In some embodiments of formula II′′′, Y ³ is CR ^a1 . In some embodiments of formula II′′′, Y ³ is NR ^a1 or CHR ^a1 . In some embodiments of formula II′′′, Y ³ is NR ^a1 . In some embodiments of formula II′′′, Y ³ is CHR ^a1 . In some embodiments of formula II′′′, Y ⁴ is NR ^a2 . In some embodiments of formula II′′′, Y ⁴ is CHR ^a2 . In some embodiments of formula II′′′, R ^a1 is hydrogen. In some embodiments of formula II′′′, R ^a1 is C _1-6 aliphatic, -CH ₂ R, or –O(C _1-4 alkyl). In some embodiments of formula II′′′, R ^a1 is C1-6 aliphatic. In some embodiments of formula II′′′, R ^a1 is C1-3 aliphatic. In some embodiments of formula II′′′, R ^a1 is methyl, ethyl, n-propyl, or isopropyl. In some embodiments of formula II′′′, R ^a1 is methyl. In some embodiments of formula II′′′, R ^a1 is ethyl. In some embodiments of formula II′′′, R ^a1 is –CH=CH2. In some embodiments of formula II′′′, R ^a1 is n- propyl. In some embodiments of formula II′′′, R ^a1 is isopropyl. In some embodiments of formula II′′′, R ^a1 is –CH ₂ C≡CH. In some embodiments of formula II′′′, R ^a1 is –CH ₂ CH=CH ₂ . In some embodiments of formula II′′′, R ^a1 is -CH2R. In some embodiments of formula II′′′, R ^a1 is –O(C1-4 alkyl). In some embodiments of formula II′′′, R ^a1 is –OMe. In some embodiments of formula II′′′, R ^a2 is hydrogen. In some embodiments of formula II′′′, R ^a2 is C _1-6 aliphatic, -CH ₂ R, or –O(C _1-4 alkyl). In some embodiments of formula II′′′, R ^a2 is C1-6 aliphatic. In some embodiments of formula II′′′, R ^a2 is C1-3 aliphatic. In some embodiments of formula II′′′, R ^a2 is methyl, ethyl, n-propyl, or isopropyl. In some embodiments of formula II′′′, R ^a2 is methyl. In some embodiments of formula II′′′, R ^a2 is ethyl. In some embodiments of formula II′′′, R ^a2 is –CH=CH ₂ . In some embodiments of formula II′′′, R ^a2 is n- propyl. In some embodiments of formula II′′′, R ^a2 is isopropyl. In some embodiments of formula II′′′, R ^a2 is –CH2C≡CH. In some embodiments of formula II′′′, R ^a2 is –CH2CH=CH2. In some embodiments of formula II′′′, R ^a2 is -CH ₂ R. In some embodiments of formula II′′′, R ^a2 is –O(C _1-4 alkyl). In some embodiments of formula II′′′, R ^a2 is –OMe. In some embodiments of formula II′′′, R ^a3 is hydrogen. In some embodiments of formula II′′′, R ^a3 is C _1-6 aliphatic, -CH ₂ R, or –O(C _1-4 alkyl). In some embodiments of formula II′′′, R ^a3 is C _1-6 aliphatic. In some embodiments of formula II′′′, R ^a3 is C _1-3 aliphatic. In some embodiments of formula II′′′, R ^a3 is methyl, ethyl, n-propyl, or isopropyl. In some embodiments of formula II′′′, R ^a3 is methyl. In some embodiments of formula II′′′, R ^a3 is ethyl. In some embodiments of formula II′′′, R ^a3 is n-propyl. In some embodiments of formula II′′′, R ^a3 is isopropyl. In some embodiments of formula II′′′, R is C1-4 aliphatic substituted with halogen. In some embodiments of formula II′′′, R is C _1-2 aliphatic substituted with halogen. In some embodiments of formula II′′′, R is –CF ₃ . Accordingly, in some embodiments of formula II′′′, R ^a1 , R ^a2 , or R ^a3 is –CH2CF3. In some embodiments of formula II′′′, R is phenyl. Accordingly, in some embodiments of formula II′′′, R ^a1 , R ^a2 , or R ^a3 is benzyl (i.e., ). In some embodiments of formula II′′′, R is a 3- to 6-membered saturated carbocyclic ring. In some embodiments of formula II′′′, R is a 3- to 4-membered saturated carbocyclic ring. In some embodiments of formula II′′′, R is a 3-membered saturated carbocyclic ring. Accordingly, in some embodiments of formula II′′′, R ^a1 , R ^a2 , or R ^a3 is . In some embodiments of formula II′′′, R is a 5- to 6-membered heteroaryl ring having 1- 3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments of formula II′′′, R is a 6-membered heteroaryl ring having 1-3 nitrogen atoms. In some embodiments of formula II′′′, R is a 6-membered heteroaryl ring having 1-2 nitrogen atoms. In some embodiments of formula II′′′, R is a 6-membered heteroaryl ring having 1 nitrogen atom. In some embodiments of formula II′′′, R is 4-pyridyl. Accordingly, in some embodiments of formula II′′′, R ^a1 , R ^a2 , or R ^a3 is . In some embodiments of formula II′′′, R ⁴ is -OH. In some embodiments of formula II′′′, R ⁴ is -OMe. In some embodiments of formula II′′ or formula II′′′, Y ³ is NR ^a1 and Y ⁴ is NH, wherein R ^a1 is C _1-6 aliphatic or -CH ₂ R. In some embodiments of formula II′′ or formula II′′′, Y ³ is NH and Y ⁴ is NR ^a2 , wherein R ^a2 is C _1-6 aliphatic or -CH ₂ R. In some embodiments of formula II′′ or formula II′′′, Y ³ is NR ^a1 and Y ⁴ is NR ^a2 , wherein each of R ^a1 and R ^a2 is independently C1-6 aliphatic or -CH ₂ R. In some embodiments, N2 is uridine, 1-methylpseudouridine, 2-thio-uridine, or 5- methyluridine. In some embodiments N ₂ is: , , , , or ; or a salt thereof, wherein # represents the point of attachment to p of N1p. In some embodiments N2 is: , , , , , , , , , , , , , , , , , or ; or a salt thereof, wherein # represents the point of attachment to p of N1p. In some embodiments N ₂ is: , , , , or ; or a salt thereof, wherein # represents the point of attachment to p of N1p. In some embodiments N ₂ is: , , , , , , , ,

or a salt thereof, wherein # represents the point of attachment to p of N ₁ p. In some embodiments, p is -P(=O)(OH)-, or a salt thereof. In some embodiments, the 5’ cap is (m ^7,2’-O )Gppp(m ^2’-O )A ₁ pU ₂ , (m ^7,3’-O )Gppp(m ^{2’- O} )A1pU2, (m ^7,2’-O )Gppp(m ^2’-O )A1pΨ2, (m ^7,3’-O )Gppp(m ^2’-O )A1pΨ2, (m ^7,2’-O )Gppp(m ^{2’- O} )A1p(m ¹ )Ψ2, (m ^7,3’-O )Gppp(m ^2’-O )A1p(m ¹ )Ψ2, (m ^7,2’-O )Gppp(m ^2’-O )A1pS ² U2, (m ^7,3’-O )Gppp(m ^{2’- O} )A ₁ pS ² U ₂ , (m ^7,2’-O )Gppp(m ^2’-O )A ₁ p(m ⁵ )U ₂ , or (m ^7,3’-O )Gppp(m ^2’-O )A ₁ p(m ⁵ )U ₂ . In some embodiments, the 5’ cap is (m ^7,2’-O )Gppp(m ^6,2’-O )A ₁ pU ₂ , (m ^7,3’-O )Gppp(m ^{6,2’- O} )A1pU2, (m ^7,2’-O )Gppp(m ^6,2’-O )A1pΨ2, (m ^7,3’-O )Gppp(m ^6,2’-O )A1pΨ2, (m ^7,2’-O )Gppp(m ^{6,2’- O} )A1p(m ¹ )Ψ2, (m ^7,3’-O )Gppp(m ^6,2’-O )A1p(m ¹ )Ψ2, (m ^7,2’-O )Gppp(m ^6,2’-O )A1pS ² U2, (m ^{7,3’- O} )Gppp(m ^6,2’-O )A1pS ² U2, (m ^7,2’-O )Gppp(m ^6,2’-O )A1p(m ⁵ )U2, or (m ^7,3’-O )Gppp(m ^6,2’-O )A1p(m ⁵ )U2. In some embodiments, the 5’ cap is (m ^7,2’-O )GpppA1(m ^2’-O )pU2, (m ^7,3’-O )GpppA1(m ^{2’- O} )pU ₂ , (m ^7,2’-O )GpppA ₁ (m ^2’-O )pΨ ₂ , (m ^7,3’-O )GpppA ₁ (m ^2’-O )pΨ ₂ , (m ^7,2’-O )GpppA ₁ (m ^2’-O )p(m ¹ )Ψ ₂ , (m ^7,3’-O )GpppA1(m ^2’-O )p(m ¹ )Ψ2, (m ^7,2’-O )Gppp(m ^2’-O )A1(m ^2’-O )pS ² U2, (m ^7,3’-O )GpppA1(m ^{2’- O} )pS ² U2, (m ^7,2’-O )GpppA1(m ^2’-O )p(m ⁵ )U2, or (m ^7,3’-O )GpppA1(m ^2’-O )p(m ⁵ )U2. In some embodiments, the 5’ cap is (m ^7,2’-O )Gppp(m ^6,2’-O )A ₁ pU ₂ , (m ^7,3’-O )Gppp(m ^{6,2’- O} )A ₁ pU ₂ , (m ^7,2’-O )Gppp(m ^6,2’-O )A ₁ pΨ ₂ , (m ^7,3’-O )Gppp(m ^6,2’-O )A ₁ pΨ ₂ , (m ^7,2’-O )Gppp(m ^{6,2’- O} )A1p(m ¹ )Ψ2, (m ^7,3’-O )Gppp(m ^6,2’-O )A1p(m ¹ )Ψ2, (m ^7,2’-O )Gppp(m ^6,2’-O )A1pS ² U2, (m ^{7,3’- O} )Gppp(m ^6,2’-O )A ₁ pS ² U ₂ , (m ^7,2’-O )Gppp(m ^6,2’-O )A ₁ p(m ⁵ )U ₂ , or (m ^7,3’-O )Gppp(m ^6,2’-O )A ₁ p(m ⁵ )U ₂ . In some embodiments, the 5’ cap is m ⁷ G( ^3’-OMe )pppA ₁ ( ^2’-OMe )pm ³ U ₂ , m ⁷ G( ^3’-OMe )pppA ₁ ( ^{2’- OMe} )pmo ⁵ U2, m ⁷ GpppA1( ^2’-OMe )pm ¹ Ψ2, m ⁷ G( ^3’-OMe )pppA1( ^2’-OMe )pm ³ Ψ2, m ⁷ G( ^3’-OMe )pppA1( ^{2’- OMe} )ptfet ¹ Ψ2, m ⁷ G( ^3’-OMe )pppA1( ^2’-OMe )p(ppg) ¹ Ψ2, m ⁷ G( ^3’-OMe )pppA1( ^2’-OMe )pbn ¹ Ψ2, m ⁷ G( ^{3’- OMe} )pppA ₁ ( ^2’-OMe )pcpm ¹ Ψ ₂ , or m ⁷ G( ^3’-OMe )pppA ₁ ( ^2’-OMe )p(4-pm) ¹ Ψ ₂ . In some embodiments, a 5’ cap provided herein is selected from those in Table 1:

or a salt thereof. In some embodiments, the 5’ cap is (m ^7,2’-O )Gppp(m ^2’-O )A ₁ pU ₂ , having a structure: or a salt thereof. In some embodiments, the 5’ cap is (m ^7,3’-O )Gppp(m ^2’-O )A ₁ pU ₂ , , or a salt thereof. In some embodiments, the 5’ cap is (m ^7,3’-O )Gppp(m ^2’-O )A1pΨ2,

or a salt thereof. In some embodiments, the 5’ cap is (m ^7,2’-O )Gppp(m ^2’-O )A1pΨ2, or a salt thereof. In some embodiments, the 5’ cap is (m ^7,2’-O )Gppp(m ^2’-O )A1p(m ¹ )Ψ2,

or a salt thereof. In some embodiments, the 5’ cap is (m ^7,3’-O )Gppp(m ^2’-O )A1p(m ¹ )Ψ2, or a salt thereof. In some embodiments, the 5’ cap is (m ^7,3’-O )Gppp(m ^2’-O )A1pS ² U2,

or a salt thereof. In some embodiments, the 5’ cap is (m ^7,2’-O )Gppp(m ^2’-O )A ₁ pS ² U ₂ , or a salt thereof. In some embodiments, the 5’ cap is (m ^7,3’-O )Gppp(m ^2’-O )A1p(m ⁵ )U2,

or a salt thereof. In some embodiments, the 5’ cap is (m ^7,2’-O )Gppp(m ^2’-O )A ₁ p(m ⁵ )U ₂ , or a salt thereof. In some embodiments, it will be appreciated that the disclosure of 5’ caps above and herein encompasses 5’ caps themselves or as part of a larger molecule (e.g., an RNA). For example, the structures drawn above encompass a 3’ ether linkage to the next nucleotid or as a free -OH. In some embodiments, the present disclosure provides a compound of formula G*N1pN2, wherein: G* is of formula I′:

Iʹ or a salt thereof, wherein each R ² , R ³ , X, N ₁ , p, and N ₂ is as defined above and described herein. In some embodimets, N2 is of formula IIʹ: IIʹ or a salt thereof, wherein each , Y ¹ , Y ² , Y ³ , Y ⁴ , R ^a1 , R ^a2 , R ⁴ , and # is as defined above and described herein. In some embodiments, N2 is of formula IIaʹ: IIaʹ or a salt thereof, wherein each Y ¹ , Y ³ , R ⁴ and # is as defined above and described herein. In some embodiments, N ₂ is of formula IIbʹ: IIbʹ or a salt thereof, wherein each of Y ¹ , Y ³ , R ⁴ and # is as defined above and described herein. In some embodiments, N ₂ is of formula IIa′ or IIb′, wherein each of Y ¹ , Y ³ , and R ⁴ is as defined above for formula II′′. In some embodiments, N2 is: or a salt thereof; wherein # represents the point of attachment to p of N1p. In some embodiments, N ₂ is:

or a salt thereof; wherein # represents the point of attachment to p of N ₁ p. In some embodiments, N ₂ is: , , , , , , , , , , , , , or ; or a salt thereof; wherein # represents the point of attachment to p of N ₁ p. In some embodiments, N1 is: , , , or ; or a salt thereof, In some embodimetns, p is -P(=O)(OH)-, or a salt thereof such as -P(=O)(O-)-,. In some embodiments, the present disclosure provides a compound (m ^7,2’-O )Gppp(m ^{2’- O} )A1pU2, having a structure: or a salt thereof. In some embodiments the present disclosure provides a compound (m ^7,3’-O )Gppp(m ^{2’- O} )A1pU2, , or a salt thereof. In some embodiments, the present disclosure provides a compound (m ^7,3’-O )Gppp(m ^{2’- O} )A ₁ pΨ ₂ , or a salt thereof. In some embodiments, the present disclosure provides a compound (m ^7,2’-O )Gppp(m ^{2’- O} )A1pΨ2, or a salt thereof. In some embodiments, the present disclosure provides a compound (m ^7,2’-O )Gppp(m ^{2’- O} )A1p(m ¹ )Ψ2,

or a salt thereof. In some embodiments, the present disclosure provides a compound (m ^7,3’-O )Gppp(m ^{2’- O} )A1p(m ¹ )Ψ2, or a salt thereof. In some embodiments, the present disclosure provides a compound (m ^7,3’-O )Gppp(m ^{2’- O} )A1pS ² U2,

or a salt thereof. In some embodiments, the present disclosure provides a compound (m ^7,2’-O )Gppp(m ^{2’- O} )A ₁ pS ² U ₂ , or a salt thereof . In some embodiments, the present disclosure provides a compound (m ^7,3’-O )Gppp(m ^{2’- O} )A1p(m ⁵ )U2,

