[0006] Despite recent advancements in the field, including the approval of four RNAi-based drugs by the U.S. Food and Drug Administration (FDA) [16-19], the delivery of siRNAs to target cells and tissues continues to be one of the major challenges in RNAi research. RNA molecules are unable to diffuse across the hydrophobic cell membrane due to their large size and polyanionic backbone [20]. Current delivery strategies include the encapsulation of siRNAs within nanoparticles or liposomes and the conjugation of siRNAs to hydrophobic molecules. Because siRNAs lack selectivity for specific cell types, receptor-targeting ligands can be used to deliver siRNAs to target cells and tissues [21]. The most successful delivery platform for siRNAs to date involves the use of a tri-GalNAc (N-acetylgalactosamine) ligand and has been employed in three out of the four FDA- approved siRNA therapeutics. GalNAc binds to the asialoglycoprotein receptor (ASGPR), a cell-surface receptor highly expressed in hepatocytes, mediating selective siRNA delivery to the liver. [0007] With the success of the receptor-targeting ligand GalNAc and the need for selective extra-hepatic RNAi delivery systems, other small bioconjugates, like folate, have gained a lot of attention. Folates are a group of essential B9 vitamins that play a key role in mammalian one-carbon metabolism. They are primarily transported by the reduced folate carrier (RFC), which is expressed ubiquitously [22,23]. However, these molecules can also bind to cell-surface glycoproteins, called folate receptors (FR), with high affinity. The FRα is expressed at very low levels in non-malignant tissues but is highly expressed on numerous cancers, including 90% of ovarian carcinomas as well as breast, endometrial, lung, brain, and kidney cancers [24,25]. Because of this, folate has been extensively studied as a drug delivery system to target tumour cells [26-28]. Unlike with the RFC, folate conjugates are recognized and internalized by FRα, making folate a promising ligand for the delivery of siRNAs. [0008] This receptor-targeting strategy has been used to deliver siRNAs by functionalizing liposomes and nanoparticles with folic acid [29-33] although selective delivery can also be achieved by direct conjugation of folic acid to siRNAs. Previous studies have successfully incorporated folic acid modifications at either the 3′ or 5′ end of siRNA and achieved selective, carrier-free delivery to target cells [34,35]. In these studies, moderate gene-silencing activity against exogenous gene targets (40–60% knockdown after 1 μM treatment) was reported. These results showed promise in the use of folic acid - 2 - 8660875

as a delivery system for siRNAs. However, there was a need to improve the gene- silencing potency of folic acid–siRNA constructs. [0009] An siRNA bearing a folate modification within the central region was previously reported [36]. Previous research had shown that modifying the central region of an siRNA, which spans the Ago2 cleavage site, can lead to enhanced gene-silencing activity [37,38]. The synthetic scheme used therein relied on copper-catalyzed azide- alkyne cycloaddition (CuAAC) to yield a library of folate-conjugated siRNAs. [0010] Research in this field has been greatly limited by sophisticated, and often expensive, synthetic approaches as well as by the lack of reliable protocols to prepare folate phosphoramidites. Notably, folate phosphoramidites are not commercially available although Berry & Associates previously offered a 5′-folate-TEG cyanoethyl phosphoramidite (BA 0349), at a cost of $843 USD for 100 µmol (Figure 1). This product has since been discontinued. Most of the current synthetic approaches of triazole-linked folate-siRNAs require the use of Cu(I) in the final synthetic step. Although effective, this approach poses challenges with scalability and potential cytotoxicity. There is a need to streamline the synthesis of folate-siRNAs. SUMMARY: [0011] It has been shown that the small molecule phosphoramidite derivatives, such as the folic acid phosphoramidite, of the present disclosure are compatible with standard solid-phase oligonucleotide synthesis protocols. Further, it has been shown that the phosphoramidite derivatives can be used to incorporate small molecule such as folic acid in an oligonucleotide in the form of a nucleotide analog. The oligonucleotides prepared by the methods of the present disclosure are taken up by cells of interest including folate receptor expressing cancer cells, in the absence of a transfection reagent, and can mediate gene-silencing activity. [0012] In an aspect, the present disclosure includes a folic acid phoshoramidite derivative of Formula I - 3 - 8660875