or a salt thereof. In some embodiments, the present disclosure provides a compound (m ^7,2’-O )Gppp(m ^{2’- O} )A1p(m ⁵ )U2, or a salt thereof. In some embodiments, a provided compound is a salt. In some embodiments, a provided compound is a pharmaceutically acceptable salt. 5’ UTR and cap proximal sequences In some embodiments, an RNA disclosed herein comprises a 5'-UTR. The term "untranslated region" or "UTR" relates to a region in a DNA molecule which is transcribed but is not translated into an amino acid sequence, or to the corresponding region in an RNA polynucleotide, such as an mRNA molecule. An untranslated region (UTR) can be present 5' (upstream) of an open reading frame (5'-UTR) and/or 3' (downstream) of an open reading frame (3'-UTR). A 5'-UTR, if present, is located at the 5' end of an RNA, upstream of the start codon of a protein-encoding region. A 5'-UTR can be downstream of the 5'-cap (if present), e.g. directly adjacent to the 5'-cap. In some embodiments, a 5’ UTR disclosed herein comprises a cap proximal sequence, e.g., as disclosed herein. In some embodiments, a cap proximal sequence comprises a sequence adjacent to a 5’ cap (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides immediately adjacent to a 5’ cap). In some embodiments, a cap proximal sequence comprises nucleotides in positions +1, +2, +3, +4, and/or +5 of an RNA polynucleotide. In some embodiments a 5’ UTR comprises a Kozak sequence (e.g., GCCACC). In some embodiments a Kozak sequence is immediately adjacent to a payload sequence (e.g., immediately upstream of a start codon). In some embodiments, a Cap structure comprises one or more polynucleotides of a cap proximal sequence. In some embodiments, a Cap structure comprises an m7 Guanosine cap and nucleotides +1 and +2 (N1 and N2) of an RNA polynucleotide. Those skilled in the art, reading the present disclosure, will appreciate that, in some embodiments, one or more residues of a cap proximal sequence (e.g., one or more of residues +1, +2, +3, +4, and/or +5) may be included in an RNA by virtue of having been included in a cap entity (e.g., a Cap1 or Cap2 structure, etc); alternatively, in some embodiments, at least some of the residues in a cap proximal sequence may be enzymatically added (e.g., by a polymerase such as a T7 polymerase). For example, in certain exemplified embodiments where a m2 ^{7,3’- O} Gppp(m ₁ ^2’-O )ApU cap is utilized, +1 (i.e., N ₁ ) and +2 (i.e. N ₂ ) are the (m ₁ ^2’-O )A and U residues of the cap, and +3, +4, and +5 are added by polymerase (e.g., T7 polymerase). In some embodiments, the 5’ cap is a trinucleotide cap structure (e.g., the trinucleotide cap structures described above and herein), wherein the cap proximal sequence comprises N1 and N ₂ of the 5’ cap, wherein N ₁ is as defined above and described herein, and N ₂ is as defined above and described herein. In some embodiments, e.g., where the 5’ cap is a trinucleotide cap structure, a cap proximal sequence comprises N ₁ and N ₂ of a the 5’ cap, and N ₃ , N ₄ and N ₅ , wherein N ₁ to N ₅ correspond to positions +1, +2, +3, +4, and/or +5 of an RNA polynucleotide. In some embodiments, N3 is A. In some embodiments, N3 is C. In some embodiments, N3 is G. In some embodiments, N3 is U. In some embodiments, N4 is A. In some embodiments, N4 is C. In some embodiments, N4 is G. In some embodiments, N4 is U. In some embodiments, N5 is A. In some embodiments, N ₅ is C. In some embodiments, N ₅ is G. In some embodiments, N ₅ is U. In some embodiments, N3 is A, N4 is A, and N5 is A. In some embodiments, N3 is A, N4 is A, and N5 is C. In some embodiments, N3 is A, N4 is A, and N5 is G. In some embodiments, N ₃ is A, N ₄ is A, and N ₅ is U. In some embodiments, N ₃ is A, N ₄ is C, and N ₅ is C. In some embodiments, N ₃ is A, N ₄ is C, and N ₅ is G. In some embodiments, N ₃ is A, N ₄ is C, and N ₅ is U. In some embodiments, N3 is A, N4 is G, and N5 is C. In some embodiments, N3 is A, N4 is G, and N ₅ is G. In some embodiments, N ₃ is A, N ₄ is G, and N ₅ is U. In some embodiments, N ₃ is A, N ₄ is U, and N ₅ is C. In some embodiments, N ₃ is A, N ₄ is U, and N ₅ is G. In some embodiments, N3 is A, N4 is U, and N5 is U. In some embodiments, N3 is C, N4 is A, and N5 is A. In some embodiments, N3 is C, N4 is A, and N ₅ is C. In some embodiments, N ₃ is C, N ₄ is A, and N ₅ is G. In some embodiments, N3 is C, N4 is A, and N5 is U. In some embodiments, N3 is C, N4 is C, and N5 is C. In some embodiments, N3 is C, N4 is C, and N5 is G. In some embodiments, N3 is C, N4 is C, and N5 is U. In some embodiments, N ₃ is C, N ₄ is G, and N ₅ is C. In some embodiments, N ₃ is C, N ₄ is G, and N ₅ is G. In some embodiments, N ₃ is C, N ₄ is G, and N ₅ is U. In some embodiments, N ₃ is C, N4 is U, and N5 is C. In some embodiments, N3 is C, N4 is U, and N5 is G. In some embodiments, N ₃ is C, N ₄ is U, and N ₅ is U. In some embodiments, N ₃ is G, N ₄ is A, and N ₅ is A. In some embodiments, N ₃ is G, N ₄ is A, and N5 is C. In some embodiments, N3 is G, N4 is A, and N5 is G. In some embodiments, N ₃ is G, N ₄ is A, and N ₅ is U. In some embodiments, N ₃ is G, N ₄ is C, and N ₅ is C. In some embodiments, N ₃ is G, N ₄ is C, and N ₅ is G. In some embodiments, N ₃ is G, N ₄ is C, and N ₅ is U. In some embodiments, N3 is G, N4 is G, and N5 is C. In some embodiments, N3 is G, N4 is G, and N5 is G. In some embodiments, N3 is G, N4 is G, and N5 is U. In some embodiments, N3 is G, N ₄ is U, and N ₅ is C. In some embodiments, N ₃ is G, N ₄ is U, and N ₅ is G. In some embodiments, N3 is G, N4 is U, and N5 is U. In some embodiments, N3 is U, N4 is A, and N5 is A. In some embodiments, N3 is U, N4 is A, and N ₅ is C. In some embodiments, N ₃ is U, N ₄ is A, and N ₅ is G. In some embodiments, N ₃ is U, N ₄ is A, and N ₅ is U. In some embodiments, N ₃ is U, N ₄ is C, and N ₅ is C. In some embodiments, N3 is U, N4 is C, and N5 is G. In some embodiments, N3 is U, N4 is C, and N5 is U. In some embodiments, N3 is U, N4 is G, and N5 is C. In some embodiments, N3 is U, N4 is G, and N5 is G. In some embodiments, N3 is U, N4 is G, and N5 is U. In some embodiments, N3 is U, N ₄ is U, and N ₅ is C. In some embodiments, N ₃ is U, N ₄ is U, and N ₅ is G. In some embodiments, N3 is U, N4 is U, and N5 is U. Exemplary 5’ UTRs include a human alpha globin (hAg) 5’UTR or a fragment thereof, a TEV 5’ UTR or a fragment thereof, a HSP705’ UTR or a fragment thereof, or a c-Jun 5’ UTR or a fragment thereof. In some embodiments, an RNA disclosed herein comprises a hAg 5’ UTR sequence or a fragment thereof. In some embodiments, an RNA disclosed herein comprises comprises a 5’ UTR comprising an AUAGU cap proximal sequence and an hAg 5’ UTR sequence (e.g., a 5’ UTR having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to a human alpha globin 5’ UTR provided in SEQ ID NO: 11). In some embodiments, an RNA disclosed herein comprises a 5’ UTR as provided in SEQ ID NO: 11). In some embodiments, an RNA disclosed herein comprises a hAg 5’ UTR having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to a human alpha globin 5’ UTR provided in SEQ ID NO: 12. In some embodiments, an RNA disclosed herein comprises a hAg 5’ UTR provided in SEQ ID NO: 12. 3’ UTR In some embodiments, an RNA disclosed herein comprises a 3'-UTR. A 3'-UTR, if present, is located at the 3' end of an RNA, downstream of the termination codon of a protein- encoding region, but the term "3'-UTR" preferably does not include a poly(A) sequence. Thus, the 3'-UTR is upstream of a poly(A) sequence (if present), e.g. directly adjacent to upstream of a poly(A) sequence. In some embodiments, an RNA disclosed herein comprises a 3’ UTR comprising a first sequence from the amino terminal enhancer of split (AES) messenger RNA (an “F element”) and/or a second sequence from the mitochondrial encoded 12S ribosomal RNA (“an I element”). In some embodiments, a 3’ UTR or a proximal sequence thereto comprises a restriction site. In some embodiments, a restriction site is a BamHI site. In some embodiments, a restriction site is a XhoI site. In some embodiments, an RNA disclosed herein comprises a 3’ UTR having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to a 3’ UTR provided in SEQ ID NO: 13. In some embodiments, an RNA disclosed herein comprises a 3’ UTR provided in SEQ ID NO: 13. PolyA In some embodiments, an RNA disclosed herein comprises a polyadenylate (PolyA) sequence, e.g., as described herein. In some embodiments, a PolyA sequence is situated downstream of a 3'-UTR, e.g., adjacent to a 3'-UTR. As used herein, the terms "poly(A) sequence" or “PolyA sequence” or "poly-A tail" refers to an uninterrupted or interrupted sequence of adenylate residues which is typically located at the 3'-end of an RNA polynucleotide. Poly(A) sequences are known to those of skill in the art and may follow the 3’-UTR in RNAs described herein. An uninterrupted poly(A) sequence is characterized by consecutive adenylate residues. In nature, an uninterrupted poly(A) sequence is typical. RNAs disclosed herein can have a poly(A) sequence attached to the free 3'-end of the RNA by a template-independent RNA polymerase after transcription or a poly(A) sequence encoded by DNA and transcribed by a template-dependent RNA polymerase. It has been demonstrated that a poly(A) sequence of about 120 A nucleotides has a beneficial influence on the levels of RNA in transfected eukaryotic cells, as well as on the levels of protein that is translated from an open reading frame that is present upstream (5’) of the poly(A) sequence (Holtkamp et al., 2006, Blood, vol.108, pp.4009-4017). A poly(A) sequence may be of any length. In some embodiments, a poly(A) sequence comprises, essentially consists of, or consists of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 A nucleotides, and, in particular, about 120 A nucleotides. In this context, "essentially consists of" means that most nucleotides in the poly(A) sequence, typically at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% by number of nucleotides in the poly(A) sequence are A nucleotides, but permits that remaining nucleotides are nucleotides other than A nucleotides, such as U nucleotides (uridylate), G nucleotides (guanylate), or C nucleotides (cytidylate). In this context, "consists of" means that all nucleotides in the poly(A) sequence, i.e., 100% by number of nucleotides in the poly(A) sequence, are A nucleotides. The term "A nucleotide" or "A" refers to adenylate. In some embodiments, a poly(A) sequence is attached during RNA transcription, e.g., during preparation of in vitro transcribed RNA, based on a DNA template comprising repeated dT nucleotides (deoxythymidylate) in the strand complementary to the coding strand. The DNA sequence encoding a poly(A) sequence (coding strand) is referred to as poly(A) cassette. In some embodiments, the poly(A) cassette present in the coding strand of a DNA template essentially consists of dA nucleotides, but is interrupted by a random sequence of the four nucleotides (dA, dC, dG, and dT). Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length. Such a cassette is disclosed in WO 2016/005324 A1, hereby incorporated by reference. Any poly(A) cassette disclosed in WO 2016/005324 A1 may be used in the present invention. A poly(A) cassette that essentially consists of dA nucleotides, but is interrupted by a random sequence having an equal distribution of the four nucleotides (dA, dC, dG, dT) and having a length of e.g., 5 to 50 nucleotides shows, on DNA level, constant propagation of plasmid DNA in E. coli and is still associated, on RNA level, with the beneficial properties with respect to supporting RNA stability and translational efficiency is encompassed. In some embodiments, the poly(A) sequence contained in an RNA polynucleotide described herein essentially consists of A nucleotides, but is interrupted by a random sequence of the four nucleotides (A, C, G, U). Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length. In some embodiments, no nucleotides other than A nucleotides flank a poly(A) sequence at its 3'-end, i.e., the poly(A) sequence is not masked or followed at its 3'-end by a nucleotide other than A. In some embodiments, the poly(A) sequence may comprise at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly(A) sequence may essentially consist of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly(A) sequence may consist of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly(A) sequence comprises at least 100 nucleotides. In some embodiments, the poly(A) sequence comprises about 150 nucleotides. In some embodiments, the poly(A) sequence comprises about 120 nucleotides. In some embodiments, an RNA disclosed herein comprises a poly(A) sequence comprising the nucleotide sequence of SEQ ID NO: 14, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 14. In some embodiments, an RNA disclosed herein comprises a poly(A) sequence of SEQ ID NO: 14. Payloads In some embodiments, an RNA polynucleotide disclosed herein comprises a sequence encoding a payload, e.g., as described herein. In some embodiments, a sequence encoding a payload comprises a promoter sequence. In some embodiments, a sequence encoding a payload comprises a sequence encoding a secretory signal peptide. In some embodiments, a payload is chosen from: a protein replacement polypeptide; an antibody agent; a cytokine; an antigenic polypeptide; a gene editing component; a regenerative medicine component or combinations thereof. In some embodiments, a payload is or comprises a protein replacement polypeptide. In some embodiments, a protein replacement polypeptide comprises a polypeptide with aberrant expression in a disease or disorder. In some embodiments, a protein replacement polypeptide comprises an intracellular protein, an extracellular protein, or a transmembrane protein. In some embodiments, a protein replacement polypeptide comprises an enzyme. In some embodiments, a disease or disorder with aberrant expression of a polypeptide includes but is not limited to: a rare disease, a metabolic disorder, a muscular dystrophy, a cardiovascular disease, or a monogenic disease. In some embodiments, a payload is or comprises an antibody agent. In some embodiments, an antibody agent binds to a polypeptide expressed on a cell. In some embodiments, an antibody agent comprises a CD3 antibody, a Claudin 6 antibody, or a combination thereof. In some embodiments, a payload is or comprises a cytokine or a fragment or a variant thereof. In some embodiments, a cytokine comprises: IL-12 or a fragment or variant or a fusion thereof, IL-15 or a fragment or a variant or a fusion thereof, GM-CSF or a fragment or a variant thereof; or IFN-alpha or a fragment or a variant thereof. In some embodiments, a payload is or comprises an antigenic polypeptide or an immunogenic variant or an immunogenic fragment thereof. In some embodiments, an antigenic polypeptide comprises one epitope from an antigen. In some embodiments, an antigenic polypeptide comprises a plurality of distinct epitopes from an antigen. In some embodiments, an antigenic polypeptide comprising a plurality of distinct epitopes from an antigen is polyepitopic. In some embodiments, an antigenic polypeptide comprises: an antigenic polypeptide from an allergen, a viral antigenic polypeptide, a bacterial antigenic polypeptide, a fungal antigenic polypeptide, a parasitic antigenic polypeptide, an antigenic polypeptide from an infectious agent, an antigenic polypeptide from a pathogen, a tumor antigenic polypeptide, or a self-antigenic polypeptide. In some embodiments, a viral antigenic polypeptide comprises an HIV antigenic polypeptide, an influenza antigenic polypeptide, a respiratory syncytial virus antigenic polypeptide, a Coronavirus antigenic polypeptide, a Rabies antigenic polypeptide, or a Zika virus antigenic polypeptide. In some embodiments, a viral antigenic polypeptide comprises an antigenic polypeptide of a virus that is associated with a respiratory infectious disease. In some embodiments, a viral antigenic polypeptide is or comprises a Coronavirus antigenic polypeptide. In some embodiments, a Coronavirus antigen is or comprises a SARS- CoV-2 protein. In some embodiments, a SARS-CoV-2 protein comprises a SARS-CoV-2 Spike (S) protein, or an immunogenic variant or an immunogenic fragment thereof. In some embodiments, a SARS-CoV-2 protein, or immunogenic variant or immunogenic fragment thereof, comprises proline residues at positions 986 and 987. In some embodiments, a SARS-CoV-2 S polypeptide has at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to a SARS-CoV-2 S polypeptide disclosed herein. In some embodiments, a SARS-CoV-2 S polypeptide has at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to SEQ ID NO: 9. In some embodiments, a SARS-CoV-2 S polypeptide is encoded by an RNA having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to a SARS-CoV-2 S polynucleotide disclosed herein. In some embodiments, a SARS-CoV-2 S polypeptide is encoded by an RNA having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to SEQ ID NO: 10. In some embodiments, a payload is or comprises a tumor antigenic polypeptide or an immunogenic variant or an immunogenic fragment thereof. In some embodiments, a tumor antigenic polypeptide comprises a tumor specific antigen, a tumor associated antigen, a tumor neoantigen, or a combination thereof. In some embodiments, a tumor antigenic polypeptide comprises p53, ART-4, BAGE, ss-catenin/m, Bcr-abL CAMEL, CAP-1, CASP-8, CDC27/m, CDK4/m, CEA, CLAUDIN-12, c-MYC, CT, Cyp-B, DAM, ELF2M, ETV6-AML1, G250, GAGE, GnT-V, Gap100, HAGE, HER-2/neu, HPV-E7, HPV-E6, HAST-2, hTERT (or hTRT), LAGE, LDLR/FUT, MAGE-A, preferably MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, or MAGE-A12, MAGE-B, MAGE-C, MART-1/Melan-A, MC1R, Myosin/m, MUC1, MUM-1, MUM-2, MUM-3, NA88-A, NF1, NY-ESO-1, NY-BR-1, p190 minor BCR-abL, Plac-1, Pm1/RARa, PRAME, proteinase 3, PSA, PSM, RAGE, RU1 or RU2, SAGE, SART-1 or SART- 3, SCGB3A2, SCP1, SCP2, SCP3, SSX, SURVIVIN, TEL/AML1, TPI/m, TRP-1, TRP-2, TRP- 2/INT2, TPTE, WT, WT-1, or a combination thereof. In some embodiments, a tumor antigenic polypeptide comprises a tumor antigen from a carcinoma, a sarcoma, a melanoma, a lymphoma, a leukemia, or a combination thereof. In some embodiments, a tumor antigenic polypeptide comprises a melanoma tumor antigen.In some embodiments, a tumor antigenic polypeptide comprises a prostate cancer antigen. In some embodiments, a tumor antigenic polypeptide comprises a HPV16 positive head and neck cancer antigen. In some embodiments, a tumor antigenic polypeptide comprises a breast cancer antigen. In some embodiments, a tumor antigenic polypeptide comprises an ovarian cancer antigen. In some embodiments, a tumor antigenic polypeptide comprises a lung cancer antigen. In some embodiments, a tumor antigenic polypeptide comprises an NSCLC antigen. In some embodiments, a payload is or comprises a self-antigenic polypeptide or an immunogenic variant or an immunogenic fragment thereof. In some embodiments, a self- antigenic polypeptide comprises an antigen that is typically expressed on cells and is recognized as a self-antigen by an immune system. In some embodiments, a self-antigenic polypeptide comprises: a multiple sclerosis antigenic polypeptide, a Rheumatoid arthritis antigenic polypeptide, a lupus antigenic polypeptide, a celiac disease antigenic polypeptide, a Sjogren’s syndrome antigenic polypeptide, or an ankylosing spondylitis antigenic polypeptide, or a combination thereof. In Vitro Synthesis of RNA Polynucleotides Commonly, in vitro transcription reactions include a double stranded DNA template comprised of a template strand (also known as a non-coding strand) and a coding strand. Those skilled in the art appreciate that a “Transcription Start Site” sequence, when presented as single stranded (SS) sequence, typically relates to the coding strand sequence and reflects the canonical position at which the relevant RNA polymerase begins transcription. Those skilled in the art, reading the present disclosure will appreciate that, in some embodiments, a cap (e.g., a co- transcriptional cap) may include one or more residues corresponding to a position of such a “transcriptional start site sequence”, such that the first residue added by the RNA polymerase may in fact represent the second (or later) residue of the canonical Transcription Start Site. In some embodiments, a DNA template is a linear DNA molecule. In some embodiments, a DNA template is a circular DNA molecule. DNA can be obtained or generated using methods known in the art, including, e.g., gene synthesis, recombinant DNA technology, or a combination thereof. In some embodiments, a DNA template comprises a nucleotide sequence coding for a transcribed region of interest (e.g., coding for a RNA described herein) and a promoter sequence that is recognized by an RNA polymerase selected for use in in vitro transcription. Various RNA polymerases are known in the art, including, e.g., DNA dependent RNA polymerases (e.g., a T7 RNA polymerase, a T3 RNA polymerase, a SP6 RNA polymerase, a N4 virion RNA polymerase, or a variant or functional domain thereof). A person skilled in the art will readily understand that an RNA polymerase utilized herein may be a recombinant RNA polymerase, and/or a purified RNA polymerase, i.e., not as part of a cell extract, which contains other components in addition to the RNA polymerases. One skilled in the art will recognize an appropriate promoter sequence for the selected RNA polymerase. In some embodiments, a DNA template can comprise a promoter sequence for a T7 RNA polymerase. In some embodiments, the present disclosure provides an insight that a double stranded DNA template containing an A and U at the +1 and +2 positions, respectively, of a Transcription Start Site downstream from a RNA polymerase promoter (e.g., T7 promoter) can be useful for improving capping efficiency (e.g., percentage of capped transcripts in an in vitro transcription reaction), quality of an RNA preparation (e.g., of an in vitro transcribed RNA, e.g., the amount of short polynucleotide byproducts produced), translation efficiency of an RNA encoding a payload, and/or expression of a polypeptide payload encoded by an RNA. In some embodiments, such improvements can be observed independent of the identity of a 5’ UTR, capping method (e.g., enzymatic capping vs. co-transcriptional capping), cap structures (e.g., Cap0, Cap1, or Cap2), coding sequences, types of ribonucleotides (e.g., modified nucleotides vs. non-modified nucleotides), formulation (e.g., lipoplex vs. lipid nanoparticles) or combinations thereof. In some particular embodiments, a double stranded DNA template comprises an A and U at the +1 and +2 positions, respectively, of a Transcription Start Site. In some embodiments, a pyrimidine base (e.g., C or U) or a purine base (e.g., G or A) can be independently present at +3, +4, or +5 positions of a Transcription Start Site of a double stranded DNA template. In some particular embodiments, such a double stranded DNA template comprises a A at +3 position of the Transcription Start Site. As appreciated by a person skilled in the art, the 3’ end of a cap structure can be extended by an RNA polymerase using naturally occurring ribonucleotides and/or modified ribonucleotides. Therefore, a person skilled in the art will understand references to A, U, G, or C throughout the specification described herein can mean a naturally occurring ribonucleotide and/or a modified ribonucleotide described herein. For example, in some embodiments, a U is uridine. In some embodiments, a U is modified uridine (e.g., pseudouridine, 1-methyl pseudouridine). In some embodiments, provided RNA polynucleotides are produced by in vitro transcription reaction described herein, e.g., using different combinations of cap structures (e.g., as described herein) and transcription start sites. AUA Transcription Start Site In some embodiments, a Transcription Start Site that may be useful in accordance with the present disclosure is AUA. In some embodiments, an in vitro transcription reaction comprises: (i) a template DNA strand comprising a polynucleotide sequence complementary to an RNA polynucleotide sequence described herein, wherein the template DNA strand comprises a sequence that is complementary to an AUA transcription start site; (ii) a polymerase (e.g., an RNA polymerase such as, e.g., T7 polymerase); (iii) ribonucleotides; and (iv) a trinucleotide cap comprising N ₁ pN ₂ ; wherein N ₁ is A or an analog thereof (e.g., as described above and herein) and N2 is U or an analog thereof (e.g., as described above and herein); and wherein the sequence in the template DNA strand that is complementary to AUA is the start site of an RNA polymerase promoter. A skilled person in the art reading the present disclosure will appreciate that when an AUA Transcription Start Site is referenced with respect to a double-stranded DNA template, a coding strand of the double-stranded DNA template comprises an AUA start sequence, while a template DNA strand of the double stranded DNA template comprises a TAT which is the start site of an RNA polymerase promoter. In some embodiments, such in vitro transcription reactions can produce an RNA polynucleotide comprising a 5’ cap, a cap proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; and a sequence encoding a payload, wherein: (i) N1 is position +1 of the RNA polynucleotide, (ii) N ₂ is position +2 of the RNA polynucleotide, wherein N ₁ is A or an analog thereof (e.g., as described above and herein), and N ₂ is U or an analog thereof (e.g., as described above and herein); and (iii) the cap proximal sequence comprises: N ₁ and N ₂ of the cap structure and a sequence comprising N ₃ N ₄ N ₅ at positions +3, +4, and +5 respectively of the RNA polynucleotide, wherein each of N ₃ , N ₄ , and N ₅ is independently selected from: A, C, G, and U (e.g., as described above and herein). By way of example only, in some embodiments, an RNA polynucleotide resulting from such an in vitro transcription reaction comprises a 5’ cap and a cap proximal sequence comprising A ₁ U ₂ A ₃ N ₄ N ₅ . In some embodiments, an RNA polynucleotide resulting from such an in vitro transcription reaction can be an RNA polynucleotide described herein. AUC Transcription Start Site In some embodiments, a Transcription Start Site that may be useful in accordance with the present disclosure is AUA. In some embodiments, an in vitro transcription reaction comprises: (i) a template DNA strand comprising a polynucleotide sequence complementary to an RNA polynucleotide sequence described herein, wherein the template DNA strand comprises a sequence that is complementary to an AUC transcription start site; (ii) a polymerase (e.g., an RNA polymerase such as, e.g., T7 polymerase); (iii) ribonucleotides; and (iv) a trinucleotide cap comprising N1pN2; wherein N1 is A or an analog thereof (e.g., as described above and herein) and N2 is U or an analog thereof (e.g., as described above and herein); and wherein the sequence in the template DNA strand that is complementary to AUC is the start site of an RNA polymerase promoter. A skilled person in the art reading the present disclosure will appreciate that when an AUC Transcription Start Site is referenced with respect to a double-stranded DNA template, a coding strand of the double-stranded DNA template comprises an AUC start sequence, while a template DNA strand of the double stranded DNA template comprises a TAG which is the start site of an RNA polymerase promoter. In some embodiments, such in vitro transcription reactions can produce an RNA polynucleotide comprising a 5’ cap, a cap proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; and a sequence encoding a payload, wherein: (i) N ₁ is position +1 of the RNA polynucleotide, (ii) N2 is position +2 of the RNA polynucleotide, wherein N1 is A or an analog thereof (e.g., as described above and herein), and N2 is U or an analog thereof (e.g., as described above and herein); and (iii) the cap proximal sequence comprises: N ₁ and N ₂ of the cap structure and a sequence comprising N ₃ N ₄ N ₅ at positions +3, +4, and +5 respectively of the RNA polynucleotide, wherein each of N3, N4, and N5 is independently selected from: A, C, G, and U (e.g., as described above and herein). By way of example only, in some embodiments, an RNA polynucleotide resulting from such an in vitro transcription reaction comprises a 5’ cap and a cap proximal sequence comprising A1U2C3N4N5. In some embodiments, an RNA polynucleotide resulting from such an in vitro transcription reaction can be an RNA polynucleotide described herein. AUG Transcription Start Site In some embodiments, a Transcription Start Site that may be useful in accordance with the present disclosure is AUA. In some embodiments, an in vitro transcription reaction comprises: (i) a template DNA strand comprising a polynucleotide sequence complementary to an RNA polynucleotide sequence described herein, wherein the template DNA strand comprises a sequence that is complementary to an AUG transcription start site; (ii) a polymerase (e.g., an RNA polymerase such as, e.g., T7 polymerase); (iii) ribonucleotides; and (iv) a trinucleotide cap comprising N ₁ pN ₂ ; wherein N ₁ is A or an analog thereof (e.g., as described above and herein) and N ₂ is U or an analog thereof (e.g., as described above and herein); and wherein the sequence in the template DNA strand that is complementary to AUG is the start site of an RNA polymerase promoter. A skilled person in the art reading the present disclosure will appreciate that when an AUG Transcription Start Site is referenced with respect to a double-stranded DNA template, a coding strand of the double-stranded DNA template comprises an AUG start sequence, while a template DNA strand of the double stranded DNA template comprises a TAC which is the start site of an RNA polymerase promoter. In some embodiments, such in vitro transcription reactions can produce an RNA polynucleotide comprising a 5’ cap, a cap proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; and a sequence encoding a payload, wherein: (i) N1 is position +1 of the RNA polynucleotide, (ii) N2 is position +2 of the RNA polynucleotide, wherein N ₁ is A or an analog thereof (e.g., as described above and herein), and N ₂ is U or an analog thereof (e.g., as described above and herein); and (iii) the cap proximal sequence comprises: N1 and N2 of the cap structure and a sequence comprising N3N4N5 at positions +3, +4, and +5 respectively of the RNA polynucleotide, wherein each of N ₃ , N ₄ , and N ₅ is independently selected from: A, C, G, and U (e.g., as described above and herein). By way of example only, in some embodiments, an RNA polynucleotide resulting from such an in vitro transcription reaction comprises a 5’ cap and a cap proximal sequence comprising A ₁ U ₂ G ₃ N ₄ N ₅ . In some embodiments, an RNA polynucleotide resulting from such an in vitro transcription reaction can be an RNA polynucleotide described herein. AUU Transcription Start Site In some embodiments, a Transcription Start Site that may be useful in accordance with the present disclosure is AUA. In some embodiments, an in vitro transcription reaction comprises: (i) a template DNA strand comprising a polynucleotide sequence complementary to an RNA polynucleotide sequence described herein, wherein the template DNA strand comprises a sequence that is complementary to an AUU transcription start site; (ii) a polymerase (e.g., an RNA polymerase such as, e.g., T7 polymerase); (iii) ribonucleotides; and (iv) a trinucleotide cap comprising N ₁ pN ₂ ; wherein N ₁ is A or an analog thereof (e.g., as described above and herein) and N2 is U or an analog thereof (e.g., as described above and herein); and wherein the sequence in the template DNA strand that is complementary to AUG is the start site of an RNA polymerase promoter. A skilled person in the art reading the present disclosure will appreciate that when an AUU Transcription Start Site is referenced with respect to a double-stranded DNA template, a coding strand of the double-stranded DNA template comprises an AUU start sequence, while a template DNA strand of the double stranded DNA template comprises a TAA which is the start site of an RNA polymerase promoter. In some embodiments, such in vitro transcription reactions can produce an RNA polynucleotide comprising a 5’ cap, a cap proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; and a sequence encoding a payload, wherein: (i) N ₁ is position +1 of the RNA polynucleotide, (ii) N2 is position +2 of the RNA polynucleotide, wherein N1 is A or an analog thereof (e.g., as described above and herein), and N2 is U or an analog thereof (e.g., as described above and herein); and (iii) the cap proximal sequence comprises: N ₁ and N ₂ of the cap structure and a sequence comprising N ₃ N ₄ N ₅ at positions +3, +4, and +5 respectively of the RNA polynucleotide, wherein each of N3, N4, and N5 is independently selected from: A, C, G, and U (e.g., as described above and herein). By way of example only, in some embodiments, an RNA polynucleotide resulting from such an in vitro transcription reaction comprises a 5’ cap and a cap proximal sequence comprising A ₁ U ₂ U ₃ N ₄ N ₅ . In some embodiments, an RNA polynucleotide resulting from such an in vitro transcription reaction can be an RNA polynucleotide described herein. Complexes In certain aspects, provided herein are complexes formed during in vitro transcription reactions described herein, e.g., using different combinations of caps (e.g., as described herein) and transcription start sites (e.g., as described herein). In some embodiments, the present disclosure provides a complex comprising a DNA template strand and a 5’ cap analog, wherein the DNA template strand comprises an RNA polymerase promoter sequence and a sequence that is complementary to a transcription start site; wherein the 5’ cap analog comprises a structure of N ₁ pN ₂ , and wherein N ₁ is A or an analog thereof (e.g., as described above and herein) and N ₂ is U or an analog thereof (e.g., as described above and herein); wherein N1 interacts with the +1 position of the DNA template strand (corresponding to the first nucleotide of the transcription start site) and N ₂ interacts with the +2 position of the DNA template strand (corresponding to the second nucleotide of the transcription start site); and wherein the sequence in the template strand that is complementary to the transcription start site is the start site of an RNA polymerase promoter. In some embodiments, N ₁ is A and N ₂ is U, and position +1 and position +2 of the DNA template strand are T and A, respectively. In various apsects described herein, one or more nucleotides of a cap (e.g., ones described herein) interact with one or more nucleotides in the RNA polymerase start site the template DNA strand via canonical Watson-Crick base pairing. In some embodiments, a provided complex comprises a DNA template strand comprises an RNA polymerase promoter sequence, which in some embodiments may be or comprise a T7 RNA polymerase promoter sequence. In some embodiments, the complexes disclosed herein further comprise an RNA polymerase (e.g., a T7 RNA polymerase). Exemplary polynucleotides In some embodiments, an RNA polynucleotide described herein or a composition or medical preparation comprising the same comprises a nucleotide sequence disclosed herein. In some embodiments, an RNA polynucleotide comprises a sequence having at least 80% identity to a nucleotide sequence disclosed herein. In some embodiments, an RNA polynucleotide comprises a sequence encoding a polypeptide having at least 80% identity to a polypeptide sequence disclosed herein. Exemplary nucleotide and polypeptide sequences are provided e.g., in Table 2 or in this section titled “Exemplary polynucleotides” or in Example 1 or 2. In some embodiments, an RNA polynucleotide described herein or a composition or medical preparation comprising the same is transcribed by a DNA template. In some embodiments, a DNA template used to transcribe an RNA polynucleotide described herein comprises a sequence complementary to an RNA polynucleotide. In some embodiments, a payload described herein is encoded by an RNA polynucleotide described herein comprising a nucleotide sequence disclosed herein, e.g., in Table 2 or in this section titled “Exemplary polynucleotides” or in Example 1 or 2. In some embodiments, an RNA polynucleotide encodes a polypeptide payload having at least 80% identity to a polypeptide payload sequence disclosed herein. In some embodiments, a payload described herein is encoded by an RNA polynucleotide transcribed by a DNA template comprising a sequence complementary to an RNA polynucleotide. Table 2: Exemplary sequences of RNA constructs disclosed herein RBL063.1 (SEQ ID NO: 28 nucleotide; SEQ ID NO: 9 amino acid) Structure beta-S-ARCA(D1)-hAg-Kozak-S1S2-PP-FI-A30L70 Encoded antigen Viral spike protein (S1S2 protein) of the SARS-CoV-2 (S1S2 full-length protein, sequence variant) SEQ ID NO: 28 Nucleic acid containing particles Nucleic acids described herein such as RNA encoding a payload may be administered formulated as particles.In the context of the present disclosure, the term "particle" relates to a structured entity formed by molecules or molecule complexes. In some embodiments, the term "particle" relates to a micro- or nano-sized structure, such as a micro- or nano-sized compact structure dispersed in a medium. In some embodiments, a particle is a nucleic acid containing particle such as a particle comprising DNA, RNA or a mixture thereof. Electrostatic interactions between positively charged molecules such as polymers and lipids and negatively charged nucleic acid are involved in particle formation. This results in complexation and spontaneous formation of nucleic acid particles. In some embodiments, a nucleic acid particle is a nanoparticle. As used in the present disclosure, "nanoparticle" refers to a particle having an average diameter suitable for parenteral administration. A "nucleic acid particle" can be used to deliver nucleic acid to a target site of interest (e.g., cell, tissue, organ, and the like). A nucleic acid particle may be formed from at least one cationic or cationically ionizable lipid or lipid-like material, at least one cationic polymer such as protamine, or a mixture thereof and nucleic acid. Nucleic acid particles include lipid nanoparticle (LNP)-based and lipoplex (LPX)-based formulations. Without intending to be bound by any theory, it is believed that the cationic or cationically ionizable lipid or lipid-like material and/or the cationic polymer combine together with the nucleic acid to form aggregates, and this aggregation results in colloidally stable particles. In some embodiments, particles described herein further comprise at least one lipid or lipid-like material other than a cationic or cationically ionizable lipid or lipid-like material, at least one polymer other than a cationic polymer, or a mixture thereof In some embodiments, nucleic acid particles comprise more than one type of nucleic acid molecules, where the molecular parameters of the nucleic acid molecules may be similar or different from each other, like with respect to molar mass or fundamental structural elements such as molecular architecture, capping, coding regions or other features. Nucleic acid particles described herein may have an average diameter that in some embodiments ranges from about 30 nm to about 1000 nm, from about 50 nm to about 800 nm, from about 70 nm to about 600 nm, from about 90 nm to about 400 nm, or from about 100 nm to about 300 nm. Nucleic acid particles described herein may exhibit a polydispersity index less than about 0.5, less than about 0.4, less than about 0.3, or about 0.2 or less. By way of example, the nucleic acid particles can exhibit a polydispersity index in a range of about 0.1 to about 0.3 or about 0.2 to about 0.3. With respect to RNA lipid particles, the N/P ratio gives the ratio of the nitrogen groups in the lipid to the number of phosphate groups in the RNA. It is correlated to the charge ratio, as the nitrogen atoms (depending on the pH) are usually positively charged and the phosphate groups are negatively charged. The N/P ratio, where a charge equilibrium exists, depends on the pH. Lipid formulations are frequently formed at N/P ratios larger than four up to twelve, because positively charged nanoparticles are considered favorable for transfection. In that case, RNA is considered to be completely bound to nanoparticles. Nucleic acid particles described herein can be prepared using a wide range of methods that may involve obtaining a colloid from at least one cationic or cationically ionizable lipid or lipid-like material and/or at least one cationic polymer and mixing the colloid with nucleic acid to obtain nucleic acid particles. The term "colloid" as used herein relates to a type of homogeneous mixture in which dispersed particles do not settle out. The insoluble particles in the mixture are microscopic, with particle sizes between 1 and 1000 nanometers. The mixture may be termed a colloid or a colloidal suspension. Sometimes the term "colloid" only refers to the particles in the mixture and not the entire suspension. For the preparation of colloids comprising at least one cationic or cationically ionizable lipid or lipid-like material and/or at least one cationic polymer methods are applicable herein that are conventionally used for preparing liposomal vesicles and are appropriately adapted. The most commonly used methods for preparing liposomal vesicles share the following fundamental stages: (i) lipids dissolution in organic solvents, (ii) drying of the resultant solution, and (iii) hydration of dried lipid (using various aqueous media). In the film hydration method, lipids are firstly dissolved in a suitable organic solvent, and dried down to yield a thin film at the bottom of the flask. The obtained lipid film is hydrated using an appropriate aqueous medium to produce a liposomal dispersion. Furthermore, an additional downsizing step may be included. Reverse phase evaporation is an alternative method to the film hydration for preparing liposomal vesicles that involves formation of a water-in-oil emulsion between an aqueous phase and an organic phase containing lipids. A brief sonication of this mixture is required for system homogenization. The removal of the organic phase under reduced pressure yields a milky gel that turns subsequently into a liposomal suspension. The term "ethanol injection technique" refers to a process, in which an ethanol solution comprising lipids is rapidly injected into an aqueous solution through a needle. This action disperses the lipids throughout the solution and promotes lipid structure formation, for example lipid vesicle formation such as liposome formation. Generally, the RNA lipoplex particles described herein are obtainable by adding RNA to a colloidal liposome dispersion. Using the ethanol injection technique, such colloidal liposome dispersion is, in some embodiments, formed as follows: an ethanol solution comprising lipids, such as cationic lipids and additional lipids, is injected into an aqueous solution under stirring. In some embodiments, the RNA lipoplex particles described herein are obtainable without a step of extrusion. The term "extruding" or "extrusion" refers to the creation of particles having a fixed, cross-sectional profile. In particular, it refers to the downsizing of a particle, whereby the particle is forced through filters with defined pores. Other methods having organic solvent free characteristics may also be used according to the present disclosure for preparing a colloid. LNPs typically comprise four components: ionizable cationic lipids, neutral lipids such as phospholipids, a steroid such as cholesterol, and a polymer conjugated lipid such as polyethylene glycol (PEG)-lipids. Each component is responsible for payload protection, and enables effective intracellular delivery. LNPs may be prepared by mixing lipids dissolved in ethanol rapidly with nucleic acid in an aqueous buffer. The term "average diameter" refers to the mean hydrodynamic diameter of particles as measured by dynamic laser light scattering (DLS) with data analysis using the so-called cumulant algorithm, which provides as results the so-called Zaverage with the dimension of a length, and the polydispersity index (PI), which is dimensionless (Koppel, D., J. Chem. Phys.57, 1972, pp 4814-4820, ISO 13321). Here "average diameter", "diameter" or "size" for particles is used synonymously with this value of the Zaverage. The "polydispersity index" is preferably calculated based on dynamic light scattering measurements by the so-called cumulant analysis as mentioned in the definition of the "average diameter". Under certain prerequisites, it can be taken as a measure of the size distribution of an ensemble of nanoparticles. Different types of nucleic acid containing particles have been described previously to be suitable for delivery of nucleic acid in particulate form (e.g. Kaczmarek, J. C. et al., 2017, Genome Medicine 9, 60). For non-viral nucleic acid delivery vehicles, nanoparticle encapsulation of nucleic acid physically protects nucleic acid from degradation and, depending on the specific chemistry, can aid in cellular uptake and endosomal escape. The present disclosure describes particles comprising nucleic acid, at least one cationic or cationically ionizable lipid or lipid-like material, and/or at least one cationic polymer which associate with nucleic acid to form nucleic acid particles and compositions comprising such particles. The nucleic acid particles may comprise nucleic acid which is complexed in different forms by non-covalent interactions to the particle. The particles described herein are not viral particles, in particular infectious viral particles, i.e., they are not able to virally infect cells. Suitable cationic or cationically ionizable lipids or lipid-like materials and cationic polymers are those that form nucleic acid particles and are included by the term "particle forming components" or "particle forming agents". The term "particle forming components" or "particle forming agents" relates to any components which associate with nucleic acid to form nucleic acid particles. Such components include any component which can be part of nucleic acid particles. Some embodiments described herein relate to compositions, methods and uses involving more than one, e.g., 2, 3, 4, 5, 6 or even more nucleic acid species such as RNA species, e.g., a) a nucleic acid comprising a first nucleotide sequence encoding an amino acid sequence comprising at least a fragment of a parental virus protein, wherein amino acid positions in the at least a fragment of a parental virus protein are modified to comprise amino acids found in the corresponding amino acid positions of one or more virus protein variants; and b) a nucleic acid comprising a second nucleotide sequence encoding an amino acid sequence comprising at least a fragment of a parental virus protein, wherein amino acid positions in the at least a fragment of a parental virus protein are modified to comprise amino acids found in the corresponding amino acid positions of one or more virus protein variants. In a particulate formulation, it is possible that each nucleic acid species is separately formulated as an individual particulate formulation. In that case, each individual particulate formulation will comprise one nucleic acid species. The individual particulate formulations may be present as separate entities, e.g. in separate containers. Such formulations are obtainable by providing each nucleic acid species separately (typically each in the form of a nucleic acid- containing solution) together with a particle-forming agent, thereby allowing the formation of particles. Respective particles will contain exclusively the specific nucleic acid species that is being provided when the particles are formed (individual particulate formulations). In some embodiments, a composition such as a pharmaceutical composition comprises more than one individual particle formulation. Respective pharmaceutical compositions are referred to as mixed particulate formulations. Mixed particulate formulations according to the invention are obtainable by forming, separately, individual particulate formulations, as described above, followed by a step of mixing of the individual particulate formulations. By the step of mixing, a formulation comprising a mixed population of nucleic acid-containing particles is obtainable. Individual particulate populations may be together in one container, comprising a mixed population of individual particulate formulations. Alternatively, it is possible that different nucleic acid species are formulated together as a combined particulate formulation. Such formulations are obtainable by providing a combined formulation (typically combined solution) of different RNA species together with a particle- forming agent, thereby allowing the formation of particles. As opposed to a mixed particulate formulation, a combined particulate formulation will typically comprise particles which comprise more than one RNA species. In a combined particulate composition different RNA species are typically present together in a single particle. Cationic polymeric materials (e.g., polymers) Given their high degree of chemical flexibility, polymeric materials are commonly used for nanoparticle-based delivery. Typically, cationic materials are used to electrostatically condense the negatively charged nucleic acid into nanoparticles. These positively charged groups often consist of amines that change their state of protonation in the pH range between 5.5 and 7.5, thought to lead to an ion imbalance that results in endosomal rupture. Polymers such as poly-L-lysine, polyamidoamine, protamine and polyethyleneimine, as well as naturally occurring polymers such as chitosan have all been applied to nucleic acid delivery and are suitable as cationic materials useful in some embodiments herein. In addition, some investigators have synthesized polymeric materials specifically for nucleic acid delivery. Poly(β-amino esters), in particular, have gained widespread use in nucleic acid delivery owing to their ease of synthesis and biodegradability. In some embodiments, such synthetic materials may be suitable for use as cationic materials herein. A "polymeric material", as used herein, is given its ordinary meaning, i.e., a molecular structure comprising one or more repeat units (monomers), connected by covalent bonds. In some embodiments, such repeat units can all be identical; alternatively, in some cases, there can be more than one type of repeat unit present within the polymeric material. In some cases, a polymeric material is biologically derived, e.g., a biopolymer such as a protein. In some cases, additional moieties can also be present in the polymeric material, for example targeting moieties such as those described herein. Those skilled in the art are aware that, when more than one type of repeat unit is present within a polymer (or polymeric moiety), then the polymer (or polymeric moiety) is said to be a "copolymer." In some embodiments, a polymer (or polymeric moiety) utilized in accordance with the present disclosure may be a copolymer. Repeat units forming the copolymer can be arranged in any fashion. For example, in some embodiments, repeat units can be arranged in a random order; alternatively or additionally, in some embodiments, repeat units may be arranged in an alternating order, or as a "block" copolymer, i.e., comprising one or more regions each comprising a first repeat unit (e.g., a first block), and one or more regions each comprising a second repeat unit (e.g., a second block), etc. Block copolymers can have two (a diblock copolymer), three (a triblock copolymer), or more numbers of distinct blocks. In certain embodiments, a polymeric material for use in accordance with the present disclosure is biocompatible. Biocompatible materials are those that typically do not result in significant cell death at moderate concentrations. In certain embodiments, a biocompatible material is biodegradable, i.e., is able to degrade, chemically and/or biologically, within a physiological environment, such as within the body. In certain embodiments, a polymeric material may be or comprise protamine or polyalkyleneimine, in particular protamine. As those skilled in the art are aware term "protamine" is often used to refer to any of various strongly basic proteins of relatively low molecular weight that are rich in arginine and are found associated especially with DNA in place of somatic histones in the sperm cells of various animals (as fish). In particular, the term "protamine" is often used to refer to proteins found in fish sperm that are strongly basic, are soluble in water, are not coagulated by heat, and yield chiefly arginine upon hydrolysis. In purified form, they are used in a long-acting formulation of insulin and to neutralize the anticoagulant effects of heparin. In some embodiments, the term "protamine" as used herein is refers to a protamine amino acid sequence obtained or derived from natural or biological sources, including fragments thereof and/or multimeric forms of said amino acid sequence or fragment thereof, as well as (synthesized) polypeptides which are artificial and specifically designed for specific purposes and cannot be isolated from native or biological sources. In some embodiments, a polyalkyleneimine comprises polyethylenimine and/or polypropylenimine, preferably polyethyleneimine. In some embodiments, a preferred polyalkyleneimine is polyethyleneimine (PEI). In some embodiments, the average molecular weight of PEI is preferably 0.75∙102 to 107 Da, preferably 1000 to 105 Da, more preferably 10000 to 40000 Da, more preferably 15000 to 30000 Da, even more preferably 20000 to 25000 Da. Preferred according to certain embodiments of the disclosure is linear polyalkyleneimine such as linear polyethyleneimine (PEI). Cationic materials (e.g., polymeric materials, including polycationic polymers) contemplated for use herein include those which are able to electrostatically bind nucleic acid. In some embodiments, cationic polymeric materials contemplated for use herein include any cationic polymeric materials with which nucleic acid can be associated, e.g. by forming complexes with the nucleic acid or forming vesicles in which the nucleic acid is enclosed or encapsulated. In some embodiments, particles described herein may comprise polymers other than cationic polymers, e.g., non-cationic polymeric materials and/or anionic polymeric materials. Collectively, anionic and neutral polymeric materials are referred to herein as non-cationic polymeric materials. Lipid and lipid-like material The terms "lipid" and "lipid-like material" are used herein to refer to molecules which comprise one or more hydrophobic moieties or groups and optionally also one or more hydrophilic moieties or groups. Molecules comprising hydrophobic moieties and hydrophilic moieties are also frequently denoted as amphiphiles. Lipids are usually poorly soluble in water. In an aqueous environment, the amphiphilic nature allows the molecules to self-assemble into organized structures and different phases. One of those phases consists of lipid bilayers, as they are present in vesicles, multilamellar/unilamellar liposomes, or membranes in an aqueous environment. Hydrophobicity can be conferred by the inclusion of apolar groups that include, but are not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted by one or more aromatic, cycloaliphatic, or heterocyclic group(s). In some embodiments, hydrophilic groups may comprise polar and/or charged groups and include carbohydrates, phosphate, carboxylic, sulfate, amino, sulfhydryl, nitro, hydroxyl, and other like groups. As used herein, the term "amphiphilic" refers to a molecule having both a polar portion and a non-polar portion. Often, an amphiphilic compound has a polar head attached to a long hydrophobic tail. In some embodiments, the polar portion is soluble in water, while the non-polar portion is insoluble in water. In addition, the polar portion may have either a formal positive charge, or a formal negative charge. Alternatively, the polar portion may have both a formal positive and a negative charge, and be a