I or a tautomer, derivative, or salt thereof, wherein Z is O, NH, or NHR ² ; L1 and L2 are each independently (CH2)2-6 or -CH2CH2OCH2CH2-; n is an integer from 2 to 6; m is an integer from 1 to 6; R ¹ is H or an acid-labile protective group; each R ² is independently H, -C(O)CH3, or -C(O)CF3; and R ³ is H, -C(O)CH3, -C(O)CF3, or C1-3alkyl. [0013] In another aspect, the present disclosure includes a phosphoramidite derivative of Formula II of a small molecule A, II or a tautomer, derivative, or salt thereof, wherein L1 and L2 are each independently (CH2)2-6 or -CH2CH2OCH2CH2-; - 4 - 8660875

n is an integer of 2 to 6; m is an integer of 1 to 6; A is the small molecule A, wherein the small molecule A comprises at least one carboxylic acid; and R ¹ is H or an acid-labile protective group. [0014] In another aspect, the present disclosure includes compound of Formula III III or a tautomer, derivative, or salt thereof, wherein L1 and L2 are each independently (CH2)2-6 or -CH2CH2OCH2CH2-; n is an integer of 2 to 6; m is an integer of 1 to 6; R ¹ is H or an acid-labile protective group; R ⁵ is H or a protective group; and R ⁶ and R ⁷ are each independently H, or an amine-protective group. [0015] In another aspect, the present disclosure includes a method of preparing a compound of Formula I of the present disclosure comprising ^ reacting a compound of Formula IIIa - 5 - 8660875

IIIa or a tautomer, derivative, or salt thereof, wherein L1 and L2 are each independently (CH2)2-6 or -CH2CH2OCH2CH2-; n is an integer of 2 to 6; m is an integer of 1 to 6; and R ¹ is H or an acid-labile protective group; with a compound of Formula IV IV, or a tautomer, derivative, or salt thereof, wherein Z, each R ² , and R ³ are each independently as defined herein, to obtain a compound of Formula V V or a tautomer, derivative, or salt thereof; and - 6 - 8660875

^ reacting the compound of Formula V or a tautomer, derivative, or salt thereof, with a phosphoramadite forming reagent, optionally 2-cyanoethyl- N,N-diisopropylchlorophosphoramidite or 2-cyanoethyl N,N,N,N- tetraisopropylphosphoramidite. [0016] In another aspect, the present disclosure includes a method of preparing a compound of Formula II of the present disclosure comprising ^ reacting a compound of Formula III of the present disclosure with the small molecule A comprising at least one COOH under conditions to form an amide bond between the small molecule A and the compound of Formula III to obtain a compound of Formula VI VI or a tautomer, derivative, or salt thereof, wherein L1 and L2 are each independently (CH2)2-6 or - CH2CH2OCH2CH2-; n is an integer of 2 to 6; m is an integer of 1 to 6; and R ¹ is H or an acid-labile protective group; and ^ reacting the compound of Formula IV with a phosphoramidite forming reagent, optionally 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite or 2-cyanoethyl N,N,N,N-tetraisopropylphosphane. [0017] In another aspect, the present disclosure includes a method of preparing an oligonucleotide comprising a folic acid nucleotide analog, the method comprising - 7 - 8660875

reacting a folic acid phosphoramidite of the present disclosure with a nucleotide having a free 3’ hydroxyl group or an oligonucleotide chain having a free 3’ hydroxyl group. [0018] In another aspect, the present disclosure includes a method of preparing an oligonucleotide comprising a small molecule nucleotide analog, the method comprising reacting a small molecule phosphoramidite of the present disclosure with a nucleotide having a free 3’ hydroxyl group or an oligonucleotide chain having a free 3’ hydroxyl group. [0019] In another aspect, the present disclosure includes an oligonucleotide prepared by a method of the present disclosure. [0020] In another aspect, the present disclosure includes an oligonucleotide comprising a folic acid nucleotide analog having the following structure * H * or a tautomer, derivative, or salt thereof, wherein Z is O or NH; L1 and L2 are each independently (CH2)2-6 or -CH2CH2OCH2CH2-; n is an integer of 2 to 6; and m is an integer of 1 to 6; and wherein one of the * is a point of attachment to an adjacent nucleotide and the remaining * is a terminus of the oligonucleotide, or wherein each * is a point of attachment to a separate adjacent nucleotide. - 8 - 8660875