zwitterion or inner salt. For purposes of the disclosure, the amphiphilic compound can be, but is not limited to, one or a plurality of natural or non- natural lipids and lipid-like compounds. The term "lipid-like material", "lipid-like compound" or "lipid-like molecule" relates to substances that structurally and/or functionally relate to lipids but may not be considered as lipids in a strict sense. For example, the term includes compounds that are able to form amphiphilic layers as they are present in vesicles, multilamellar/unilamellar liposomes, or membranes in an aqueous environment and includes surfactants, or synthesized compounds with both hydrophilic and hydrophobic moieties. Generally speaking, the term refers to molecules, which comprise hydrophilic and hydrophobic moieties with different structural organization, which may or may not be similar to that of lipids. As used herein, the term "lipid" is to be construed to cover both lipids and lipid-like materials unless otherwise indicated herein or clearly contradicted by context. Specific examples of amphiphilic compounds that may be included in an amphiphilic layer include, but are not limited to, phospholipids, aminolipids and sphingolipids. In certain embodiments, the amphiphilic compound is a lipid. The term "lipid" refers to a group of organic compounds that are characterized by being insoluble in water, but soluble in many organic solvents. Generally, lipids may be divided into eight categories: fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, polyketides (derived from condensation of ketoacyl subunits), sterol lipids and prenol lipids (derived from condensation of isoprene subunits). Although the term "lipid" is sometimes used as a synonym for fats, fats are a subgroup of lipids called triglycerides. Lipids also encompass molecules such as fatty acids and their derivatives (including tri-, di-, monoglycerides, and phospholipids), as well as sterol- containing metabolites such as cholesterol. Fatty acids, or fatty acid residues are a diverse group of molecules made of a hydrocarbon chain that terminates with a carboxylic acid group; this arrangement confers the molecule with a polar, hydrophilic end, and a nonpolar, hydrophobic end that is insoluble in water. The carbon chain, typically between four and 24 carbons long, may be saturated or unsaturated, and may be attached to functional groups containing oxygen, halogens, nitrogen, and sulfur. If a fatty acid contains a double bond, there is the possibility of either a cis or trans geometric isomerism, which significantly affects the molecule's configuration. Cis-double bonds cause the fatty acid chain to bend, an effect that is compounded with more double bonds in the chain. Other major lipid classes in the fatty acid category are the fatty esters and fatty amides. Glycerolipids are composed of mono-, di-, and tri-substituted glycerols, the best-known being the fatty acid triesters of glycerol, called triglycerides. The word "triacylglycerol" is sometimes used synonymously with "triglyceride". In these compounds, the three hydroxyl groups of glycerol are each esterified, typically by different fatty acids. Additional subclasses of glycerolipids are represented by glycosylglycerols, which are characterized by the presence of one or more sugar residues attached to glycerol via a glycosidic linkage. The glycerophospholipids are amphipathic molecules (containing both hydrophobic and hydrophilic regions) that contain a glycerol core linked to two fatty acid-derived "tails" by ester linkages and to one "head" group by a phosphate ester linkage. Examples of glycerophospholipids, usually referred to as phospholipids (though sphingomyelins are also classified as phospholipids) are phosphatidylcholine (also known as PC, GPCho or lecithin), phosphatidylethanolamine (PE or GPEtn) and phosphatidylserine (PS or GPSer). Sphingolipids are a complex family of compounds that share a common structural feature, a sphingoid base backbone. The major sphingoid base in mammals is commonly referred to as sphingosine. Ceramides (N-acyl-sphingoid bases) are a major subclass of sphingoid base derivatives with an amide-linked fatty acid. The fatty acids are typically saturated or mono- unsaturated with chain lengths from 16 to 26 carbon atoms. The major phosphosphingolipids of mammals are sphingomyelins (ceramide phosphocholines), whereas insects contain mainly ceramide phosphoethanolamines and fungi have phytoceramide phosphoinositols and mannose- containing headgroups. The glycosphingolipids are a diverse family of molecules composed of one or more sugar residues linked via a glycosidic bond to the sphingoid base. Examples of these are the simple and complex glycosphingolipids such as cerebrosides and gangliosides. Sterol lipids, such as cholesterol and its derivatives, or tocopherol and its derivatives, are an important component of membrane lipids, along with the glycerophospholipids and sphingomyelins. Saccharolipids describe compounds in which fatty acids are linked directly to a sugar backbone, forming structures that are compatible with membrane bilayers. In the saccharolipids, a monosaccharide substitutes for the glycerol backbone present in glycerolipids and glycerophospholipids. The most familiar saccharolipids are the acylated glucosamine precursors of the Lipid A component of the lipopolysaccharides in Gram-negative bacteria. Typical lipid A molecules are disaccharides of glucosamine, which are derivatized with as many as seven fatty- acyl chains. The minimal lipopolysaccharide required for growth in E. coli is Kdo2-Lipid A, a hexa-acylated disaccharide of glucosamine that is glycosylated with two 3-deoxy-D-manno- octulosonic acid (Kdo) residues. Polyketides are synthesized by polymerization of acetyl and propionyl subunits by classic enzymes as well as iterative and multimodular enzymes that share mechanistic features with the fatty acid synthases. They comprise a large number of secondary metabolites and natural products from animal, plant, bacterial, fungal and marine sources, and have great structural diversity. Many polyketides are cyclic molecules whose backbones are often further modified by glycosylation, methylation, hydroxylation, oxidation, or other processes. According to the disclosure, lipids and lipid-like materials may be cationic, anionic or neutral. Neutral lipids or lipid-like materials exist in an uncharged or neutral zwitterionic form at a selected pH. Cationic or cationically ionizable lipids or lipid-like materials In some embodiments, nucleic acid particles described and/or utilized in accordance with the present disclosure may comprise at least one cationic or cationically ionizable lipid or lipid- like material as particle forming agent. Cationic or cationically ionizable lipids or lipid-like materials contemplated for use herein include any cationic or cationically ionizable lipids or lipid-like materials which are able to electrostatically bind nucleic acid. In some embodiments, cationic or cationically ionizable lipids or lipid-like materials contemplated for use herein can be associated with nucleic acid, e.g. by forming complexes with the nucleic acid or forming vesicles in which the nucleic acid is enclosed or encapsulated. As used herein, a "cationic lipid" or "cationic lipid-like material" refers to a lipid or lipid- like material having a net positive charge. Cationic lipids or lipid-like materials bind negatively charged nucleic acid by electrostatic interaction. Generally, cationic lipids possess a lipophilic moiety, such as a sterol, an acyl chain, a diacyl or more acyl chains, and the head group of the lipid typically carries the positive charge. In certain embodiments, a cationic lipid or lipid-like material has a net positive charge only at certain pH, in particular acidic pH, while it has preferably no net positive charge, preferably has no charge, i.e., it is neutral, at a different, preferably higher pH such as physiological pH. This ionizable behavior is thought to enhance efficacy through helping with endosomal escape and reducing toxicity as compared with particles that remain cationic at physiological pH. For purposes of the present disclosure, such "cationically ionizable" lipids or lipid-like materials are comprised by the term "cationic lipid or lipid-like material" unless contradicted by the circumstances. In some embodiments, a cationic or cationically ionizable lipid or lipid-like material comprises a head group which includes at least one nitrogen atom (N) which is positive charged or capable of being protonated. Examples of cationic lipids include, but are not limited to: ((4- hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecano ate); 1,2-dioleoyl-3- trimethylammonium propane (DOTAP); N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 3-(N—(N′,N′- dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), dimethyldioctadecylammonium (DDAB); 1,2-dioleoyl-3-dimethylammonium-propane (DODAP); 1,2-diacyloxy-3- dimethylammonium propanes; 1,2-dialkyloxy-3-dimethylammonium propanes; dioctadecyldimethyl ammonium chloride (DODAC), 1,2-distearyloxy-N,N-dimethyl-3- aminopropane (DSDMA), 2,3-di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium (DMRIE), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), l,2-dimyristoyl-3- trimethylammonium propane (DMTAP), 1,2-dioleyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DORIE), and 2,3-dioleoyloxy- N-[2(spermine carboxamide)ethyl]-N,N- dimethyl-l-propanamium trifluoroacetate (DOSPA), 1,2-dilinoleyloxy-N,N- dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), dioctadecylamidoglycyl spermine (DOGS), 3-dimethylamino-2-(cholest-5-en-3- beta-oxybutan-4-oxy)-1-(cis,cis-9,12-oc-tadecadienoxy)propan e (CLinDMA), 2-[5′-(cholest-5- en-3-beta-oxy)-3′-oxapentoxy)-3-dimethyl-1-(cis,cis-9′,1 2′-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N′-dioleylcarbamyl- 3-dimethylaminopropane (DOcarbDAP), 2,3-Dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP), 1,2-N,N′-Dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1,2- Dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl-4- dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleyl-4-dimethylaminoethyl- [1,3]-dioxolane (DLin-K-XTC2-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)buta noate (DLin- MC3-DMA), N-(2-Hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-pro panaminium bromide (DMRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(cis-9-tetradecen yloxy)-1- propanaminium bromide (GAP-DMORIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3- bis(dodecyloxy)-1-propanaminium bromide (GAP-DLRIE), (±)-N-(3-aminopropyl)-N,N- dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (GAP-DMRIE), N-(2-Aminoethyl)- N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (βAE-DMRIE), N-(4- carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-amini um (DOBAQ), 2-({8-[(3β)- cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-oct adeca-9,12-dien-1-yloxy]propan- 1-amine (Octyl-CLinDMA), 1,2-dimyristoyl-3-dimethylammonium-propane (DMDAP), 1,2- dipalmitoyl-3-dimethylammonium-propane (DPDAP), N1-[2-((1S)-1-[(3-aminopropyl)amino]- 4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[ol eyloxy]-benzamide (MVL5), 1,2- dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), 2,3-bis(dodecyloxy)-N-(2-hydroxyethyl)- N,N-dimethylpropan-1-amonium bromide (DLRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3- bis(tetradecyloxy)propan-1-aminium bromide (DMORIE), di((Z)-non-2-en-1-yl) 8,8'- ((((2(dimethylamino)ethyl)thio)carbonyl)azanediyl)dioctanoat e (ATX), N,N-dimethyl-2,3- bis(dodecyloxy)propan-1-amine (DLDMA), N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1- amine (DMDMA), Di((Z)-non-2-en-1-yl)-9-((4-(dimethylaminobutanoyl)oxy)hepta decanedioate (L319), N-Dodecyl-3-((2-dodecylcarbamoyl-ethyl)-{2-[(2-dodecylcarbam oyl-ethyl)-2-{(2- dodecylcarbamoyl-ethyl)-[2-(2-dodecylcarbamoyl-ethylamino)-e thyl]-amino}- ethylamino)propionamide (lipidoid 98N12-5), 1-[2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2- [bis(2 hydroxydodecyl)amino]ethyl]piperazin-1-yl]ethyl]amino]dodeca n-2-ol (lipidoid C12- 200); or heptadecan-9-yl 8-((2-hydroxyethyl) (6-oxo-6-(undecyloxy) hexyl) amino) octanoate (SM-102). In some embodiments, a cationic lipid is or comprises heptadecan-9-yl 8-((2- hydroxyethyl) (6-oxo-6-(undecyloxy) hexyl) amino) octanoate (SM-102). In some embodiments, a cationic lipid is or comprises a cationic lipid shown in the structure below. In some embodiments, a cationic lipid is or comprises ((4- hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecano ate) which is also referred to as ALC-0315 herein. In some embodiments, a cationic lipid may comprise from about 10 mol % to about 100 mol %, about 20 mol % to about 100 mol %, about 30 mol % to about 100 mol %, about 40 mol % to about 100 mol %, or about 50 mol % to about 100 mol % of the total lipid present in the particle. In some particular embodiments, a particle for use in accordance with the present disclosure includes ALC-0315, for example in a weight percent within a range of about 40-55 mol percent of total lipids. Additional lipids or lipid-like materials In some embodiments, particles described herein comprise (e.g., in addition to a cationic lipid such as ALC315), one or more lipids or lipid-like materials other than cationic or cationically ionizable lipids or lipid-like materials, e.g., non-cationic lipids or lipid-like materials (including non-cationically ionizable lipids or lipid-like materials). Collectively, anionic and neutral lipids or lipid-like materials are referred to herein as non-cationic lipids or lipid-like materials. Optimizing the formulation of nucleic acid particles by addition of other hydrophobic moieties, such as cholesterol and lipids, in addition to an ionizable/cationic lipid or lipid-like material may enhance particle stability and efficacy of nucleic acid delivery. An additional lipid or lipid-like material may be incorporated which may or may not affect the overall charge of the nucleic acid particles. In certain embodiments, the additional lipid or lipid-like material is a non-cationic lipid or lipid-like material. The non-cationic lipid may comprise, e.g., one or more anionic lipids and/or neutral lipids. As used herein, an "anionic lipid" refers to any lipid that is negatively charged at a selected pH. As used herein, a "neutral lipid" refers to any of a number of lipid species that exist either in an uncharged or neutral zwitterionic form at a selected pH. In preferred embodiments, the additional lipid comprises one of the following neutral lipid components: (1) a phospholipid, (2) cholesterol or a derivative thereof; or (3) a mixture of a phospholipid and cholesterol or a derivative thereof. Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'- hydroxybutyl ether, tocopherol and derivatives thereof, and mixtures thereof. Specific phospholipids that can be used include, but are not limited to, phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acids, phosphatidylserines or sphingomyelin. Such phospholipids include in particular diacylphosphatidylcholines, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), palmitoyloleoyl-phosphatidylcholine (POPC), 1,2-di-O- octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2- cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero- 3-phosphocholine (C16 Lyso PC) and phosphatidylethanolamines, in particular diacylphosphatidylethanolamines, such as dioleoylphosphatidylethanolamine (DOPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), dilauroyl-phosphatidylethanolamine (DLPE), diphytanoyl-phosphatidylethanolamine (DPyPE), and further phosphatidylethanolamine lipids with different hydrophobic chains. In certain preferred embodiments, the additional lipid is DSPC or DSPC and cholesterol. In certain embodiments, the nucleic acid particles include both a cationic lipid and an additional lipid. In some embodiments, particles described herein include a polymer conjugated lipid such as a pegylated lipid. The term "pegylated lipid" refers to a molecule comprising both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art. In some embodiments, a pegylated lipid is ALC-0159, also referred to herein as (2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide). Without wishing to be bound by theory, the amount of the at least one cationic lipid compared to the amount of the at least one additional lipid may affect important nucleic acid particle characteristics, such as charge, particle size, stability, tissue selectivity, and bioactivity of the nucleic acid. Accordingly, in some embodiments, the molar ratio of the at least one cationic lipid to the at least one additional lipid is from about 10:0 to about 1:9, about 4:1 to about 1:2, or about 3:1 to about 1:1. In some embodiments, the non-cationic lipid, in particular neutral lipid, (e.g., one or more phospholipids and/or cholesterol) may comprise from about 0 mol % to about 90 mol %, from about 0 mol % to about 80 mol %, from about 0 mol % to about 70 mol %, from about 0 mol % to about 60 mol %, or from about 0 mol % to about 50 mol %, of the total lipid present in the particle. In some embodiments, particles for use in accordance with the present disclosure may include, for example, ALC-0315, DSPC, CHOL, and ALC-0159, for example, wherein ALC- 0315 is at about 40 to 55 mol percent; DSPC is at about 5 to 15 mol percent; CHOL is at about 30 to 50 mol percent; and ALC-0159 is at about 1 to 10 mol percent. Lipoplex Particles In certain embodiments of the present disclosure, an RNA may be present in RNA lipoplex particles. In the context of the present disclosure, the term "RNA lipoplex particle" relates to a particle that contains lipid, in particular cationic lipid, and RNA. Electrostatic interactions between positively charged liposomes and negatively charged RNA results in complexation and spontaneous formation of RNA lipoplex particles. Positively charged liposomes may be generally synthesized using a cationic lipid, such as DOTMA, and additional lipids, such as DOPE. In some embodiments, a RNA lipoplex particle is a nanoparticle. In certain embodiments, the RNA lipoplex particles include both a cationic lipid and an additional lipid. In an exemplary embodiment, the cationic lipid is DOTMA and the additional lipid is DOPE. In some embodiments, the molar ratio of the at least one cationic lipid to the at least one additional lipid is from about 10:0 to about 1:9, about 4:1 to about 1:2, or about 3:1 to about 1:1. In specific embodiments, the molar ratio may be about 3:1, about 2.75:1, about 2.5:1, about 2.25:1, about 2:1, about 1.75:1, about 1.5:1, about 1.25:1, or about 1:1. In an exemplary embodiment, the molar ratio of the at least one cationic lipid to the at least one additional lipid is about 2:1. RNA lipoplex particles described herein have an average diameter that in some embodiments ranges from about 200 nm to about 1000 nm, from about 200 nm to about 800 nm, from about 250 to about 700 nm, from about 400 to about 600 nm, from about 300 nm to about 500 nm, or from about 350 nm to about 400 nm. In specific embodiments, the RNA lipoplex particles have an average diameter of about 200 nm, about 225 nm, about 250 nm, about 275 nm, about 300 nm, about 325 nm, about 350 nm, about 375 nm, about 400 nm, about 425 nm, about 450 nm, about 475 nm, about 500 nm, about 525 nm, about 550 nm, about 575 nm, about 600 nm, about 625 nm, about 650 nm, about 700 nm, about 725 nm, about 750 nm, about 775 nm, about 800 nm, about 825 nm, about 850 nm, about 875 nm, about 900 nm, about 925 nm, about 950 nm, about 975 nm, or about 1000 nm. In an embodiment, the RNA lipoplex particles have an average diameter that ranges from about 250 nm to about 700 nm. In another embodiment, the RNA lipoplex particles have an average diameter that ranges from about 300 nm to about 500 nm. In an exemplary embodiment, the RNA lipoplex particles have an average diameter of about 400 nm. In some embodiments, RNA lipoplex particles andor compositions comprising RNA lipoplex particles described herein are useful for delivery of RNA to a target tissue after parenteral administration, in particular after intravenous administration. In some embodiments, RNA lipoplex particles may be prepared using liposomes that may be obtained by injecting a solution of the lipids in ethanol into water or a suitable aqueous phase. In some embodiments, the aqueous phase has an acidic pH. In some embodiments, the aqueous phase comprises acetic acid, e.g., in an amount of about 5 mM. Liposomes may be used for preparing RNA lipoplex particles by mixing the liposomes with RNA. In some embodiments, the liposomes and RNA lipoplex particles comprise at least one cationic lipid and at least one additional lipid. In some embodiments, the at least one cationic lipid comprises 1,2-di-O-octadecenyl-3- trimethylammonium propane (DOTMA) and/or 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). In some embodiments, the at least one additional lipid comprises 1,2-di-(9Z- octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol (Chol) and/or 1,2- dioleoyl-sn-glycero-3-phosphocholine (DOPC). In some embodiments, the at least one cationic lipid comprises 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and the at least one additional lipid comprises 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE). In some embodiments, the liposomes and RNA lipoplex particles comprise 1,2-di-O- octadecenyl-3-trimethylammonium propane (DOTMA) and 1,2-di-(9Z-octadecenoyl)-sn- glycero-3-phosphoethanolamine (DOPE). Spleen targeting RNA lipoplex particles are described in WO 2013/143683, herein incorporated by reference. It has been found that RNA lipoplex particles having a net negative charge may be used to preferentially target spleen tissue or spleen cells such as antigen- presenting cells, in particular dendritic cells. Accordingly, following administration of the RNA lipoplex particles, RNA accumulation and/or RNA expression in the spleen occurs. Thus, RNA lipoplex particles of the disclosure may be used for expressing RNA in the spleen. In an embodiment, after administration of the RNA lipoplex particles, no or essentially no RNA accumulation and/or RNA expression in the lung and/or liver occurs. In some embodiments, after administration of the RNA lipoplex particles, RNA accumulation and/or RNA expression in antigen presenting cells, such as professional antigen presenting cells in the spleen occurs. Thus, RNA lipoplex particles of the disclosure may be used for expressing RNA in such antigen presenting cells. In some embodiments, the antigen presenting cells are dendritic cells and/or macrophages. Lipid nanoparticles (LNPs) In some embodiments, nucleic acid such as RNA described herein is administered in the form of lipid nanoparticles (LNPs). The LNP may comprise any lipid capable of forming a particle to which the one or more nucleic acid molecules are attached, or in which the one or more nucleic acid molecules are encapsulated. In some embodiments, the LNP comprises one or more cationic lipids, and one or more stabilizing lipids. Stabilizing lipids include neutral lipids and pegylated lipids. In some embodiments, the LNP comprises a cationic lipid, a neutral lipid, a steroid, a polymer conjugated lipid; and the RNA, encapsulated within or associated with the lipid nanoparticle. In some embodiments, an LNP comprises from 40 to 55 mol percent, from 40 to 50 mol percent, from 41 to 49 mol percent, from 41 to 48 mol percent, from 42 to 48 mol percent, from 43 to 48 mol percent, from 44 to 48 mol percent, from 45 to 48 mol percent, from 46 to 48 mol percent, from 47 to 48 mol percent, or from 47.2 to 47.8 mol percent of the cationic lipid. In some embodiments, the LNP comprises about 47.0, 47.1, 47.2, 47.3, 47.4, 47.5, 47.6, 47.7, 47.8, 47.9 or 48.0 mol percent of the cationic lipid. In some embodiments, the neutral lipid is present in a concentration ranging from 5 to 15 mol percent, from 7 to 13 mol percent, or from 9 to 11 mol percent. In some embodiments, the neutral lipid is present in a concentration of about 9.5, 10 or 10.5 mol percent. In some embodiments, the steroid is present in a concentration ranging from 30 to 50 mol percent, from 35 to 45 mol percent or from 38 to 43 mol percent. In some embodiments, the steroid is present in a concentration of about 40, 41, 42, 43, 44, 45 or 46 mol percent. In some embodiments, the LNP comprises from 1 to 10 mol percent, from 1 to 5 mol percent, or from 1 to 2.5 mol percent of the polymer conjugated lipid. In some embodiments, the LNP comprises from 40 to 50 mol percent a cationic lipid; from 5 to 15 mol percent of a neutral lipid; from 35 to 45 mol percent of a steroid; from 1 to 10 mol percent of a polymer conjugated lipid; and the RNA, encapsulated within or associated with the lipid nanoparticle. In some embodiments, the mol percent is determined based on total mol of lipid present in the lipid nanoparticle. In some embodiments, the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, DOPG, DPPG, POPE, DPPE, DMPE, DSPE, and SM. In some embodiments, the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In some embodiments, the neutral lipid is DSPC. In some embodiments, the steroid is cholesterol. In some embodiments, the polymer conjugated lipid is a pegylated lipid. In some embodiments, the pegylated lipid has the following structure: or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: R ¹² and R ¹³ are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and w has a mean value ranging from 30 to 60. In some embodiments, R ¹² and R ¹³ are each independently straight, saturated alkyl chains containing from 12 to 16 carbon atoms. In some embodiments, w has a mean value ranging from 40 to 55. In some embodiments, the average w is about 45. In some embodiments, R ¹² and R ¹³ are each independently a straight, saturated alkyl chain containing about 14 carbon atoms, and w has a mean value of about 45. In some embodiments, the pegylated lipid is DMG-PEG 2000, e.g., having the following structure: In some embodiments, the cationic lipid component of the LNPs has the structure of Formula (III): or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein: one of L ¹ or L ² is –O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)x-, -S-S-, -C(=O)S-, SC(=O)-, - NR ^a C(=O)-, -C(=O)NR ^a -, NR ^a C(=O)NR ^a -, -OC(=O)NR ^a - or -NR ^a C(=O)O-, and the other of L ¹ or L ² is –O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) _x -, -S-S-, -C(=O)S-, SC(=O)-, -NR ^a C(=O)-, - C(=O)NR ^a -, NR ^a C(=O)NR ^a -, -OC(=O)NR ^a - or -NR ^a C(=O)O- or a direct bond; G ¹ and G2 are each independently unsubstituted C1-C12 alkylene or C1-C12 alkenylene; G ³ is C ₁ -C ₂₄ alkylene, C ₁ -C ₂₄ alkenylene, C ₃ -C ₈ cycloalkylene, C ₃ -C ₈ cycloalkenylene; R ^a is H or C ₁ -C ₁₂ alkyl; R ¹ and R ² are each independently C6-C24 alkyl or C6-C24 alkenyl; R ³ is H, OR ⁵ , CN, -C(=O)OR ⁴ , -OC(=O)R ⁴ or –NR ⁵ C(=O)R ⁴ ; R ⁴ is C ₁ -C ₁₂ alkyl; R ⁵ is H or C1-C6 alkyl; and x is 0, 1 or 2. In some of the foregoing embodiments of Formula (III), the lipid has one of the following structures (IIIA) or (IIIB): wherein: A is a 3 to 8-membered cycloalkyl or cycloalkylene ring; R ⁶ is, at each occurrence, independently H, OH or C1-C24 alkyl; n is an integer ranging from 1 to 15. In some of the foregoing embodiments of Formula (III), the lipid has structure (IIIA), and in other embodiments, the lipid has structure (IIIB). In other embodiments of Formula (III), the lipid has one of the following structures (IIIC) or (IIID): wherein y and z are each independently integers ranging from 1 to 12. In any of the foregoing embodiments of Formula (III), one of L ¹ or L ² is -O(C=O)-. For example, in some embodiments each of L ¹ and L ² are -O(C=O)-. In some different embodiments of any of the foregoing, L ¹ and L ² are each independently -(C=O)O- or -O(C=O)-. For example, in some embodiments each of L ¹ and L ² is -(C=O)O-. In some different embodiments of Formula (III), the lipid has one of the following structures (IIIE) or (IIIF): In some of the foregoing embodiments of Formula (III), the lipid has one of the following structures (IIIG), (IIIH), (IIII), or (IIIJ):

In some of the foregoing embodiments of Formula (III), n is an integer ranging from 2 to 12, for example from 2 to 8 or from 2 to 4. For example, in some embodiments, n is 3, 4, 5 or 6. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6. In some other of the foregoing embodiments of Formula (III), y and z are each independently an integer ranging from 2 to 10. For example, in some embodiments, y and z are each independently an integer ranging from 4 to 9 or from 4 to 6. In some of the foregoing embodiments of Formula (III), R ⁶ is H. In other of the foregoing embodiments, R ⁶ is C ₁ -C ₂₄ alkyl. In other embodiments, R ⁶ is OH. In some embodiments of Formula (III), G ³ is unsubstituted. In other embodiments, G ³ is substituted. In various different embodiments, G ³ is linear C ₁ -C ₂₄ alkylene or linear C ₁ -C ₂₄ alkenylene. In some other foregoing embodiments of Formula (III), R ¹ or R ² , or both, is C6-C24 alkenyl. For example, in some embodiments, R ¹ and R ² each, independently have the following structure: wherein: R ^7a and R ^7b are, at each occurrence, independently H or C ₁ -C ₁₂ alkyl; and a is an integer from 2 to 12, wherein R ^7a , R ^7b and a are each selected such that R ¹ and R ² each independently comprise from 6 to 20 carbon atoms. For example, in some embodiments a is an integer ranging from 5 to 9 or from 8 to 12. In some of the foregoing embodiments of Formula (III), at least one occurrence of R ^7a is H. For example, in some embodiments, R ^7a is H at each occurrence. In other different embodiments of the foregoing, at least one occurrence of R ^7b is C ₁ -C ₈ alkyl. For example, in some embodiments, C1-C8 alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert- butyl, n-hexyl or n-octyl. In different embodiments of Formula (III), R ¹ or R ² , or both, has one of the following structures: In some of the foregoing embodiments of Formula (III), R ³ is OH, CN, -C(=O)OR ⁴ , -OC(=O)R ⁴ or –NHC(=O)R ⁴ . In some embodiments, R ⁴ is methyl or ethyl. In various different embodiments, the cationic lipid of Formula (III) has one of the structures set forth in the table below. Representative Compounds of Formula (III). In some embodiments, an LNP comprises a lipid of Formula (III), RNA, a neutral lipid, a steroid and a pegylated lipid. In some embodiments, a lipid of Formula (III) is compound III-3. In some embodiments, a neutral lipid is DSPC. In some embodiments, a steroid is cholesterol. In some embodiments, a pegylated lipid is ALC-0159. In some embodiments, the cationic lipid is present in the LNP in an amount from about 40 to about 50 mole percent. In some embodiments, the neutral lipid is present in the LNP in an amount from about 5 to about 15 mole percent. In some embodiments, the steroid is present in the LNP in an amount from about 35 to about 45 mole percent. In some embodiments, the pegylated lipid is present in the LNP in an amount from about 1 to about 10 mole percent. In some embodiments, the LNP comprises compound III-3 in an amount from about 40 to about 50 mole percent, DSPC in an amount from about 5 to about 15 mole percent, cholesterol in an amount from about 35 to about 45 mole percent, and ALC-0159 in an amount from about 1 to about 10 mole percent. In some embodiments, the LNP comprises compound III-3 in an amount of about 47.5 mole percent, DSPC in an amount of about 10 mole percent, cholesterol in an amount of about 40.7 mole percent, and ALC-0159 in an amount of about 1.8 mole percent. In various different embodiments, the cationic lipid has one of the structures set forth in the table below.