[0021] In another aspect, the present disclosure includes an oligonucleotide comprising a small molecule nucleotide analog having the following structure or a tautomer, derivative, or salt thereof, wherein A is a small molecule as defined herein; L1 and L2 are each independently (CH2)2-6 or -CH2CH2OCH2CH2-; n is an integer of 2 to 6; and m is an integer of 1 to 6; and wherein at least one of the * is a point of attachment to an adjacent nucleotide and the remaining * if present is a terminus of the oligonucleotide. [0022] In another aspect, the present disclosure includes a method of treating or preventing cancer comprising administering an oligonucleotide comprising a folic acid nucleotide analogue of the present disclosure to a subject in need thereof. [0023] In another aspect, the present disclosure includes a use of an oligonucleotide comprising a folic acid nucleotide analogue of the present disclosure for treating or preventing cancer. [0024] In another aspect, the present disclosure includes a use of an oligonucleotide comprising a folic acid nucleotide analogue of the present disclosure in the preparation of a medication for treating or preventing cancer. [0025] In another aspect, the present disclosure includes an oligonucleotide comprising a folic acid nucleotide analogue of the present disclosure for use in the treatment or prevention of cancer. - 9 - 8660875

[0026] In another aspect, the present disclosure includes a kit for oligonucleotide synthesis comprising a folic acid phosphoramidite derivative of the present disclosure in a container. [0027] In another aspect, the present disclosure includes a kit for oligonucleotide synthesis comprising a small molecule phosphoramidite derivative of the present disclosure in a container. [0028] In another aspect, the present disclosure includes a use of a compound of Formula I of the present disclosure in the synthesis of an oligonucleotide. [0029] In another aspect, the present disclosure includes a use of a compound of Formula II of the present disclosure in the synthesis of an oligonucleotide. [0030] In another aspect, the present disclosure includes a use of a compound of Formula III of the present disclosure in the synthesis of a phosphoramidite derivative. [0031] Other features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the disclosure are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS: [0032] Figure 1 is a structure of 5′-folate-TEG cyanoethyl phosphoramidite (BA 0349) previously offered by Berry & Associates. [0033] Figure 2 shows a circular dichroism spectra of anti-luciferase folate siRNAs. [0034] Figure 3 is a graph showing relative expression of firefly luciferase in HeLa cells 24 hours after siRNA transfection using Lipofectamine 2000 ^TM . Firefly luciferase expression was normalized to Renilla luciferase. [0035] Figure 4 is a graph showing relative expression of firefly luciferase in HeLa cells 16 hours after carrier-free siRNA transfection. Firefly luciferase expression was normalized to Renilla luciferase. - 10 - 8660875

[0036] Figure 5 is a graph showing relative expression of firefly luciferase in HT- 29 cells 24 hours after siRNA transfection using Lipofectamine LTX. Firefly luciferase expression was normalized to Renilla luciferase. [0037] Figure 6 is a graph showing relative expression of firefly luciferase in HeLa cells 16 hours after carrier-free siRNA transfection. Firefly luciferase expression was normalized to Renilla luciferase. [0038] Figure 7 is a graph showing relative viability of HeLa cells after treatment with wild-type (wt) and folate siRNAs (25-3000 nM). DETAILED DESCRIPTION OF THE DISCLOSURE: [0039] The following is a detailed description provided to aid those skilled in the art in practicing the present disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting of the disclosure. All publications, patent applications, patents, figures and other references mentioned herein are expressly incorporated by reference in their entirety. I. Definitions [0040] Unless otherwise indicated, the definitions and embodiments described in this and other sections are intended to be applicable to all embodiments and aspects of the application herein described for which they are suitable as would be understood by a person skilled in the art. Unless otherwise specified within this application or unless a person skilled in the art would understand otherwise, the nomenclature used in this application generally follows the examples and rules stated in “Nomenclature of Organic Chemistry” (Pergamon Press, 1979), Sections A, B, C, D, E, F, and H. Optionally, a name of a compound may be generated using a chemical naming program: ACD/ChemSketch, Version 5.09/September 2001, Advanced Chemistry Development, Inc., Toronto, Canada. [0041] The term “compound of the disclosure” or “compound of the present disclosure” and the like as used herein refers to a phosphoramidite derivative of the disclosure or an oligonucleotide of the disclosure, or a compound of Formula III, IV, or V as described herein. - 11 - 8660875