In some embodiments, the LNP comprises a cationic lipid shown in the above table, e.g., a cationic lipid of Formula (B) or Formula (D), in particular a cationic lipid of Formula (D), RNA, a neutral lipid, a steroid and a pegylated lipid. In some embodiments, the neutral lipid is DSPC. In some embodiments, the steroid is cholesterol. In some embodiments, the pegylated lipid is DMG-PEG 2000. In some embodiments, the LNP comprises a cationic lipid that is an ionizable lipid-like material (lipidoid). In some embodiments, the cationic lipid has the following structure: The N/P value is preferably at least about 4. In some embodiments, the N/P value ranges from 4 to 20, 4 to 12, 4 to 10, 4 to 8, or 5 to 7. In some embodiments, the N/P value is about 6. LNP described herein may have an average diameter that in some embodiments ranges from about 30 nm to about 200 nm, or from about 60 nm to about 120 nm. Pharmaceutical compositions In some embodiments, a pharmaceutical composition comprises an RNA polynucleotide disclosed herein formulated as a particle. In some embodiments, a particle is or comprises a lipid nanoparticle (LNP) or a lipoplex (LPX) particle. In some embodiments, an RNA polynucleotide disclosed herein may be administered in a pharmaceutical composition or a medicament and may be administered in the form of any suitable pharmaceutical composition. In some embodiments, a pharmaceutical composition described herein is an immunogenic composition for inducing an immune response. For example, in some embodiments, an immunogenic composition is a vaccine. In some embodiments, an RNA polynucleotide disclosed herein may be administered in a pharmaceutical composition which may comprise a pharmaceutically acceptable carrier and may optionally comprise one or more adjuvants, stabilizers etc. In some embodiments, a pharmaceutical composition is for therapeutic or prophylactic treatments. The term "adjuvant" relates to a compound which prolongs, enhances or accelerates an immune response. Adjuvants comprise a heterogeneous group of compounds such as oil emulsions (e.g., Freund's adjuvants), mineral compounds (such as alum), bacterial products (such as Bordetella pertussis toxin), or immune-stimulating complexes. Examples of adjuvants include, without limitation, LPS, GP96, CpG oligodeoxynucleotides, growth factors, and cytokines, such as monokines, lymphokines, interleukins, chemokines. The cytokines may be IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL12, IFNα, IFNγ, GM-CSF, LT-a. Further known adjuvants are aluminium hydroxide, Freund's adjuvant or oil such as Montanide® ISA51. Other suitable adjuvants for use in the present disclosure include lipopeptides, such as Pam3Cys. The pharmaceutical compositions according to the present disclosure are generally applied in a "pharmaceutically effective amount" and in "a pharmaceutically acceptable preparation". The term "pharmaceutically acceptable" refers to the non-toxicity of a material which does not interact with the action of the active component of the pharmaceutical composition. The term "pharmaceutically effective amount" or "therapeutically effective amount" refers to the amount which achieves a desired reaction or a desired effect alone or together with further doses. In the case of the treatment of a particular disease, the desired reaction preferably relates to inhibition of the course of the disease. This comprises slowing down the progress of the disease and, in particular, interrupting or reversing the progress of the disease. The desired reaction in a treatment of a disease may also be delay of the onset or a prevention of the onset of said disease or said condition. An effective amount of the compositions described herein will depend on the condition to be treated, the severeness of the disease, the individual parameters of the patient, including age, physiological condition, size and weight, the duration of treatment, the type of an accompanying therapy (if present), the specific route of administration and similar factors. Accordingly, the doses administered of the compositions described herein may depend on various of such parameters. In the case that a reaction in a patient is insufficient with an initial dose, higher doses (or effectively higher doses achieved by a different, more localized route of administration) may be used. In some embodiments, a pharmaceutical composition disclosed herein may contain salts, buffers, preservatives, and optionally other therapeutic agents. In some embodiments, a pharmaceutical composition disclosed herein comprises one or more pharmaceutically acceptable carriers, diluents and/or excipients. Suitable preservatives for use in a pharmaceutical compositions of the present disclosure include, without limitation, benzalkonium chloride, chlorobutanol, paraben and thimerosal. The term "excipient" as used herein refers to a substance which may be present in a pharmaceutical composition of the present disclosure but is not an active ingredient. Examples of excipients, include without limitation, carriers, binders, diluents, lubricants, thickeners, surface active agents, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, or colorants. The term "diluent" relates a diluting and/or thinning agent. Moreover, the term "diluent" includes any one or more of fluid, liquid or solid suspension and/or mixing media. Examples of suitable diluents include ethanol, glycerol and water. The term "carrier" refers to a component which may be natural, synthetic, organic, inorganic in which the active component is combined in order to facilitate, enhance or enable administration of the pharmaceutical composition. A carrier as used herein may be one or more compatible solid or liquid fillers, diluents or encapsulating substances, which are suitable for administration to subject. Suitable carrier include, without limitation, sterile water, Ringer, Ringer lactate, sterile sodium chloride solution, isotonic saline, polyalkylene glycols, hydrogenated naphthalenes and, in particular, biocompatible lactide polymers, lactide/glycolide copolymers or polyoxyethylene/polyoxy-propylene copolymers. In some embodiments, the pharmaceutical composition of the present disclosure includes isotonic saline. Pharmaceutically acceptable carriers, excipients or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R Gennaro edit.1985). Pharmaceutical carriers, excipients or diluents can be selected with regard to the intended route of administration and standard pharmaceutical practice. In some embodiments, a pharmaceutical composition described herein may be administered intravenously, intraarterially, subcutaneously, intradermally or intramuscularly. In certain embodiments, the pharmaceutical composition is formulated for local administration or systemic administration. Systemic administration may include enteral administration, which involves absorption through the gastrointestinal tract, or parenteral administration. As used herein, "parenteral administration" refers to the administration in any manner other than through the gastrointestinal tract, such as by intravenous injection. In a preferred embodiment, the pharmaceutical composition is formulated for intramuscular administration. In another embodiment, the pharmaceutical composition is formulated for systemic administration, e.g., for intravenous administration. Characterization In some embodiments, an RNA polynucleotide disclosed herein is characterized in that, when assessed in an organism administered a composition or medical preparation comprising an RNA polynucleotide, elevated expression of a payload is observed relative to an appropriate reference comparator. In some embodiments, an RNA polynucleotide disclosed herein is characterized in that, when assessed in an organism administered a composition or medical preparation comprising an RNA polynucleotide, increased duration of expression (e.g., prolonged expression) of a payload is observed relative to an appropriate reference comparator. In some embodiments, an RNA polynucleotide disclosed herein is characterized in that, when assessed in an organism administered a composition or medical preparation comprising an RNA polynucleotide, decreased interaction with IFIT1 of an RNA polynucleotide is observed relative to an appropriate reference comparator. In some embodiments, an RNA polynucleotide disclosed herein is characterized in that, when assessed in an organism administered a composition or medical preparation comprising an RNA polynucleotide, increased translation an RNA polynucleotide is observed relative to an appropriate reference comparator. In some embodiments, a reference comparator comprises an organism administered an otherwise similar RNA polynucleotide without a cap described herein. In some embodiments, a reference comparator comprises an organism administered an otherwise similar RNA polynucleotide without a cap proximal sequence disclosed herein. In some embodiments, a reference comparator comprises an organism administered an otherwise similar RNA polynucleotide with a self-hybridizing sequence. In some embodiments, an RNA polynucleotide disclosed herein is characterized in that, when assessed in an organism administered a composition or medical preparation comprising an RNA polynucleotide, elevated expression and increased duration of expression (e.g., prolonged expression) of a payload is observed relative to an appropriate reference comparator. In some embodiments, elevated expression is determined at least 24 hours, at least 48 hours at least 72 hours, at least 96 hours or at least 120 hours after administration of a composition or medical preparation comprising an RNA polynucleotide. In some embodiments, elevated expression is determined at least 24 hours after administration of a composition or medical preparation comprising an RNA polynucleotide.. In some embodiments, elevated expression is determined at least 48 hours after administration of a composition or medical preparation comprising an RNA polynucleotide.. In some embodiments, elevated expression is determined at least 72 hours after administration of a composition or medical preparation comprising an RNA polynucleotide. In some embodiments, elevated expression is determined at least 96 hours after administration of a composition or medical preparation comprising an RNA polynucleotide. In some embodiments, elevated expression is determined at least 120 hours after administration of a composition or medical preparation comprising an RNA polynucleotide. In some embodiments, elevated expression is determined at about 24-120 hours after administration of a composition or medical preparation comprising an RNA polynucleotide. In some embodiments, elevated expression is determined at about 24-110 hours, about 24-100 hours, about 24-90 hours, about 24-80 hours, about 24-70 hours, about 24-60 hours, about 24-50 hours, about 24-40 hours, about 24-30 hours, about 30-120 hours, about 40-120 hours, about 50- 120 hours, about 60-120 hours, about 70-120 hours, about 80-120 hours, about 90-120 hours, about 100-120 hours, or about 110-120 hours after administration of a composition or medical preparation comprising an RNA polynucleotide. In some embodiments, elevated expression of a payload is at least 2-fold to at least 10- fold. In some embodiments, elevated expression of a payload is at least 2-fold. In some embodiments, elevated expression of a payload is at least 3-fold. In some embodiments, elevated expression of a payload is at least 4-fold. In some embodiments, elevated expression of a payload is at least 6-fold. In some embodiments, elevated expression of a payload is at least 8- fold. In some embodiments, elevated expression of a payload is at least 10-fold. In some embodiments, elevated expression of a payload is about 2-fold to about 50-fold. In some embodiments, elevated expression of a payload is about 2-fold to about 45-fold, about 2- fold to about 40-fold, about 2-fold to about 30-fold, about 2-fold to about 25-fold, about 2-fold to about 20-fold, about 2-fold to about 15-fold, about 2-fold to about 10-fold, about 2-fold to about 8-fold, about 2-fold to about 5-fold, about 5-fold to about 50-fold, about 10-fold to about 50- fold, about 15-fold to about 50-fold, about 20-fold to about 50-fold, about 25-fold to about 50- fold, about 30-fold to about 50-fold, about 40-fold to about 50-fold, or about 45-fold to about 50- fold. In some embodiments, elevated expression (e.g., increased duration of expression) of a payload persists for at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours after administration of a composition or a medical preparation comprising an RNA polynucleotide. In some embodiments, elevated expression of a payload persists for at least 24 hours after administration. In some embodiments, elevated expression of a payload persists for at least 48 hours after administration. In some embodiments, elevated expression of a payload persists for at least 72 hours after administration. In some embodiments, elevated expression of a payload persists for at least 96 hours after administration. In some embodiments, elevated expression of a payload persists for at least 120 hours after administration of a composition or medical preparation comprising an RNA polynucleotide. In some embodiments, elevated expression of a payload persists for at about 24-120 hours after administration of a composition or medical preparation comprising an RNA polynucleotide. In some embodiments, elevated expression persists for about 24-110 hours, about 24-100 hours, about 24-90 hours, about 24-80 hours, about 24-70 hours, about 24-60 hours, about 24-50 hours, about 24-40 hours, about 24-30 hours, about 30-120 hours, about 40- 120 hours, about 50-120 hours, about 60-120 hours, about 70-120 hours, about 80-120 hours, about 90-120 hours, about 100-120 hours, or about 110-120 hours after administration of a composition or medical preparation comprising an RNA polynucleotide. Uses Disclosed herein, among other things, are methods of making and methods of using an RNA polynucleotide comprising a 5’cap; a 5’ UTR comprising a cap proximal structure; and a sequence encoding a payload. In some embodiments, disclosed herein is a method of producing a polypeptide comprising a step of: providing an RNA polynucleotide that comprises a 5’ cap (e.g., as described herein), a cap proximal sequence that comprises positions +1, +2, +3, +4, and +5 of an RNA polynucleotide, and a sequence encoding a payload; wherein an RNA polynucleotide is characterized in that when assessed in an organism administered an RNA polynucleotide or a composition comprising the same, elevated expression and/or increased duration of expression of an payload is observed relative to an appropriate reference comparator. In some embodiments, disclosed herein is a method comprising: administering to a subject, a pharmaceutical composition comprising an RNA polynucleotide formulated in a lipid nanoparticle (LNP) or a lipoplex (LPX) particle disclosed herein. In some embodiments, disclosed herein is a method of inducing an immune response in a subject, comprising administering to a subject, a pharmaceutical composition comprising an RNA polynucleotide formulated in a lipid nanoparticle (LNP) or a lipoplex (LPX) particle disclosed herein. In some embodiments, disclosed herein is a method of vaccination of a subject, comprising administering to a subject, a pharmaceutical composition comprising an RNA polynucleotide formulated in a lipid nanoparticle (LNP) or a lipoplex (LPX) particle disclosed herein. In some embodiments, provided herein is a methood of decreasing interaction with IFIT1 of an RNA polynucleotide that comprises a 5’ cap and a cap proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide, the method comprising a step of: providing a variant of an RNA polynucleotide that differs from a parental RNA polynucleotide by substitution of one or more residues within the cap proximal sequence, and determining that interaction of a variant with IFIT1 is decreased relative to that of a parental RNA polynucleotide. In some embodiments, determining comprises administering the RNA polynucleotide or a composition comprising the same to a cell or an organism. In some embodiments, disclosed herein is a method of increasing translatability of an RNA polynucleotide that comprises a 5’ cap, a cap proximal sequence that comprises positions +1, +2, +3, +4, and +5 of the RNA polynucleotide and a sequence encoding a payload, the method comprising a step of: providing a variant of an RNA polynucleotide that differs from a parental RNA polynucleotide by substitution of one or more residues within a cap proximal sequence; and determining that expression of a variant is increased relative to that of a parental RNA polynucleotide. In some embodiments, determining comprises administering the RNA polynucleotide or a composition comprising the same to a cell or an organism. In some embodiments, increased translatability is assessed by increased expression and/or a persistence of expression of the payload. In some embodiments, increased expression is determined at least 6 hours, at least 24 hours, at least 48 hours at least 72 hours, at least 96 hours or at least 120 hours after administering. In some embodiments, increase in expression is at least 2-fold to 10-fold. In some embodiments, increase in expression is about 2-fold to 50-fold. In some embodiments, elevated expression persists for at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours after administration. In some embodiments of any of the methods disclosed herein, an immune response is induced in a subject. In some embodiments of any of the methods disclosed herein, an immune response is a prophylactic immune response or a therapeutic immune response. In some embodiments of any of the methods disclosed herein, a subject is a mammal. In some embodiments of any of the methods disclosed herein, a subject is a human. In some embodiments of any of the methods disclosed herein, a subject has a disease or disorder disclosed herein. In some embodiments of any of the methods disclosed herein, vaccination generates an immune response to an agent. In some embodiments, an immune response is a prophylactic immune response. In some embodiments of any of the methods disclosed herein, a subject has a disease or disorder disclosed herein. In some embodiments of any of the methods disclosed herein, one dose of a pharmaceutical composition is administered. In some embodiments of any of the methods disclosed herein, a plurality of doses of a pharmaceutical composition is administered. In some embodiments of any of the methods disclosed herein, the method further comprises administration of one or more therapeutic agents. In some embodiments, one or more therapeutic agents are administered before, after, or concurrently with administration of a pharmaceutical composition comprising an RNA polynucleotide. Also provided herein is a method of improving capping efficiency (e.g., percentage of capped transcripts in an in vitro transcription reaction) of RNA transcripts, the improvement that comprises including A or an analog thereof and U or an analog thereof at the +1 and +2 positions, respectively, of a transcription start site in a coding strand of a double-stranded DNA template for in vitro transcription. In some embodiments, a transcription start site may be AUA, AUC, AUG, or AUU. In some embodiments, such improvements can be observed independent of the identity of a 5’ UTR, capping method (e.g., enzymatic capping vs. co-transcriptional capping), cap structures (e.g., Cap0, Cap1, or Cap2), coding sequences, types of ribonucleotides (e.g., modified nucleotides vs. non-modified nucleotides), formulation (e.g., lipoplex vs. lipid nanoparticles) or combinations thereof. Also provided herein in some embodiments, is a method of providing a framework for an RNA polynucleotide that comprises a 5’ cap, a cap proximal sequence, and a payload sequence, the method comprising a step of: assessing at least two variants of an RNA polynucleotide, wherein: each variant includes a same 5’ cap and payload sequence; and the variants differ from one another at one or more specific residues of a cap proximal sequence; wherein the assessing comprises determining expression levels and/or duration of expression of a payload; and selecting at least one combination of 5’ cap and a cap proximal sequence that displays elevated expression relative to at least one other combination. In some embodiments, assessing comprises administering an RNA construct or a composition comprising the same to a cell or an organism: In some embodiments, elevated expression of a payload is detected at a time point at least 6 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours after administering. In some embodiments, elevated expression is at least 2-fold to 10-fold. In some embodiments, elevated expression is about 2-fold to about 50-fold. In some embodiments, elevated expression of a payload persists for at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours after administering. In some embodiments of any of the methods disclosed herein, an RNA polynucleotide comprises one or more features of an RNA polynucleotide provided herein. In some embodiments of any of the methods disclosed herein, a composition comprising an RNA polynucleotide comprises a pharmaceutical composition provided herein. ENUMERATED EMBODIMENTS 1. A composition or medical preparation comprising an RNA polynucleotide comprising: a 5’ cap; a cap proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; and a sequence encoding a payload, wherein: (i) the 5’ cap is a trinucleotide cap structure comprises N ₁ pN ₂ , wherein N ₁ is position +1 of the RNA polynucleotide and N ₂ is position +2 of the RNA polynucleotide, and wherein N ₁ is A or an analog thereof; and N ₂ is U or an analog thereof; and (ii) the cap proximal sequence comprises: N ₁ and N ₂ of the trinucleotide cap structure and a sequence comprising N ₃ N ₄ N ₅ at positions +3, +4, and +5 respectively of the RNA polynucleotide, wherein N ₃ , N ₄ , and N ₅ are each independently selected from: A, C, G, and U. 2. The composition or medical preparation of embodiment 1, wherein N ₃ is A. 3. The composition or medical preparation of embodiment 1 or 2, wherein N5 is U. 4. The composition or medical preparation of any one of embodiments 1-3, wherein N ₄ is A. 5. The composition or medical preparation of any one of embodiments 1-3, wherein N4 is C. 6. The composition or medical preparation of any one of embodiments 1-3, wherein N ₄ is G. 7. The composition or medical preparation of any one of embodiments 1-3, wherein N4 is U. 8. The composition or medical preparation of any one of embodiments 1-7, wherein the trinucleotide cap structure has a structure: G*N1pN2, wherein G* comprises a structure of formula I: I or a salt thereof, wherein each R ² and R ³ is -OH or -OCH3; and X is OH or SH (e.g., O- or S-). 9. The composition or medical preparation of embodiment 8, wherein R ² is -OH. 10. The composition or medical preparation of embodiment 8, wherein R ² is -OCH ₃ . 11. The composition or medical preparation of any one of embodiments 8-10, wherein R ³ is - OH. 12. The composition or medical preparation of any one of embodiments 8-10, wherein R ³ is - OCH3. 13 The composition or medical preparation of any one of embodiments 8-12 wherein X is OH (e.g., O-). 14. The composition or medical preparation of any one of embodiments 1-13, wherein the trinucleotide cap structure comprises a Cap1 structure. 15. The composition or medical preparation of any one of embodiments 1-14, wherein N ₂ is of formula II: II or a salt thereof, wherein: each is independently a single or double bond, as allowed by valency; Y ¹ is O or S; Y ² is N, C, or CH; Y ³ is N, NR ^a1 , CR ^a1 , or CHR ^a1 ; Y ⁴ is NR ^a2 or CHR ^a2 ; each of R ^a1 or R ^a2 is independently hydrogen or C1-6 aliphatic; R ⁴ is -OH or -OMe; and # represents the point of attachment to p of N ₁ p. 16. The composition or medical preparation of embodiment 15, wherein N2 is of formula IIa: IIa or a salt thereof. 17. The composition or medical preparation of embodiment 15, wherein N ₂ is of formula IIb: IIb or a salt thereof. 18. The composition or medical preparation of any one of embodiments 15-17, wherein Y ¹ is O. 19. The composition or medical preparation of any one of embodiments 15-17, wherein Y ¹ is S. 20. The composition or medical preparation of embodiment 15 or 16, wherein Y ³ is N. 21. The composition or medical preparation of embodiment 15 or 16, wherein Y ³ is CR ^a1 . 22. The composition or medical preparation of embodiment 15 or 17, wherein Y ³ is NR ^a1 . 23. The composition or medical preparation of embodiment 15 or 17, wherein Y ³ is CHR ^a1 . 24. The composition or medical preparation of any one of embodiments 15-23, wherein each R ^a1 or R ^a2 is independently hydrogen or methyl. 25. The composition or medical preparation of any one of embodiments 15-24, wherein R ⁴ is -OH. 26. The composition or medical preparation of any one of embodiments 15-24, wherein R ⁴ is -OMe. 27. The composition or medical preparation of any one of embodiments 1-14, wherein N2 is uridine, or a modified uridine (e.g., m1ψ, 2-thio-uridine, or 5-methyluridine). 28. The composition or medical preparation of any one of embodiments 1-14, wherein N ₂ is: , , , , or ; or a salt thereof; wherein # represents the point of attachment to p of N ₁ p. 29. The composition or medical preparation of any one of embodiments 1-28, wherein N ₁ is adenosine or a modified adenosine (e.g., 6-methyladenosine). 30. The composition or medical preparation of any one of embodiments 1-7, wherein the 5’ cap is (m ^7,2’-O )Gppp(m ^2’-O )A ₁ pU ₂ , (m ^7,3’-O )Gppp(m ^2’-O )A ₁ pU ₂ , (m ^7,2’-O )Gppp(m ^2’-O )A ₁ pΨ ₂ , (m ^{7,3’- O} )Gppp(m ^2’-O )A1pΨ2, (m ^7,2’-O )Gppp(m ^2’-O )A1p(m ¹ )Ψ2, (m ^7,3’-O )Gppp(m ^2’-O )A1p(m ¹ )Ψ2, (m ^{7,2’- O} )Gppp(m ^2’-O )A1pS ² U2, (m ^7,3’-O )Gppp(m ^2’-O )A1pS ² U2, (m ^7,2’-O )Gppp(m ^2’-O )A1p(m ⁵ )U2, or (m ^{7,3’- O} )Gppp(m ^2’-O )A ₁ p(m ⁵ )U ₂ . 31. The composition or medical preparation of any one of embodiments 1-7, wherein the 5’ cap is (m ^7,2’-O )Gppp(m ^6,2’-O )A1pU2, (m ^7,3’-O )Gppp(m ^6,2’-O )A1pU2, (m ^7,2’-O )Gppp(m ^6,2’-O )A1pΨ2, (m ^7,3’-O )Gppp(m ^6,2’-O )A1pΨ2, (m ^7,2’-O )Gppp(m ^6,2’-O )A1p(m ¹ )Ψ2, (m ^7,3’-O )Gppp(m ^6,2’-O )A1p(m ¹ )Ψ2, (m ^7,2’-O )Gppp(m ^6,2’-O )A ₁ pS ² U ₂ , (m ^7,3’-O )Gppp(m ^6,2’-O )A ₁ pS ² U ₂ , (m ^7,2’-O )Gppp(m ^6,2’-O )A ₁ p(m ⁵ )U ₂ , or (m ^7,3’-O )Gppp(m ^6,2’-O )A1p(m ⁵ )U2. 32. An in vitro transcription reaction comprising: (i) a template DNA strand comprising a polynucleotide sequence complementary to an RNA polynucleotide sequence provided in any one of embodiments 1-31, wherein the template DNA strand comprises a sequence that is complementary to a AUA, AUC, AUG, or AUU transcription start site; (ii) a polymerase; (iii) ribonucleotides; and (iv) a 5’ cap comprising N ₁ pN ₂ ; wherein N1 is A or an analog thereof, and N2 is U or an analog thereof; wherein the sequence in the template strand that is complementary to AUA, AUC, AUG, or AUU is the start site of an RNA polymerase promoter. 33. The in vitro transcription reaction of embodiment 32, wherein the template DNA strand comprises a sequence that is complementary to a transcription start site comprising AUA. 34. The in vitro transcription reaction of embodiment 32, wherein the template DNA strand comprises a sequence that is complementary to a transcription start site comprising AUC. 35. The in vitro transcription reaction of embodiment 32, wherein the template DNA strand comprises a sequence that is complementary to a transcription start site comprising AUG. 36. The in vitro transcription reaction of embodiment 32, wherein the template DNA strand comprises a sequence that is complementary to a transcription start site comprising AUU. 37. The in vitro transcription reaction of any one of embodiments 32-36, wherein the template DNA strand comprises: a sequence encoding a 5' UTR, a sequence encoding a payload, a sequence encoding a 3' UTR, and a sequence encoding a polyA sequence. 38. The in vitro transcription reaction of any one of embodiments 32-37, wherein N ₂ is uridine or a modified uridine (e.g., m1ψ, 2-thio-uridine, or 5-methyluridine). 39. An RNA polynucleotide produced from an in vitro transcription reaction provided in any one of embodiments 32-38. 40. A method of making a capped RNA polynucleotide comprising a 5’ cap comprising N ₁ pN ₂ ; a cap proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; and a sequence encoding a payload, wherein: the cap proximal sequence comprises N1 and N2 of the 5’ cap, and N3, N4, and N5, wherein N ₁ to N ₅ correspond to positions +1, +2, +3, +4, and +5 of the RNA polynucleotide, wherein N1 is A or an analog thereof, N2 is U or an analog thereof, and N3, N4, and N5 are each independently chosen from: A, C, G, and U; and wherein the method comprises transcribing a template DNA strand in the presence of the 5’ cap and an RNA polymerase, wherein the template DNA strand comprises an RNA polymerase promoter sequence and a sequence that is complementary to a transcription start site, wherein the sequence that is complementary to the transcription start site is the start site of the RNA polymerase promoter. 41. The method of embodiment 39, wherein N ₁ is complementary to position +1 of the template DNA strand (corresponding to the first nucleotide of the transcription start site), and N ₂ is complementary to position +2 of the template DNA strand (corresponding to the second nucleotide of the transcription start site). 42. The method of embodiment 39 or 40, wherein the RNA polymerase is T7 RNA polymerase. 43. The method of any one of embodiments 39-41, wherein N ₂ is uridine or modified uridine (e.g., m1ψ, 2-thio-uridine, or 5-methyluridine). 44. A method of making a capped RNA polynucleotide comprising: transcribing a DNA template strand in the presence of a 5’ cap, wherein the 5’ cap comprises the structure N ₁ pG ₂ , wherein the DNA template strand comprises an RNA polymerase promoter sequence and a sequence that is complementary to a AUA, AUC, AUG, or AUU transcription start site; wherein N ₁ is A or an analog thereof, and N ₂ is U or an analog thereof. 45. The method of embodiment 44, wherein N2 is uridine or modified uridine (e.g., m1ψ, 2- thio-uridine, or 5-methyluridine). 46. A complex comprising a DNA template strand and a 5’ cap analog comprising a structure of N1pN2, wherein the DNA template strand comprises an RNA polymerase promoter sequence and a sequence that is complementary to a transcription start site; wherein N1 is A or an analog thereof, and N2 is U or an analog thereof; wherein N1 interacts with the +1 position of the DNA template strand (corresponding to the first nucleotide of the transcription start site) and N ₂ interacts with the +2 position of the DNA template strand (corresponding to the second nucleotide of the transcription start site); and wherein the sequence in the template strand that is complementary to the transcription start site is the start site of an RNA polymerase promoter. 47 The complex of embodiment 46, wherein position +1 and position +2 of the DNA template strand are T and A, respectively. 48. The complex of embodiment 46 or 47, wherein the nucleotides of the cap interact with the nucleotides of the template DNA strand via canonical Watson-Crick base pairing. 49. The complex of any one of embodiments 46-48, wherein the RNA polymerase promoter sequence is a T7 RNA polymerase promoter sequence. 50. The complex of any one of embodiments 46-49, wherein the complex further comprises an RNA polymerase (e.g., a T7 RNA polymerase). 51. The complex of any one of embodiments 46-50, wherein N2 is uridine or modified uridine (e.g., m1ψ, 2-thio-uridine, or 5-methyluridine). 52. A method of formulating a pharmaceutical composition, the method comprising combining a preparation comprising an RNA polynucleotide of any one of embodiments 1 to 31 with a preparation comprising lipids. 53. The method of embodiment 52, wherein the method comprises combining the preparation comprising RNA polynucleotide with the preparation comprising lipids to form lipid nanoparticles that encapsulate the RNA polynucleotide. 54. The method of embodiment 52, wherein the method comprises combining the preparation comprising RNA polynucleotide with the preparation comprising lipids to form RNA lipoplexes. 55. A compound of formula G*N ₁ pN ₂ , wherein: G* is of formula Iʹ: Iʹ or a salt thereof, wherein: each R ² and R ³ is -OH or -OCH3; and X is OH or SH (e.g., O- or S-); p is a phosphate linker; N1 is A or an analog thereof; and N2 is U or an analog thereof. 56 The compound of embodiment 55 wherein R ² is -OH 57. The compound of embodiment 55, wherein R ² is -OCH3. 58. The compound of any one of embodiments 55-57, wherein R ³ is -OH. 59. The compound of any one of embodiments 55-57, wherein R ³ is -OCH3. 60. The compound of any one of embodiments 55-59, wherein X is OH (e.g., O-). 61. The compound of any one of embodiments 55-60, wherein N ₂ is of formula IIʹ: IIʹ or a salt thereof, wherein: each is independently a single or double bond, as allowed by valency; Y ¹ is O or S; Y ² is N, C, or CH; Y ³ is N, NR ^a1 , CR ^a1 , or CHR ^a1 ; Y ⁴ is NR ^a2 or CHR ^a2 ; each of R ^a1 or R ^a2 is independently hydrogen or C _1-6 aliphatic; R ⁴ is -OH or -OMe; and # represents the point of attachment to p of N1p. 62. The compound of embodiment 15, wherein N ² is of formula IIa: IIa or a salt thereof. 63. The compound of embodiment 62, wherein N ² is of formula IIb: IIb or a salt thereof. 64. The compound of any one of embodiments 61-63, wherein Y ¹ is O. 65. The compound of any one of embodiments 61-63, wherein Y ¹ is S. 66. The compound of embodiment 61 or 62, wherein Y ³ is N. 67. The compound of embodiment 61 or 62, wherein Y ³ is CR ^a1 . 68. The compound of embodiment 61 or 63, wherein Y ³ is NR ^a1 . 69. The compound of embodiment 61 or 63, wherein Y ³ is CHR ^a1 . 70. The compound of any one of embodiments 61-69, wherein each R ^a1 or R ^a2 is independently hydrogen or methyl. 71. The compound of any one of embodiments 61-70, wherein R ⁴ is -OH. 72. The compound of any one of embodiments 61-70, wherein R ⁴ is -OMe. 73. The compound of any one of embodiments 55-60, wherein N2 is: or a salt thereof; wherein # represents the point of attachment to p of N1p. 74. The compound of any one of embodiments 55-73, wherein N ₁ is adenosine or 6- methyladenosine. 75. The compound of any one of embodiments 55-73, wherein N ₁ is: , , , or ; or a salt thereof, 76. The compound of any one of embodiments 55-75, wherein p is -P(=O)(OH)-, or a salt thereof. 77. The compound of embodiment 55, wherein the compound is (m ^7,2’-O )Gppp(m ^2’-O )A ₁ pU ₂ , (m ^7,3’-O )Gppp(m ^2’-O )A ₁ pU ₂ , (m ^7,2’-O )Gppp(m ^2’-O )A ₁ pΨ ₂ , (m ^7,3’-O )Gppp(m ^2’-O )A ₁ pΨ ₂ , (m ^{7,2’- O} )Gppp(m ^2’-O )A1p(m ¹ )Ψ2, (m ^7,3’-O )Gppp(m ^2’-O )A1p(m ¹ )Ψ2, (m ^7,2’-O )Gppp(m ^2’-O )A1pS ² U2, (m ^{7,3’- O} )Gppp(m ^2’-O )A1pS ² U2, (m ^7,2’-O )Gppp(m ^2’-O )A1p(m ⁵ )U2, or (m ^7,3’-O )Gppp(m ^2’-O )A1p(m ⁵ )U2, or a salt thereof. 78. The compound of embodiment 55, wherein the compound is (m ^7,2’-O )Gppp(m ^{6,2’- O} )A1pU2, (m ^7,3’-O )Gppp(m ^6,2’-O )A1pU2, (m ^7,2’-O )Gppp(m ^6,2’-O )A1pΨ2, (m ^7,3’-O )Gppp(m ^6,2’-O )A1pΨ2, (m ^7,2’-O )Gppp(m ^6,2’-O )A ₁ p(m ¹ )Ψ ₂ , (m ^7,3’-O )Gppp(m ^6,2’-O )A ₁ p(m ¹ )Ψ ₂ , (m ^7,2’-O )Gppp(m ^{6,2’- O} )A ₁ pS ² U ₂ , (m ^7,3’-O )Gppp(m ^6,2’-O )A ₁ pS ² U ₂ , (m ^7,2’-O )Gppp(m ^6,2’-O )A ₁ p(m ⁵ )U ₂ , or (m ^{7,3’- O} )Gppp(m ^6,2’-O )A1p(m ⁵ )U2, or a salt thereof. 79. The composition or medical preparation of embodiment 15 or 17, wherein Y ³ is N. 80. A method of making a capped RNA polynucleotide comprising: transcribing a DNA template strand in the presence of a 5’ cap, wherein the 5’ cap comprises the structure N1pN2, wherein the DNA template strand comprises an RNA polymerase promoter sequence and a sequence that is complementary to a AUA, AUC, AUG, or AUU transcription start site; wherein N ₁ is A or an analog thereof, and N ₂ is U or an analog thereof. EXEMPLIFICATION Example 1 - Evaluation of (m ₂ ^7,3’-O )Gppp(m ^2’-O )ApU and (m ^7,3’-O )Gppp(m ^2’-O )A ₁ p(m ¹ )Ψ ₂ , For templates, linearized plasmid encoding codon-optimized murine erythropoietin (EPO) were used. The mRNAs starting with AUAAU, AUACU, AUAGU or AUAUU were designed to contain the 5’ untranslated region (5’UTR) sequences of human α-globin (hAg) mRNA, an FI element as the 3’UTR, and an interrupted 100 nt-long 3’ poly(A) tail flanking the coding sequence. The MEGAscript T7 Transcription kit (Thermo Fisher Scientific, Waltham, MA, USA) was used for transcription, and UTP was kept or was replaced with N1- methylpseudouridine (m1Ψ) triphosphate (TriLink, San Diego, CA, USA). Capping of in vitro- transcribed mRNAs was performed co-transcriptionally using trinucleotide cap analogs (Cap 1 corresponds to compound I′-1 ((m ₂ ^7,3’-O )Gppp(m ^2’-O )ApU); Cap 2 corresponds to compound I′-6 ((m ₂ ^7,3’-O )Gppp(m ^2’-O )Ap(m ¹ )Ψ); CC114 corresponds to (m ⁷ )Gppp(m ^2'-O )ApU (TriLink, USA); CC413 corresponds to (m2 ^7,3'-O )Gppp(m ^2'-O )ApG (TriLink, USA). To obtain the desired transcripts generated with cap analogs, the initial GTP and UTP or m1ѰTP concentration in the transcription reaction was reduced from 7.5 mM to 1.5 mM and the 1.5 mL tubes were incubated at 37°C for 30 min in a hybridization chamber. Sequential additions of 1.5 mM GTP and UTP or m1ѰTP were required to supplement the reaction at 30, 60, 90 and 120 min. incubated further at 37°C for 30 min. To remove template DNA, Turbo DNase (Thermo Fisher Scientific, USA) was added to the reaction mix after the transcription reaction was completed and incubated for 15 min at 37°C. The synthesized mRNA was precipitated by adding a half volume of 8 M LiCl solution (Merck, Darmstadt, Germany) to the reaction mix and then pelleted by centrifugation. After dissolving in nuclease free water, mRNAs were cellulose-purified to remove double- stranded RNA contaminants, as described in Baiersdörfer, M., et al. (2019) Molecular therapy. Nucleic acids, 15, 26–35. The mRNA concentration and quality were measured on a NanoDropTM 2000c spectrophotometer (Thermo Fisher Scientific, USA). Small aliquots of mRNA samples were stored in siliconized tubes at -20°C. These findings demonstrated that (m2 ^7,3’-O )Gppp(m ^2’-O )ApU paried with an AUAGU transcriptional start site generated EPO- encoding mRNAs (EPO mRNA) with the highest RNA yield (64 µg/Unit) (Figure 2A). In order to determine the capping efficiency of mRNAs, in vitro transcription reaction followed by a Ribozyme assay was performed in a cap analog concentration-dependent manner (1, 3, 6, 9 and 12 mM). The Ribozyme cleavage reaction contained 0.45 µM mRNA. A 3-fold molar excess of ribozyme over mRNA substrate was added in aqueous solution containing 30 mM HEPES and 150 mM NaCl. The ribozyme cleavage reaction was performed on a PCR machine utilizing the following program: 95°C for 2 min, chill the mixture up to 37°C by ramping rate of 0.1 °C/sec, 37 °C for 5 min; after adding 30 mM MgCl ₂ solution to each sample, the mixtures were maintained at 37 °C for 60 min followed by stopping the annealing at 80°C for 2 min and transferring to ice for 5 min. Then, the short and long RNA fragments were separated using the RNA Clean & Concentrator-5 kit (Zymo Research Europe, Freiburg, Germany) according to the manufacturer’s instructions. In this study, the following custom-designed hammerhead ribozyme specific for the hAg 5’UTR was used: 5’-UGU GGG CUG AUG AGG CCG UGA GGC CGA AAC CAG AAG AAU-3’ (SEQ ID NO: 44) (synthesized by Metabion International AG, Planegg, Germany). To detect the short fragments, the samples (30 ng) were resolved on a 21% (vol/vol) 19:1 acrylamide:bisacrylamide denaturing gel supplemented with 8 M urea (Merck, Germany). Before loading, the samples were denatured by incubation at 75 °C for 5 min in the presence of 2x RNA loading buffer (New England Biolabs, Germany). The gel was pre-run at 180 V for 60 min. When the pre-run was finished, the pockets were rinsed with 1x TBE buffer. Immediately afterwards, the samples were loaded, and the gel was run at 200 V constantly until the dye front reached the end of the gel. To identify the short, cleaved products, the gel was incubated with 1x TBE buffer containing 0.01% SYBR Gold nucleic acid stain (Thermo Fisher Scientific, USA) and the fluorescent signals were captured using a Gel Doc EZ Imager (Bio-Rad, Hercules, CA, USA). High yield is observed when the (m ₂ ^7,3’-O )Gppp(m ^{2’- O} )ApU was used in the range of 3-6 mM concentration (Figure 2B), accordingly in further tests, the concentration of 4 mM was applied. The capping efficiency of (m ₂ ^7,3’-O )Gppp(m ^2’-O )ApU is close to 100% regardless of the concentration was used (Figure 2B). Using the optimized conditions, in vitro-transcribed (IVT) EPO mRNA containing U or m1Ѱ was generated with trinucleotide cap analogs CC114, CC413, compound I′-1, or compound I′-6. High yield as well as capping efficiency is observed in terms of each tested mRNA regardless of cap analog. According to Ribozyme assay analysis, the capping efficiency of compound I′-1 and compound I′-6 is close to 100% and it is comparable to the commercially available CC114 and CC413 cap analogs (Figure 3). For detection of short byproducts, IVT mRNAs (1.5-2 µg) were resolved on a 21% (vol/vol) 19:1 acrylamide:bisacrylamide denaturing gel supplemented with 8 M urea (Merck, Germany). bothWhen the pre-run was finished, the pockets were rinsed with 1x TBE buffer. Immediately afterwards, the samples were applied, and the gel was run at 180 V constantly until the dye front has reached the end of the gel. For identification of the short byproducts, the gel was incubated with 1x TBE buffer containing 0.01% SYBR Gold nucleic acid stain (Thermo Fisher Scientific, USA) and the fluorescent signals were captured using a Gel Doc EZ Imager (Bio-Rad, USA). Denaturing Urea Polyacrylamide Gel Electrophoresis showed minimal amount of short contaminants for compound I′-6 and CC413 for EPO m1Ѱ-mRNAs, while for unmodified ones significant amount was observed independently of cap analogs (Figure 4). Human buffy coats from healthy individuals were obtained from the Faculty of Medicine of Johannes Gutenberg University, Mainz and used to isolate peripheral blood mononuclear cells (PBMCs) on a Ficoll-Paque™ PLUS (Cytiva, Marlborough, MA, USA) density gradient. In preparation for mRNA transfection, cryopreserved PBMCs were thawed and seeded into 96-well plates at a density of 5 × 10 ⁵ cells per well in 190 µL RPMI medium supplemented with 1% non- essential amino acids (NEAA), 1% sodium pyruvate and 10% Fetal Bovine Serum (Merck, Germany). Cells were maintained at 37°C with 5% CO2 until transfection with 0.5, 1.5, 5.0 and 15 µg/ml lipoplex-formulated EPO mRNA (LPX-RNA), as described in Kranz, L.M., et al. (2016) Nature, 534, 396–401. The complexed RNA (10 µl per well) was added in triplicates and supernatants were collected at 24 h after transfection to perform single cell cytotoxicity assay and to measure the cytokine/chemokine profile. To determine the cell viability and the production of selected cytokines/chemokines, supernatants from human PBMCs transfected with LPX-RNA were subjected to XTT cytotoxicity test using Cell Proliferation Kit II (Sigma- Aldrich) and cytokine/chemokine profile analysis using the Meso Scale Discovery V-PLEX Custom Human Biomarkers Proinflammatory and Chemokine Panel (Meso Scale Diagnostics - MSD, Rockville, MD, USA) according to the manufacturer’s instructions, respectively. In regards of cytokine/chemokine profile analysis, a sample dilution of 1:5 (supernatant:MSD diluent) was used in each experiment. The levels of IL-6 (Interleukin 6), TNF-α (Tumor necrosis factor alpha), IL-1β (Interleukin 1 beta), IFN-γ (Interferon gamma), MCP-1 (Monocyte Chemoattractant Protein-1), MIP-1β (Macrophage inflammatory protein 1 beta) and IL-10 (Interleukin 10) were quantified 24 h after the mRNA transfection. These results demonstrated that no toxic effect on cell viability originating from compound I′-1 as well as compound I′-6 was observed (Figure 5). No toxic effect on PBMCs up to 1 µg/well mRNA for m1Ѱ-modified EPO mRNA (Figure 5). Transfection of unmodified mRNAs leads to decrease in cellular viability starting at dose 1.5 µg/ml, however this effect does not depend on cap used but on mRNA modification (Figure 5). Levels of each cytokine/chemokine showed zero to moderate increases in each sample measured (Figure 6). Compound I′-6 was comparable to CC413 in regard to amount of cytokines/chemokines secreted by human PBMCs after transfection of EPO m1Ѱ-mRNA (Figure 6). This low immunogenicity can be directly attributed to the nucleoside modifications incorporated into the mRNA. To illustrate this, cells treated with lipid-complexed U-containing mRNA capped with compound I′-1, CC114 and CC413 induced much higher proinflammatory cytokine/chemokine responses even at their lowest dose (0.5 µg/ml) when compared to m1Ѱ-modified mRNA (Figure 6). In regard to cytokines/chemokines for unmodified mRNA, compound I′-1 is comparable to CC114 and leads to significantly higher cytokines/chemokines in comparison to CC413 (Figure 6). To measure the translational efficiency of EPO mRNA capped with compound I′-1, compound I′-6, CC114, and CC413 in vitro, human primary hepatocytes were seeded into 96- well plates at a density of 2.5 × 10 ⁴ cells per well and were transfected with mRNA samples (0.1 µg) complexed with TransIT reagent (Mirus Bio, Madison, WI, USA) in a final volume of 200 µL in InVitroGRO CP Medium (Sigma-Aldrich) supplemented with Thorpedo Antibiotic Mix (Sigma-Aldrich). To quantify EPO levels, the supernatant was collected at 1, 2, 3, 4, 5 and 6 day after transfection and EPO levels were analyzed by mouse Erythropoietin DuoSet ELISA kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. Compound I′-1 resulted in higher translation compared to CC413 at each time points after transfection in a context of unmodified RNA (Figure 7). However, compound I′-6 in m1Ѱ- mRNA context leads to lower translation at later time points but in close to comparable translation to standard CC413 cap analog in human primary hepatocytes. To confirm in vitro data, female BALB/c mice from Jackson Laboratory (Bar Harbor, ME, USA) at the age of eight to ten weeks were used for in vivo experiment in accordance with federal policies on animal research (Ethics approval number: G18-12-027). Mice (n=3/group) were injected intravenously (i.v.) with 3 µg TransIT-complexed (Mirus Bio) EPO mRNAs in a final volume of 200 µL Dulbecco’s modified Eagle medium (DMEM). Mice used as controls were injected with TransIT-reagent diluted in DMEM but without RNA. Mice were injected with EPO mRNA complexed with TransIT-reagent and EPO levels in plasma were measured using ELISA (Figure 8A). Hematocrit was measured from 18µl of blood that was collected at the indicated times using centrifugation in Drummond microcaps glass capillary tubes (20 µl volume, Merck, Germany) as described (20) (Figure 8B).. After determination of the hematocrit, capillary tubes were snapped open, and the plasma was collected to measure EPO levels and analyzed for mouse Erythropoietin DuoSet ELISA kit (R&D Systems) according to the manufacturer’s instructions. These results demonstrated again that mRNA capped with ARCA cap analog translated much better compared to those capped with non-ARCA version regardless of the cap initiators (Figure 1, Figure 8A, 8B). EPO RNA containing m1Ѱ translated two-fold more than U-containing mRNA at each time points (Figure 8A). Both compound I′-1 and compound I′-6 are suitable for translation of the encoded protein and each cap analog can be used for synthesizing of non-replicating functional mRNAs (Figure 8A, 8B). Interestingly, compound I′-6 has a beneficial effect on translational efficiency of mRNA, meaning it showed 1.5-3 fold more translation compared to those capped with CC413 at 48 and 72 h after injection, respectively (Figure 8A). This is confirmed by the fact that the 24-hour EPO values are higher than the 6-hour values, which is surprising after IV injection, and it never experienced before (Figure 8A). The positive effect of compound I′-6 on the translational capacity of the mRNA is also expressed in the biological activities of the mRNA because hematocrit values remained very high level in mice injected with compound I′-6-capped EPO mRNA even at day 21 after injection (Figure 8B). Hematocrit values started to decrease at D14 after injection of U- containing mRNA capped with CC114 and CC413, but it was not observed in regards to compound I′-1-capped mRNA (Figure 8B). Hematocrit value in mice injected with compound I′-6-capped EPO mRNA at 21 days after administration is at the same level as the values of D7 and D14 in mice injected with CC413-capped mRNA (Figure 8B). In conclusion, the presented data demonstrated that m1Ѱ-containing cap analog can be adapted to synthesis of non-replicating mRNA. These studies evaluated a unique, nucleoside modification-containing anti-reverse trinucleotide cap1 analog, compound I′-6, that was used to generate a functional mRNA with 5’ cap1 structure resulting in a long-term maintenance of the encoded protein. These data showed that an appropriate combination of initial sequence and compound I′-6 leads to a superior mRNA that significantly surpasses the translational capacity and biological activity of IVT mRNA capped with the commonly used trinucleotide cap analogs that has already been used successfully, e.g., in an mRNA-based vaccine against SARS-CoV-2.

Example 2. Synthesis of 5’ caps. General trinucleotide cap1 structure: wherein: R2/R3: OH/OMe N1 and N2: A, U, G, C, Ψ, m ⁶ A, modified U, modified Ψ, any natural/ unnatural nucleoside, any modified nucleoside General trinucleotide cap1 structure containing 2’-OMe-Ψ analogs at N ₁ and other nucleosides at N ₂ : wherein: R2/R3: OH/OMe R3*, R4*, R5*: H, Me, any alkyl, aryl, benzyl, naphthyl, vinyl, allyl, propargyl, carbocycles, heterocycles N ₂ : A, G, m ⁶ A General trinucleotide cap1 structure containing 2’-OMe-A at N ₁ and U/Ψ analogs at N ₂ :

wherein: R2/R3: OH/OMe N ₂ : U, Ψ, modified U, modified Ψ General synthetic route of different cap analogs

General synthetic route of different cap analogs containing 2’-OMe-A at N ₁ and various Ψ derivatives at N ₂ General synthetic route of different m ⁷ GDP derivatives (1-3)

Synthesis of 7-methyl-guanosine 5’-diphosphate derivatives (m ⁷ GDP derivatives, 1-3) m ⁷ GDP derivative synthesis was performed according to modified published procedures (Patent US 2003/0194759 A1; Patent WO 2008/016473 A2; Patent WO2017053297A1; Bioorg. Med. Chem. Lett.2007, 17, 5295.). 7-methylguanosine 5’-diphosphate (m ⁷ GDP, 1): ¹ H NMR (300 MHz, D2O): δ 6.04 (d, J = 3.4 Hz, 1H), 4.66 (dd, J = 4.8, 3.4 Hz, 1H), 4.50 (t, J = 5.2 Hz, 1H), 4.42 – 4.28 (m, 2H), 4.20 (ddd, J = 11.9, 5.3, 2.1 Hz, 1H), 4.10 (s, 3H), 3.19 (q, J = 7.3 Hz, 22H), 1.26 (t, J = 7.3 Hz, 33H). ³¹ P NMR (121 MHz, D2O): δ -10.40 (d, J = 20.9 Hz), -11.38 (d, J = 20.7 Hz). MS (ESI-): Exact mass calculated for C11H16N5O11P2 [M - H]-, 456.03. Found 456.01. 7-methyl-2’-O-methylguanosine 5’-diphosphate (m ⁷ GDP(2’-OMe), 2): ¹ H NMR (300 MHz, D2O): δ 6.21 (d, J = 3.0 Hz, 1H), 4.69 (dd, J = 6.1, 4.9 Hz, 1H), 4.43 – 4.34 (m, 3H), 4.31 – 4.22 (m, 1H), 4.17 (s, 3H), 3.65 (s, 3H), 3.25 (q, J = 7.4 Hz, 9H, Et ₃ NH ⁺ ), 1.33 (t, J = 7.3 Hz, 14H, Et ₃ NH ⁺ ). ³¹ P NMR (121 MHz, D2O): δ -8.98 (d, J = 21.7 Hz), -11.03 – -11.36 (m). MS (ESI-): Exact mass calculated for C12H18N5O11P2 [M - H]-, 470.05. Found 470.02. 7-methyl-3’-O-methylguanosine 5’-diphosphate (m ⁷ GDP(3’-OMe), 3): ¹ H NMR (300 MHz, D2O): δ 6.11 (d, J = 4.0 Hz, 1H), 4.90 (t, J = 4.5 Hz, 1H), 4.54 (dq, J = 5.1, 2.6 Hz, 1H), 4.39 (ddd, J = 11.9, 4.5, 2.6 Hz, 1H), 4.30 – 4.19 (m, 2H), 4.18 (s, 3H), 3.55 (s, 3H), 3.25 (q, J = 7.4 Hz, 8H, Et3NH ⁺ ), 1.33 (t, J = 7.4 Hz, 12H, Et3NH ⁺ ). ³¹ P NMR (121 MHz, D2O): δ -8.95 (d, J = 21.5 Hz), -11.20 (dq, J = 21.2, 4.3 Hz). MS (ESI-): Exact mass calculated for C12H18N5O11P2 [M - H]-, 470.05. Found 470.09. General scheme for the synthesis of different cap analogs General Procedure (I) Cap synthesis involving multiple steps was performed according to modified published procedures. (Patent WO2017053297A1; WO2021162567A1, Nucleic Acids Res.2020, 48, 1607.). Step I: Coupling + Oxidation 2’,3’-N-protected nucleoside derivative (SM-1, 5 mmol), 5-Ethylthio-1H-tetrazole (activator, 25 mmol) and 2′-OMe phosphoramidite derivative (SM-2, 5 mmol) were taken in a 100 mL Schlenk flask and 50 mL dry ACN was added. The solution was stirred for 30 min at RT. Later tert-Butyl hydroperoxide solution (to oxidize P(III) to P(V) state, 25 mmol) was added dropwise, solution turned yellow, stirred at RT for further 30 min. Solvent was evaporated and the residue was redissolved in DCM (300 mL), extracted with water (3 x 100 mL) and brine (100 mL). The organic phase was dried over anhydrous Na2SO4, filtered and the solvent was evaporated in vacuo, yellow sticky solid was obtained. Crude residue was purified by flash chromatography (0-5% MeOH/DCM) and coupling product (Int-1) was achieved as yellowish-white solid foam (yield 84- 92%). Step II: Detritylation Coupling product (Int-1, 4 mmol) was taken in a 250 mL Schlenk flask and 150 mL dry DCM was added, formed colorless solution. Then dichloroacetic acid (40 mmol) was added dropwise at RT over 5 min, the solution turned dark red. The reaction was stirred for 15 min at RT. TLC was performed to check complete deprotection of the DMT group. Later MeOH was added until faint red color persisted. Solvent was evaporated and the residue was dissolved in 300 mL DCM, again MeOH was added until only a faint red color persisted. The solution was extracted with saturated aqueous NaHCO3 solution (3 x 100 mL), followed by extraction with brine (100 mL). The organic phase was dried over anhydrous Na2SO4, filtered and the solvent was evaporated in vacuo, yellow foam was obtained. Crude residue was purified by flash chromatography (0-10% MeOH/DCM) and compound (Int-2) was yielded as yellowish-white solid foam (yield 86-91%). Step III: Coupling + Oxidation Compound (Int-2, 3 mmol), 5-Ethylthio-1H-tetrazole (activator, 15 mmol) were taken in a 100 mL Schlenk flask and 30 mL dry ACN was added. After that, 3-({[bis(propan-2-yl)amino](2- cyanoethoxy)phosphanyl}oxy)propanenitrile (4.5 mmol) was added dropwise. The solution was stirred for 30 min at RT. Later tert-Butyl hydroperoxide solution (15 mmol) was added dropwise, solution turned yellow, stirred at RT for further 30 min. Solvent was evaporated and the residue was redissolved in DCM (200 mL), extracted with water (3 x 50 mL) and brine (50 mL). The organic phase was dried over anhydrous Na ₂ SO ₄ , filtered and the solvent was evaporated in vacuo, yellow sticky residue was obtained. Crude residue was purified by flash chromatography (0-10% MeOH/DCM) and compound (Int-3) was obtained as yellowish-white solid foam (yield 72-80%). Step IV: Deprotection Compound (Int-3, 2 mmol) was taken in an autoclave and 20 mL absolute EtOH was added, followed by the addition of 60 mL aq. NH3 solution (32%). The reaction was stirred at 60 °C for 6 h, showed complete deprotection of the substrate in LCMS studies. The solution was concentrated in vacuo, redissolved in water/absolute ethanol (1:1, 50 mL) and evaporated under reduced pressure to obtain yellow residue. when R* = Bz, the residue was purified directly by anion exchange chromatography, followed by reversed phase chromatography. Compound (pN1(2’-OMe)pN2, Int-4) was obtained as white solid foam as triethylammonium salt (yield 77-89%). when R* = TBS, the residue was further treated with 1M TBAF in THF solution. Reaction was stirred at 40 °C for 18 h. Deprotection of the TBS group was monitored by LCMS studies. After complete deprotection, 100 mM TEAB (pH = 7.5) was added and purified directly by anion exchange chromatography and reversed phase chromatography. Compound (pN ₁ (2’-OMe)pN ₂ , Int-4) was obtained as white solid foam as triethylammonium salt (yield 65-72%). Anion exchange chromatography: XK 50/30 column filled with DEAE Sephadex A-25 (~75 g resin, column volume ~550 mL, Cytiva. Detection wavelengths: 220, 260 nm; Solvent systems: buffer A: 100 mM TEAB (pH = 7.5); buffer B: 1.0 M TEAB (pH = 7.5); Flow rate: 30 ml/min; Gradient: 0% B for 30 min, 0–80% B in 160 min, 80–100% B in 5 min,100% B for 25 min). Product obtained after Anion exchange chromatography was further purified by reversed phase chromatography. Reversed phase chromatography: Column: FP Select C18330 g; Detection wavelengths: 220 nm, 260 nm; Solvent systems: solvent A: 100 mM TEAB (pH = 7.5), solvent B: acetonitrile; Flow Rate: 60 mL/min; Gradient: 5% B for 6 min, 5-20% B in 24 min, 20-50% B in 5 min, 50% B for 5 min). Product containing fractions were collected and the solvents were evaporated and lyophilized. Step V: Phosphorimidazolide formation Compound (pN ₁ (2’-OMe)pN ₂ , Int-4, 1 mmol) was taken in a 100 mL RB flask and co- evaporated with dry DMF (2 x 15 mL). After that, 35 mL dry DMF was added. In another dry 50 mL Schlenk flask, triphenylphosphine (2 mmol), 2,2’-dipyridyldisulfide (2 mmol), and imidazole (2.5 mmol) were dissolved in 20 mL dry DMF and Et ₃ N (10 mmol). The solution was then dropwise added to the solution of dinucleotide derivative, turned to yellow solution. The reaction was stirred for 18 h at RT. The solution was then poured onto 1 L 2% anhydrous NaClO4 in dry acetone (w/v) solution, white precipitate formation was observed. Filtration was performed and the residue was washed with 1 L dry acetone. The product was dried over anhydrous P2O5 under reduced pressure. Dinucleotide phosphorimidazolide (Im-pN1(2’-OMe)pN2, Int-5) was obtained as disodium salt (91-98%). Step VI: Final coupling 7-methyl-guanosine 5’-diphosphate derivative (m ⁷ GDP derivative, 0.75 mmol) and dinucleotide phosphorimidazolide (Im-pN1(2’-OMe)pN2, Int-5, 0.9 mmol) were taken in a 50 mL RB flask and 15 mL dry DMF was added under argon atmosphere, formed suspension. Then anhydrous ZnCl ₂ (7.5 mmol) was added under argon flow. The reaction mixture was stirred for 1 day at RT under argon atmosphere. Later the reaction mixture was added to aqueous Na2EDTA solution (1.5 g in 75 mL water), and pH was adjusted to 7.0 using saturated sodium bicarbonate solution. The solution containing crude product was purified was purified directly by anion exchange chromatography, followed by reversed phase chromatography. Anion exchange chromatography: XK 50/30 column filled with DEAE Sephadex A-25 (~75 g resin, column volume ~550 mL, Cytiva. Detection wavelengths: 220, 260 nm; Solvent systems: buffer A: 100 mM TEAB (pH = 7.5), buffer B: 1.0 M TEAB (pH = 7.5); Flow rate: 30 ml/min; Gradient: 0% B for 30 min, 0–80% B in 200 min, 80–100% B in 5 min,100% B for 25 min). Product obtained after anion exchange chromatography was further purified by reversed phase chromatography. Reversed phase chromatography: Column: Atlantis T3 OBD Prep Column, 100Å, 5 µm, 50 x 100 mm, Waters; Detection wavelengths: 220 nm, 260 nm; Solvent systems: solvent A: 50 mM NH ₄ OAc in water (pH 7.0), solvent B: 100 mM NH ₄ OAc in water (pH 7.0)/ACN (1:1); Flow Rate: 90 mL/min; Gradient: 2-15% B in 45 min, 15-50% B in 5 min, 50% B for 5 min, 50-2% B in 2 min, 2% B for 10 min). Product containing fractions were collected and the solvents were evaporated and lyophilized. The product after reversed phase chromatography was obtained as ammonium salt which was converted to sodium salt using 2% NaClO4 in acetone (w/v) solution. Sodium salt of the desired cap analogs was obtained as white solid foam (yield 40-50%).