[0042] The term “phosphoramidite derivative of the disclosure” or “phosphoramidite derivative of the present disclosure” and the like as used herein refers to a compound of Formula I or II, and pharmaceutically acceptable salts, solvates and/or prodrugs thereof. [0043] The term “oligonucleotide of the disclosure or “oligonucleotide of the present disclosure” and the like as used herein refers to an oligonucleotide comprising a small molecule nucleotide analog or a folic acid nucleotide analog, and pharmaceutically acceptable salts, solvates and/or prodrugs thereof. [0044] The term “composition of the disclosure” or “composition of the present disclosure” and the like as used herein refers to a composition comprising one or more phosphoramidite derivatives or oligonucleotides of the application and at least one additional ingredient. [0045] As used in the present application, the singular forms “a”, “an” and “the” include plural references unless the content clearly dictates otherwise. For example, an embodiment including “a compound” should be understood to present certain aspects with one compound, or two or more additional compounds. [0046] In embodiments comprising an “additional” or “second” component, such as an additional or second compound, the second component as used herein is chemically different from the other components or first component. A “third” component is different from the other, first, and second components, and further enumerated or “additional” components are similarly different. [0047] In understanding the scope of the present application, the term “comprising” and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and/or steps. The foregoing also applies to words having similar meanings such as the terms, “including”, “having” and their derivatives. [0048] The term “consisting” and its derivatives, as used herein, are intended to be closed terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but exclude the presence of other unstated features, elements, components, groups, integers and/or steps. - 12 - 8660875

[0049] The term “consisting essentially of”, as used herein, is intended to specify the presence of the stated features, elements, components, groups, integers, and/or steps as well as those that do not materially affect the basic and novel characteristic(s) of features, elements, components, groups, integers, and/or steps. [0050] The term “suitable” as used herein means that the selection of the particular compound or conditions would depend on the specific synthetic manipulation to be performed, and the identity of the species to be transformed, but the selection would be well within the skill of a person trained in the art. All method steps described herein are to be conducted under conditions sufficient to provide the desired product. A person skilled in the art would understand that all reaction conditions, including, for example, reaction solvent, reaction time, reaction temperature, reaction pressure, reactant ratio and whether or not the reaction should be performed under an anhydrous or inert atmosphere, can be varied to optimize the yield of the desired product and it is within their skill to do so. [0051] In embodiments of the present application, the compounds described herein may have at least one asymmetric center. Where compounds possess more than one asymmetric center, they may exist as diastereomers. It is to be understood that all such isomers and mixtures thereof in any proportion are encompassed within the scope of the present application. It is to be further understood that while the stereochemistry of the compounds may be as shown in any given compound listed herein, such compounds may also contain certain amounts (for example, less than 20%, suitably less than 10%, more suitably less than 5%) of compounds of the present application having alternate stereochemistry. It is intended that any optical isomers, as separated, pure or partially purified optical isomers or racemic mixtures thereof are included within the scope of the present application. [0052] The compounds of the present application may also exist in different tautomeric forms and it is intended that any tautomeric forms which the compounds form are included within the scope of the present application. [0053] The compounds of the present application may further exist in varying polymorphic forms and it is contemplated that any polymorphs which form are included within the scope of the present application. [0054] Terms of degree such as “substantially”, “about” and “approximately” as used herein mean a reasonable amount of deviation of the modified term such that the - 13 - 8660875

end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ^5% of the modified term if this deviation would not negate the meaning of the word it modifies or unless the context suggests otherwise to a person skilled in the art. [0055] The term “alkyl” as used herein, whether it is used alone or as part of another group, means straight or branched chain, saturated alkyl groups. The number of carbon atoms that are possible in the referenced alkyl group are indicated by the prefix “Cn1-n2”. For example, the term C1-6alkyl means an alkyl group having 1, 2, 3, 4, 5 or 6 carbon atoms. All alkyl groups are optionally fluorosubstituted unless otherwise stated. [0056] All cyclic groups, including aryl and cyclo groups, contain one or more than one ring (i.e. are polycyclic). When a cyclic group contains more than one ring, the rings may be fused, bridged, spirofused or linked by a bond. [0057] A first ring being “fused” with a second ring means the first ring and the second ring share two adjacent atoms there between. [0058] A first ring being “bridged” with a second ring means the first ring and the second ring share two non-adjacent atoms there between. [0059] A first ring being “spirofused” with a second ring means the first ring and the second ring share one atom there between. [0060] The term “halo” as used herein refers to a halogen atom and includes fluoro, chloro, bromo and iodo. [0061] The term “optionally substituted” refers to groups, structures, or molecules that are either unsubstituted or are substituted with one or more substituents. [0062] The term “protecting group” or “PG” and the like as used herein refers to a chemical moiety which protects or masks a reactive portion of a molecule to prevent side reactions in those reactive portions of the molecule, while manipulating or reacting a different portion of the molecule. After the manipulation or reaction is complete, the protecting group is removed under conditions that do not degrade or decompose the remaining portions of the molecule. The selection of a suitable protecting group can be made by a person skilled in the art. Many conventional protecting groups are known in the art, for example as described in “Protective Groups in Organic Chemistry” McOmie, J.F.W. Ed., Plenum Press, 1973, in Greene, T.W. and Wuts, P.G.M., “Protective Groups - 14 - 8660875