m ⁷ G(3’-OMe)pppA(2’-OMe)pU (I′-1): Compound I′-1 was synthesized according to the general procedure (I). 1H NMR (300 MHz, D ₂ O): δ 9.15 (s, 1H), 8.60 (s, 1H), 8.34 (s, 1H), 7.89 (d, J = 8.2 Hz, 1H), 6.16 (d, J = 5.6 Hz, 1H), 5.93 (d, J = 4.5 Hz, 1H), 5.86 (d, J = 4.2 Hz, 1H), 5.82 (d, J = 8.1 Hz, 1H), 4.98 (dt, J = 8.2, 4.3 Hz, 1H), 4.73 (t, J = 4.5 Hz, 1H), 4.63 – 4.56 (m, 1H), 4.50 (t, J = 4.9 Hz, 1H), 4.46 – 4.11 (m, 11H), 4.06 (s, 3H), 3.51 (s, 3H), 3.48 (s, 3H). 3 ¹ P NMR (121 MHz, D ₂ O): δ -1.01 (d, J = 7.4 Hz, 1P), -11.23 – -11.78 (m, 2P), -22.76 (t, J = 18.5 Hz, 1P). MS (ESI-): Exact mass calculated for C32H43N12O25P4 [M - H]-, 1119.14. Found 1119.07. m ⁷ G(2’-OMe)pppA(2’-OMe)pU (I′-2): Compound I′-2 was synthesized according to the general procedure (I). 1H NMR (300 MHz, D2O): δ 9.11 (s, 1H), 8.56 (s, 1H), 8.32 (s, 1H), 7.93 (d, J = 8.1 Hz, 1H), 6.16 (d, J = 5.8 Hz, 1H), 5.99 – 5.95 (m, 2H), 5.87 (d, J = 8.1 Hz, 1H), 5.00 (dt, J = 8.0, 4.1 Hz, 1H), 4.63 – 4.51 (m, 3H), 4.48 – 4.16 (m, 11H), 4.10 (s, 3H), 3.60 (s, 3H), 3.54 (s, 3H). 3 ¹ P NMR (121 MHz, D2O): δ -1.00 (d, J = 7.2 Hz, 1P), -11.22 – -11.82 (m, 2P), -22.83 (t, J = 18.4 Hz, 1P). MS (ESI-): Exact mass calculated for C ₃₂ H ₄₃ N ₁₂ O ₂₅ P ₄ [M - H]-, 1119.14. Found 1119.07. m ⁷ G(3’-OMe)pppA(2’-OMe)pm ³ U (I′-11): Compound I′-11 was synthesized according to the general procedure (I). 1H NMR (300 MHz, D ₂ O): δ 8.46 (s, 1H), 8.19 (s, 1H), 7.88 (d, J = 8.1 Hz, 1H), 6.10 (d, J = 5.7 Hz, 1H), 5.98 (d, J = 4.0 Hz, 1H), 5.89 – 5.84 (m, 2H), 4.97 (dt, J = 8.2, 4.2 Hz, 1H), 4.71 (t, J = 4.7 Hz, 1H), 4.62 – 4.57 (m, 1H), 4.50 (t, J = 5.4 Hz, 1H), 4.46 – 4.17 (m, 10H), 4.13 (t, J = 4.7 Hz, 1H), 4.08 (s, 3H), 3.54 (s, 3H), 3.51 (s, 3H), 3.30 (s, 3H). 3 ¹ P NMR (121 MHz, D ₂ O): δ -1.05 (s, 1P), -11.55 (d, J = 18.6 Hz, 2P), -22.92 (t, J = 18.6 Hz, 1P). MS (ESI-): Exact mass calculated for C33H45N12O25P4 [M - H]-, 1133.16. Found 1133.19. m ⁷ G(3’-OMe)pppA(2’-OMe)pm ⁵ U (I′-9): Compound I′-9 was synthesized according to the general procedure (I). 1H NMR (300 MHz, D2O): δ 9.20 (s, 1H), 8.65 (s, 1H), 8.38 (s, 1H), 7.75 (s, 1H), 6.21 (d, J = 5.1 Hz, 1H), 5.97 (d, J = 4.9 Hz, 1H), 5.92 (d, J = 4.2 Hz, 1H), 5.00 (dt, J = 8.5, 4.4 Hz, 1H), 4.65 – 4.60 (m, 1H), 4.55 (t, J = 5.0 Hz, 1H), 4.49 – 4.15 (m, 11H), 4.11 (s, 3H), 3.57 (s, 3H), 3.53 (s, 3H), 1.87 (s, 3H). 3 ¹ P NMR (121 MHz, D2O): δ -1.25 (d, J = 7.5 Hz, 1P), -11.54 (t, J = 20.6 Hz, 2P), -22.76 (t, J = 18.4 Hz, 1P). MS (ESI-): Exact mass calculated for C ₃₃ H ₄₅ N ₁₂ O ₂₅ P ₄ [M - H]-, 1133.16. Found 1133.12. m ⁷ G(3’-OMe)pppA(2’-OMe)pmo ⁵ U (I′-12): Compound I′-12 was synthesized according to the general procedure (I). 1H NMR (300 MHz, D ₂ O): δ 9.11 (s, 1H), 8.52 (s, 1H), 8.28 (s, 1H), 7.34 (s, 1H), 6.15 (d, J = 5.6 Hz, 1H), 6.07 – 6.02 (m, 1H), 5.90 (d, J = 4.3 Hz, 1H), 5.01 (dt, J = 8.0, 3.9 Hz, 1H), 4.74 (t, J = 4.6 Hz, 1H), 4.63 – 4.59 (m, 1H), 4.56 (t, J = 5.4 Hz, 1H), 4.48 – 4.19 (m, 10H), 4.15 (t, J = 4.6 Hz, 1H), 4.09 (s, 3H), 3.80 (s, 3H), 3.52 (s, 3H), 3.51 (s, 3H). 3 ¹ P NMR (121 MHz, D ₂ O): δ -1.24 (s, 1P), -11.34 – -11.81 (m, 2P), -22.90 (t, J = 18.5 Hz, 1P). MS (ESI-): Exact mass calculated for C33H45N12O26P4 [M - H]-, 1149.15. Found 1149.17. m ⁷ GpppA(2’-OMe)pm ¹ Ψ (I′-13): Compound I′-13 was synthesized according to the general procedure (I). 1H NMR (300 MHz, D2O): δ 9.18 (s, 1H), 8.65 (s, 1H), 8.36 (s, 1H), 7.75 (d, J = 1.0 Hz, 1H), 6.20 (d, J = 5.5 Hz, 1H), 5.94 (d, J = 3.7 Hz, 1H), 5.00 (dt, J = 8.2, 4.2 Hz, 1H), 4.66 – 4.57 (m, 3H), 4.52 (t, J = 5.0 Hz, 1H), 4.47 – 4.37 (m, 3H), 4.36 – 4.11 (m, 7H), 4.09 (s, 3H), 3.55 (s, 3H), 3.38 (s, 3H). 3 ¹ P NMR (121 MHz, D2O): δ -0.72 (d, J = 6.8 Hz, 1P), -11.44 (t, J = 20.3 Hz, 2P), -22.73 (t, J = 18.5 Hz, 1P). MS (ESI-): Exact mass calculated for C32H43N12O25P4 [M - H]-, 1119.14. Found 1119.27. m ⁷ G(3’-OMe)pppA(2’-OMe)pm ¹ Ψ (I′-6): Compound I′-6 was synthesized according to the general procedure (I). 1H NMR (300 MHz, D2O): δ 9.08 (s, 1H), 8.57 (s, 1H), 8.27 (s, 1H), 7.63 (d, J = 1.0 Hz, 1H), 6.10 (d, J = 5.3 Hz, 1H), 5.77 (d, J = 4.1 Hz, 1H), 4.87 (dq, J = 9.3, 4.6 Hz, 1H), 4.66 – 4.61 (m, 2H), 4.57 – 4.44 (m, 2H), 4.36 – 4.28 (m, 3H), 4.24 – 3.98 (m, 8H), 3.96 (s, 3H), 3.43 (s, 3H), 3.39 (s, 3H), 3.25 (s, 3H). 3 ¹ P NMR (121 MHz, D2O): δ -1.82 (d, J = 7.4 Hz, 1P), -12.61 (t, J = 17.9 Hz, 2P), -23.86 (t, J = 19.4 Hz, 1P). MS (ESI-): Exact mass calculated for C ₃₃ H ₄₅ N ₁₂ O ₂₅ P ₄ [M - H]-, 1133.16. Found 1133.18. m ⁷ G(2’-OMe)pppA(2’-OMe)pm ¹ Ψ (I′-5): Compound I′-5 was synthesized according to the general procedure (I). 1H NMR (300 MHz, D2O): δ 9.15 (s, 1H), 8.63 (s, 1H), 8.35 (s, 1H), 7.75 (d, J = 1.0 Hz, 1H), 6.18 (d, J = 5.6 Hz, 1H), 5.98 (d, J = 3.6 Hz, 1H), 5.00 (dt, J = 8.2, 4.1 Hz, 1H), 4.68 – 4.56 (m, 3H), 4.48 – 4.38 (m, 2H), 4.37 – 4.12 (m, 9H), 4.10 (s, 3H), 3.60 (s, 3H), 3.55 (s, 3H), 3.38 (s, 3H). 3 ¹ P NMR (121 MHz, D ₂ O): δ -0.74 (d, J = 6.9 Hz, 1P), -11.50 (t, J = 19.0 Hz, 2P), -22.78 (t, J = 18.5 Hz, 1P). MS (ESI-): Exact mass calculated for C33H45N12O25P4 [M - H]-, 1133.16. Found 1133.18. m ⁷ G(3’-OMe)pppA(2’-OMe)pΨ (I′-3): Compound I′-3 was synthesized according to the general procedure (I). 1H NMR (300 MHz, D ₂ O): δ 9.19 (s, 1H), 8.62 (s, 1H), 8.35 (s, 1H), 7.60 (s, 1H), 6.21 (d, J = 4.8 Hz, 1H), 5.92 (d, J = 4.3 Hz, 1H), 4.96 (dt, J = 8.7, 4.6 Hz, 1H), 4.77 – 4.73 (m, 2H), 4.67 – 4.58 (m, 2H), 4.47 – 4.39 (m, 3H), 4.34 – 4.14 (m, 8H), 4.11 (s, 3H), 3.59 (s, 3H), 3.52 (s, 3H). 3 ¹ P NMR (121 MHz, D ₂ O): δ -0.70 (d, J = 6.7 Hz, 1P), -11.33 – -11.74 (m, 2P), -22.79 (t, J = 18.5 Hz, 1P). MS (ESI-): Exact mass calculated for C32H43N12O25P4 [M - H]-, 1119.14. Found 1119.04. m ⁷ G(3’-OMe)pppA(2’-OMe)pm ³ Ψ (I′-14): Compound I′-14 was synthesized according to the general procedure (I). 1H NMR (300 MHz, D ₂ O): δ 9.24 (s, 1H), 8.70 (s, 1H), 8.41 (s, 1H), 7.60 (d, J = 1.1 Hz, 1H), 6.24 (d, J = 4.9 Hz, 1H), 5.93 (d, J = 4.3 Hz, 1H), 4.98 (dt, J = 8.6, 4.5 Hz, 1H), 4.66 – 4.61 (m, 2H), 4.50 – 4.39 (m, 3H), 4.38 – 4.13 (m, 8H), 4.12 (s, 3H), 3.60 (s, 3H), 3.53 (s, 3H), 3.27 (s, 3H). 3 ¹ P NMR (121 MHz, D ₂ O): δ -0.70 (d, J = 6.5 Hz, 1P), -11.27 – -11.74 (m, 2P), -22.75 (t, J = 18.4 Hz, 1P). MS (ESI-): Exact mass calculated for C33H45N12O25P4 [M - H]-, 1133.16. Found 1133.22. m ⁷ G(3’-OMe)pppA(2’-OMe)ptfet ¹ Ψ (I′-15): Compound I′-15 was synthesized according to the general procedure (I). 1H NMR (300 MHz, D ₂ O): δ 9.10 (s, 1H), 8.54 (s, 1H), 8.29 (s, 1H), 7.86 (s, 1H), 6.15 (d, J = 6.3 Hz, 1H), 5.89 (d, J = 4.3 Hz, 1H), 5.01 (dt, J = 8.0, 4.1 Hz, 1H), 4.83 (d, J = 3.2 Hz, 1H), 4.74 (t, J = 4.7 Hz, 1H), 4.69 – 4.52 (m, 4H), 4.47 – 4.12 (m, 11H), 4.10 (s, 3H), 3.52 (s, 3H), 3.51 (s, 3H). 3 ¹ P NMR (121 MHz, D ₂ O): δ -0.81 (s, 1P), -11.54 (d, J = 18.6 Hz, 2P), -22.89 (t, J = 18.5 Hz, 1P). MS (ESI-): Exact mass calculated for C34H44F3N12O25P4 [M - H]-, 1201.14. Found 1201.05. m ⁷ G(3’-OMe)pppA(2’-OMe)p(ppg) ¹ Ψ (I′-16): Compound I′-16 was synthesized according to the general procedure (I). 1H NMR (300 MHz, D ₂ O): δ 8.48 (s, 1H), 8.21 (s, 1H), 7.84 (s, 1H), 6.12 (d, J = 6.2 Hz, 1H), 5.86 (d, J = 4.3 Hz, 1H), 5.00 (dt, J = 7.9, 4.5 Hz, 1H), 4.70 (t, J = 4.6 Hz, 1H), 4.66 – 4.61 (m, 1H), 4.60 – 4.54 (m, 3H), 4.45 – 4.10 (m, 12H), 4.09 (s, 3H), 3.51 (s, 6H), 2.85 (t, J = 2.5 Hz, 1H). 3 ¹ P NMR (121 MHz, D ₂ O): δ -0.73 (d, J = 7.2 Hz, 1P), -11.26 – -11.78 (m, 2P), -22.88 (t, J = 18.6 Hz, 1P). MS (ESI-): Exact mass calculated for C35H45N12O25P4 [M - H]-, 1157.16. Found 1157.12. m ⁷ G(3’-OMe)pppA(2’-OMe)pbn ¹ Ψ (I′-17): Compound I′-17 was synthesized according to the general procedure (I). 1H NMR (300 MHz, D ₂ O): δ 9.13 (s, 1H), 8.53 (s, 1H), 8.24 (s, 1H), 7.90 (s, 1H), 7.35 – 7.18 (m, 5H), 6.12 (d, J = 6.5 Hz, 1H), 5.90 (d, J = 4.6 Hz, 1H), 5.08 – 5.00 (m, 1H), 4.96 – 4.80 (m, 3H), 4.76 – 4.67 (m, 2H), 4.56 (t, J = 5.6 Hz, 1H), 4.48 – 4.12 (m, 11H), 4.09 (s, 3H), 3.52 (s, 3H), 3.45 (s, 3H). 3 ¹ P NMR (121 MHz, D ₂ O): δ -0.86 (s, 1P), -11.34 – -11.79 (m, 2P), -22.86 (t, J = 18.6 Hz, 1P). MS (ESI-): Exact mass calculated for C39H49N12O25P4 [M - H]-, 1209.19. Found 1209.18. m ⁷ G(3’-OMe)pppA(2’-OMe)pcpm ¹ Ψ (I′-18): Compound I′-18 was synthesized according to the general procedure (I). 1H NMR (300 MHz, D2O): δ 8.49 (s, 1H), 8.22 (s, 1H), 7.82 (d, J = 1.0 Hz, 1H), 6.12 (d, J = 6.4 Hz, 1H), 5.87 (d, J = 4.4 Hz, 1H), 5.00 (ddd, J = 7.8, 4.8, 2.9 Hz, 1H), 4.85 – 4.80 (m, 1H), 4.72 (t, J = 4.7 Hz, 1H), 4.66 – 4.60 (m, 1H), 4.56 (ddd, J = 6.5, 4.9, 1.4 Hz, 1H), 4.46 – 4.10 (m, 11H), 4.09 (s, 3H), 3.66 (h, J = 7.0 Hz, 2H), 3.52 (s, 3H), 3.50 (s, 3H), 1.17 (ddt, J = 10.3, 7.7, 3.8 Hz, 1H), 0.63 – 0.54 (m, 2H), 0.40 – 0.32 (m, 2H). 3 ¹ P NMR (121 MHz, D ₂ O): δ -0.80 (d, J = 6.4 Hz, 1P), -11.53 (dt, J = 18.7, 6.7 Hz, 2P), - 22.90 (t, J = 18.7 Hz, 1P). MS (ESI-): Exact mass calculated for C36H49N12O25P4 [M - H]-, 1173.19. Found 1173.07. m ⁷ G(3’-OMe)pppA(2’-OMe)p(4-pm) ¹ Ψ (I′-19): Compound I′-19 was synthesized according to the general procedure (I). 1H NMR (300 MHz, D ₂ O): δ 8.53 – 8.49 (m, 2H), 8.48 (s, 1H), 8.20 (s, 1H), 7.91 (d, J = 1.2 Hz, 1H), 7.45 – 7.40 (m, 2H), 6.08 (d, J = 7.0 Hz, 1H), 5.91 (d, J = 4.4 Hz, 1H), 5.08 (s, 2H), 5.01 (ddd, J = 7.2, 4.7, 2.2 Hz, 1H), 4.90 – 4.84 (m, 1H), 4.77 – 4.74 (m, 1H), 4.71 – 4.66 (m, 1H), 4.58 (ddd, J = 6.8, 4.8, 1.7 Hz, 1H), 4.50 – 4.38 (m, 2H), 4.32 – 4.11 (m, 9H), 4.09 (s, 3H), 3.52 (s, 3H), 3.45 (s, 3H). 3 ¹ P NMR (121 MHz, D2O): δ -0.86 (d, J = 6.7 Hz, 1P), -11.30 – -11.83 (m, 2P), -22.92 (t, J = 18.7 Hz, 1P). MS (ESI-): Exact mass calculated for C38H48N13O25P4 [M - H]-, 1210.18. Found 1210.16. Example 3. Extended translation of non-replicating mRNA by novel cap analogs containing nucleoside modification Background One of the most defining characteristic features of mRNAs is a multifunctional cap at the five-prime end (5’) that was first discovered in the 1970s (1). For several cellular processes including nuclear transport (2), mRNA splicing (3,4), regulation of mRNA decay (5), and robust translation (6) mRNA requires a functional 5’ cap structure. Naturally occurring eukaryotic mRNA possesses a 7-methylguanosine (m7G) cap linked to the mRNA via a 5´ to 5´ triphosphate bridge resulting in what is termed as the Cap0 structure (m7GpppN). In most eukaryotic and some viral mRNA, further modifications can occur at the 2’-hydroxy-group (2’-OH) in the first and subsequent nucleotides, thereby producing Cap1 or Cap2 structures, respectively. Cap0-mRNA cannot be translated as efficiently as cap1 mRNA, where the role of 2’-O-met in the penultimate position at the mRNA 5’ end is determinant (7). The use of co-transcriptional cap analogs offers a cost-effective and time-saving process for imitating the natural 5’ cap structure of endogenous mRNA. Dinucleotide cap analogs for capping of in vitro-transcribed (IVT) self-amplifying RNA (saRNA) and conventional messenger RNA (mRNA) are adequate substitutes for enzymatic capping and still widely used because of their many advantages including resistance to human decapping enzymes and to interferon- induced proteins with tetratricopeptide repeats (IFITs), which inhibit cap0-dependent translation (8,9). However, the main shortcoming is that GTP competes with the dinucleotide cap analogs during transcription, leading to poor capping efficiency and hence weak translational capacity. Nevertheless, cap1 analogs are commercially available that can be incorporated into both IVT saRNA (CleanCap AU, #N7-114, TriLink BioTechnologies) and IVT mRNA (CleanCap 3’OMe AG, #N7-413, TriLink BioTechnologies) in the correct orientation to produce cap1 mRNA with a high capping efficiency, all in a rapid co-transcriptional reaction (10). The most appealing feature is that this trinucleotide cap1 analog requires an A initiator, avoiding the slippage of RNA polymerases on the DNA template strand as opposed to those containing a G triplet as a transcriptional start site (11). Enzymatic capping can also yield cap1 mRNA, but it is time- consuming since it requires an extra purification step. Moreover, it demands a heating step to improve the accessibility of structured 5’ ends, thereby risking RNA degradation. In addition to this, the 5' cap of enzymatically capped mRNA is not modified at the C2’ or C3’ position and it is well known that anti reverse cap analog (ARCA)-capped mRNA possesses higher translation efficiency compared to conventional cap analogs (12,13). There is also a variant of the CleanCap AG cap1 analog with a modification at the C3’ position of 7-methylguanosine (CleanCap AG 3’ OMe) which played an important role in the progress of immunotherapeutic vaccination strategy against SARS-CoV-2 (14). It is also well known that incorporation of nucleoside modifications including N1-methylpseudouridine (m1Ψ) (15) or N6-methyladenine (m6A) (16) into IVT mRNAs increases biological stability and thereby enhances the durability of the encoded protein compared to unmodified RNAs. Due to the fact that saRNA cannot contain modified nucleosides, its use is primarily limited to preventive vaccines against infectious diseases (17) in contrast to non-replicating mRNAs. The latter also have great potential in research areas such as gene editing, protein replacement therapy, where the reduction and elimination of immunomodulation is indispensable for reaching the appropriate therapeutic goal. The advantages of using various modified nucleosides are indisputable in the mRNA research field. Nevertheless, the effects of cap analogs containing modified nucleosides on quality, translational efficiency and biological activity or immunogenicity of a long mRNA that encodes a potentially therapeutic protein is not understood. To answer this question, trinucleotide cap1 analogs containing various nucleoside modifications (i.e., compounds of formula I′) were synthesized and tested in vitro and in vivo using IVT mRNAs encoding murine erythropoietin. Our findings on the biological activity and immunogenicity of mRNAs capped with I′-6 (m ₂ ^7,3’O )Gppp(m ^2’O )Ap(m1)Ѱ) cap analog suggest that this co-transcriptional trinucleotide cap analog containing modified nucleoside is a promising alternative to current capping strategies in mRNA vaccines and especially in RNA-based therapeutics. Evaluating the impact of I′-6 cap analog containing N1-methylpseudouridine For templates, linearized plasmid encoding codon-optimized murine erythropoietin (EPO) was used. The mRNAs starting with AUAGU was designed to contain the 5’ untranslated region (5’UTR) sequences of human α-globin (hAg) mRNA, an FI element as the 3’UTR, and an interrupted 100 nt-long 3’ poly(A) tail flanking the coding sequence. The MEGAscript T7 Transcription kit (Thermo Fisher Scientific, Waltham, MA, USA) was used for transcription, and UTP was kept or was replaced with N1-methylpseudouridine (m1Ψ) triphosphate (TriLink, San Diego, CA, USA). Capping of in vitro-transcribed mRNAs was performed co-transcriptionally using 4 mM of self-designed trinucleotide cap analogs which either contained a nucleoside modification (I′-3, I′-5, I′-6, I′-9, and I′-11 to I′-19) or not (I′-1 or I′-2) and commercially available reference cap analogs (CC114, CC413). To obtain the desired transcripts generated with these cap analogs, the initial GTP and UTP or m1ѰTP concentration in the transcription reaction was reduced from 7.5 mM to 1.5 mM and the 1.5 mL tubes were incubated at 37°C for 30 min in a hybridization chamber. Sequential additions of 1.5 mM GTP and UTP or m1ѰTP were required to supplement the reaction at 30, 60, 90 and 120 min and incubated further at 37°C for 30 min. To remove template DNA, Turbo DNase (Thermo Fisher Scientific, USA) was added to the reaction mix after the transcription reaction was completed and incubated for 15 min at 37°C. The synthesized mRNA was precipitated by adding a half volume of 8 M LiCl solution (Merck, Darmstadt, Germany) to the reaction mix and then pelleted by centrifugation. After dissolving in nuclease free water, mRNAs were cellulose-purified to remove double-stranded RNA contaminants, as described (18). The mRNA concentration and quality were measured on a NanoDropTM 2000c spectrophotometer (Thermo Fisher Scientific, USA). Small aliquots of mRNA samples were stored in RNase-free tubes at -20°C. In order to determine the capping efficiency of mRNAs capped with m1Ѱ-containing I′-6 and cap analogs that do not contain uridine modifications (I′-1, CC413, and CC114), in vitro transcription reaction followed by a Ribozyme assay was performed. The Ribozyme cleavage reaction contained 0.45 µM mRNA. A 3-fold molar excess of ribozyme over mRNA substrate was added in an aqueous solution containing 30 mM HEPES and 150 mM NaCl. The ribozyme cleavage reaction was performed on a PCR machine utilizing the following program: 95°C for 2 min, chill the mixture up to 37°C by ramping rate of 0.1°C/sec, 37°C for 5 min; after adding 30 mM MgCl2 solution to each sample, the mixtures were maintained at 37°C for 60 min followed by stopping the annealing at 80°C for 2 min and transferring to ice for 5 min. Then, the short and long RNA fragments were separated using the RNA Clean & Concentrator-5 kit (Zymo Research Europe, Freiburg, Germany) according to the manufacturer’s instructions. In our study, the following custom-designed hammerhead ribozyme specific for the hAg 5’UTR was used: 5’-UGU GGG CUG AUG AGG CCG UGA GGC CGA AAC CAG AAG AAU-3’ (synthesized by Metabion International AG, Planegg, Germany). To detect the short fragments, the samples (30 ng) were resolved on a 21% (vol/vol) 19:1 acrylamide:bisacrylamide denaturing gel supplemented with 8 M urea (Merck, Germany). Before loading the samples denatured by incubation at 75°C for 5 min in the presence of 2x RNA loading buffer (New England Biolabs, Germany), the gel was pre-run at 180 V for 60 min. When the pre-run was finished, the pockets were rinsed with 1x TBE buffer. Immediately afterwards, the samples were loaded and the gel was run at 200 V constantly until the dye front reached the end of the gel. To identify the short, cleaved products, the gel was incubated with 1x TBE buffer containing 0.01% SYBR Gold nucleic acid stain (Thermo Fisher Scientific, USA) and the fluorescent signals were captured using a Gel Doc EZ Imager (Bio-Rad, Hercules, CA, USA). High capping efficiency is observed in terms of each tested mRNA regardless of cap analog (Figure 3). According to Ribozyme assay analysis, the capping efficiency of novel m1Ѱ-modified cap1 analog I′-6 is close to 100% and is comparable to the reference cap analogs (I′-1, CC413, and CC114) (Figure 3). For detection of short byproducts, IVT mRNAs (1.5-2 µg) were resolved on a 21% (vol/vol) 19:1 acrylamide:bisacrylamide denaturing gel supplemented with 8 M urea (Merck, Germany). Before loading the mRNA samples denatured by incubation at 75°C for 10 min in the presence of 2x RNA loading buffer (New England Biolabs, Germany), the gel was pre-run at 180 V for 60 min. When the pre-run was finished, the pockets were rinsed with 1x TBE buffer. Immediately afterwards the samples were applied, and the gel was run at 180 V constantly until the dye front has reached the end of the gel. For identification of the short byproducts, the gel was incubated with 1x TBE buffer containing 0.01% SYBR Gold nucleic acid stain (Thermo Fisher Scientific, USA) and the fluorescent signals were captured using a Gel Doc EZ Imager (Bio-Rad, USA). Denaturing Urea Polyacrylamide Gel Electrophoresis showed minimal amount of short contaminants for I′-6 similar to CC413, while for unmodified mRNA samples a significant amount of contaminants was observed independent of cap analogs (Figure 3). In each in vivo experiment in order to measure the translational efficiency and to determine the functionality of EPO mRNA capped with I′-1, I′-2, I′-3, I′-5, I′-6, I′-9, I′-12, I′-13, I′-16, CC114, and CC413 in vivo, female BALB/c mice from Jackson Laboratory (Bar Harbor, ME, USA) at the age of eight to ten weeks were used for in accordance with federal policies on animal research (Ethics approval number: G19-12-074). In each case, mice (n=3/group) were injected intravenously (i.v.) with 3 µg TransIT-complexed (Mirus Bio) EPO mRNAs in a final volume of 200 µL Dulbecco’s modified Eagle medium (DMEM). Mice used as controls were injected with TransIT-reagent diluted in DMEM but without RNA. To measure hematocrits and EPO levels in the individual mice that were injected with EPO mRNA capped with I′-6, I′-1, CC114, and CC413 and complexed with TransIT-reagent, 18 µL of blood was collected at the indicated times (Figure 8B) and centrifuged in Drummond microcaps glass capillary tubes (20 µl volume, Merck, Germany) as described (19). After determination of the hematocrit, capillary tubes were snapped open, and the plasma was collected to measure EPO levels and analyzed for mouse Erythropoietin DuoSet ELISA kit (R&D Systems) according to the manufacturer’s instructions. First of all, these results demonstrated that mRNA capped with I′-6 (anti-reverse (ARCA) cap1 analog (m2(7,3'OMe)G(5')ppp(5')(2'OMe)Apm1Ѱ)) outperformed each reference sample regardless of the cap initiators (Figure 8A). EPO RNA containing m1Ѱ modifications translated 2-3-fold, 5- fold, and 10-fold more than U-containing mRNAs at 6-24, 48 and 72 hours after injections, respectively. (Figure 8A). I′-6 cap1 analog has a beneficial effect on long-term translational efficiency of m1Ѱ-modified mRNA, meaning it showed 1.5-3-fold more plasma EPO level compared to those capped with CC413 at 48 and 72 h after injection, respectively (Figure 8A). The positive effect of ARCA 3'-OMe I′-6 on the translational capacity of the mRNA is also expressed in the biological activities of the mRNA because hematocrit values remained very high level in mice injected with I′-6-capped EPO mRNA even at day 21 after injection (Figure 8B). Hematocrit values started to decrease at D14 after injection of each mRNA capped with CC114 and CC413 but it was not observed in regards to I′-6-capped mRNA (Figure 8B). Hematocrit value in mice injected with m1Ѱ-mRNA capped with I′-6 at 21 days after administration is at the same level as the values of D7 and D14 in mice injected with CC413-capped mRNA (Figure 8B). To address translation of I′-6-capped mRNA in human primary hepatocytes, EPO level was measured from supernatants transfected using 0.1µg/well TransIT-formulated I′-6- or CC114-capped uRNA (Figure 13). Increased secretion of EPO in human primary cells was detected at all three tested time points: 24h, 48h and 144h (Figure 13). These results suggest that cap1 analogs employing nucleoside modification (e.g., I′-6) are suitable for translation of the encoded protein and can be used for synthesizing non-replicating functional mRNAs. To test the effect of the cap types and mRNA 5´end on immunogenicity in human peripheral blood monocyte cells (PBMCs) we compared changes in levels of cytokines and chemokines after application of Lipoplex (LPX)-formulated EPO mRNAs. First, we compared the impact of m1Ѱ-mRNA capped with I′-6 containing a UAGU 5´end vs. CC413 (AGAAU 5´end) (Figure 12). Tested cytokines (IL-6, TNF-α, IL-1β, IFN-γ) showed significant decrease when I′-6 capped mRNA was used after application of 1 and 3 µg/well, corresponding to 5 and 15 µg LPX- m1Ѱ-mRNA/ml respectively. MIP-1β showed decrease when I′-6 was used after application of 0.333 and 1 µg/well, corresponding to 1.7 and 5 µg LPX-m1Ѱ-mRNA/ml respectively. Second, we compared capped EPO uRNA with I′-6 or I′-1 (Figure 14). In this case both mRNAs had the same TAGT 5´end. Still, I′-6 showed benefit leading to significantly lower cytokines (IL-6, TNF- α, IL-1β and IFN-γ) 24h after application to human PBMCs. Thus, benefit of I′-6 in decreasing the immunogenicity and thus increasing safety was found when I′-6 was used. ARCA trinucleotide caps are not only much more effective than dinucleotide cap analogs and attributed to the correct orientation, but the additional methyl group on the m7G moiety might impact translation of the mRNA. To this end, we generated U-containing as well as m1Ѱ-modified EPO RNA capped with cap analogs which belong to either the non-ARCA (I′-13) or ARCA (I′-6) classes. Moreover, the latter ones contain 2'-OMe (I′-5) or 3'-OMe (I′-6) at the ribose of the m7G moiety. Three µg of TransIT-complexed EPO mRNA capped with nucleoside modification- containing NeoCaps I′-13, I′-5 and I′-6 cap analogs were injected into mice intravenously. After that EPO level of plasma collected from mice was measured at 6, 24 and 48 hours after administration to compare the translational activity of mRNA capped with NeoCaps with U- containing or m1Ѱ-modified mRNA capped with non-modified ARCA 3`OMe I′-1. EPO ELISA demonstrated that mRNA capped with m1Ѱ-modified cap analogs, even non-ARCA I′-13 translated at least two-fold better at each tested time point compared to those capped with non- modified ARCA (3′-OMe I′-13), regardless of the mRNA modification (Figure 9). In order to investigate the importance of the presence of nucleoside modification (such as m1Ѱ) in the cap analog on the long-term translational capacity of the m1Ѱ-modified mRNA, two different combinations of cap analog were transcribed, one of which contained nucleoside modification (m1Ѱ) at the N2 position (I′-6), while the other did not (I′-1). Plasma EPO level of mice injected with 3 µg of each TransIT-complexed mRNA was determined using murine EPO- specific ELISA at 6, 24, 48 and 72 hours after intravenous administration. According to our in vivo result, the nucleoside modification, in this case m1Ѱ, is determinant and very important to incorporate m1Ѱ into the cap analog (I′-6) because the combination of m1Ѱ-m1Ѱ translated 2-3- fold and 4-5 fold more than those capped with unmodified I′-16-24 hours and 48-72 hours after administration, respectively (Figure 10). Taken together, these findings suggested that the incorporation of nucleoside modification into cap analogs has the beneficial effect on short and long-term translational activity of mRNA m1Ѱ-modified ARCA 3’ OMe I′-6, which markedly stands out from other cap analogs. To test whether the incorporation of various uridine (U) or pseudouridine (Ѱ) derivatives into the cap analogs improve the performance of IVT mRNA, EPO-encoding mRNA capped with cap analogs bearing N3-methyluridine (I′-11), N5-methyluridine (I′-9), N5-methoxyuridine (I′- 12), N1-methylpseudouridine (I′-6), pseudouridine (I′-3), N3-methylpseudouridine I′-14), N1- trifluoroethylpseudouridine (I′-15), N1-propargylpseudouridine (I′-16), N1-benzylpseudouridine (I′-17), N1-cyclopropylmethyl-pseudouridine (I′-18) and N1-4-pyridylmethylpseudouridine (I′- 19) were prepared using the same IVT conditions as described above. After purification of preselected mRNAs, 3 µg of each was complexed with TransIT-mRNA reagent and injected into Balb/c mice intravenously. At 6, 24, 48 and 72 hours after injection of mRNA capped with I′-9, I′- 12, I′-6, I′-3, I′-16, and I′-1 into mice, murine EPO-specific ELISA was performed. For all cap analogs comprising a modified nucleoside at position N2, EPO levels were higher as compared to unmodified cap analog I′-1. EPO level in mice injected with mRNA capped with Ѱ-containing cap analog (I′-3 - m2(7,3'OMe)G(5′)ppp(5′)(2'OMe)ApѰ) is equal or slightly less to those bearing m1Ѱ (I′-6 - m2(7,3'OMe)G(5′)ppp(5′)(2'OMe)Apm1Ѱ) (Figure 11). Our finding showed that neither uridine (U) and its derivatives (5-methylU, 5-methoxyU) nor pseudouridine (Ѱ) and its derivatives (1-propargylѰ) could improve the potency of N1-methylpseudouridine (1-methylѰ)- containing cap (I′-6). I′-6 cap1 analog incorporated into m1Ѱ-modified mRNA surpasses the performance of other cap analogs with other modification despite of the same capping efficiency. To test if various uridine (U) or pseudouridine (Ѱ) derivatives of cap analogues can improve translation in human primary hepatocytes, we first measured EPO secretion after application of EPO-encoding Ѱ-mRNA capped with compounds desctribed herein analog bearing pseudouridine (I′-3) or CC413 (no modification). We found that level of EPO was higher at 24h and 48h when I′-3 was used compared to CC413 (Figure 15). Comparison of EPO-encoding mRNA capped with cap1 analogs bearing N5-methyluridine (I′-9), N5-methoxyuridine (I′-12), N1-methylpseudouridine (I′-6) and N1-propargylpseudouridine (I′-16) showed increased level of secreted EPO at 24h when I′-6 was used compared to other listed caps (Figure 16). In addition, all tested modification bearing caps led to increase in EPO secretion at 24h and 48h when compared to the unmodified I′-1 (Figure 16). Conclusion In conclusion, the presented data demonstrated that m1Ѱ-containing cap analog (I′-6 - m2(7,3'OMe)G(5′)ppp(5′)(2'OMe)Apm1Ѱ) bearing 3'-OMe at the ribose of the m7G moiety can be adapted to synthesis of non-replicating mRNA. We evaluated a unique, nucleoside modification-containing anti-reverse trinucleotide cap1 analog I′-6 that was used to generate a functional mRNA with 5' cap1 structure resulting in a long-term maintenance of the encoded protein. We showed that using trinucleotide caps comprising a modified base at position N2, such as in particular I′-6 leads to a superior mRNA that significantly surpasses the translational capacity and biological activity of IVT mRNA capped with cap analogs that do not contain any nucleoside modifications (Figures 10, 11). 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Nucleic acids, 15, 26–35. First published on Feb 27, 2019. 19. Mahiny, A.J. and Karikó, K. (2016) Measuring Hematocrit in Mice Injected with In Vitro- Transcribed Erythropoietin mRNA, Methods in molecular biology (Clifton, N.J.), 1428, 297– 306. EQUIVALENTS Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is to be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the listed claims is introduced into another claim dependent on the same base claim (or, as relevant, any other claim) unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. Further, it should also be understood that any embodiment or aspect of the invention can be explicitly excluded from the claims, regardless of whether the specific exclusion is recited in the specification. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the claims that follow.