in Organic Synthesis”, John Wiley & Sons, 3 ^rd Edition, 1999 and in Kocienski, P. Protecting Groups, ^3r d Edition, 2003, Georg Thieme Verlag (The Americas). Examples of suitable protective groups include, but are not limited to t-Boc, Ac, Ts, Ms, silyl ethers such as TMS, TBDMS, TBDPS, Tf, Ns, benzyl (Bn), Fmoc, benzoyl, dimethoxytrityl, methoxyethoxymethyl ether, methoxymethyl ether, pivaloyl, p-methyoxybenzyl ether, tetrahydropyranyl, trityl, ethoxyethyl ethers, carbobenzyloxy, benzoyl and the like. For example, the term “acid labile protective group” as used herein refers to a protective group that can be deprotected under suitable acidic conditions and is stable under basic and/or neutral conditions. It can be appreciated that some protective groups are more suitable for or are commonly used in the field with amines, or preferentially protect amines. They are referred to as “amine-protective groups”. Examples of amine protective groups include, but are not limited to t-Boc, Fmoc, Cbz, and benzyl. [0063] The term “inert organic solvent” as used herein refers to a solvent that is generally considered as non-reactive with the functional groups that are present in the compounds to be combined together in any given reaction so that it does not interfere with or inhibit the desired synthetic transformation. Organic solvents are typically non- polar and dissolve compounds that are non soluble in aqueous solutions. [0064] The term “cell” as used herein refers to a single cell or a plurality of cells and includes a cell either in a cell culture or in a subject. [0065] The term “subject” as used herein includes all members of the animal kingdom including mammals, and suitably refers to humans. Thus, the methods and uses of the present application are applicable to both human therapy and veterinary applications. [0066] The term “pharmaceutically acceptable” means compatible with the treatment of subjects, for example humans. [0067] The term “pharmaceutically acceptable carrier” means a non-toxic solvent, dispersant, excipient, adjuvant or other material which is mixed with the active ingredient in order to permit the formation of a pharmaceutical composition, i.e., a dosage form capable of administration to a subject. [0068] The term “pharmaceutically acceptable salt” means either an acid addition salt or a base addition salt which is suitable for, or compatible with the treatment of subjects. - 15 - 8660875

[0069] An acid addition salt suitable for, or compatible with, the treatment of subjects is any non-toxic organic or inorganic acid addition salt of any basic compound. Basic compounds that form an acid addition salt include, for example, compounds comprising an amine group. Illustrative inorganic acids which form suitable salts include hydrochloric, hydrobromic, sulfuric, nitric and phosphoric acids, as well as acidic metal salts such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate. Illustrative organic acids which form suitable salts include mono-, di- and tricarboxylic acids. Illustrative of such organic acids are, for example, acetic, trifluoroacetic, propionic, glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, hydroxymaleic, benzoic, hydroxybenzoic, phenylacetic, cinnamic, mandelic, salicylic, 2-phenoxybenzoic, p-toluenesulfonic acid and other sulfonic acids such as methanesulfonic acid, ethanesulfonic acid and 2-hydroxyethanesulfonic acid. Either the mono- or di-acid salts can be formed, and such salts can exist in either a hydrated, solvated or substantially anhydrous form. In general, acid addition salts are more soluble in water and various hydrophilic organic solvents, and generally demonstrate higher melting points in comparison to their free base forms. The selection criteria for the appropriate salt will be known to one skilled in the art. Other non-pharmaceutically acceptable salts such as but not limited to oxalates may be used, for example in the isolation of compounds of the application for laboratory use, or for subsequent conversion to a pharmaceutically acceptable acid addition salt. [0070] A base addition salt suitable for, or compatible with, the treatment of subjects is any non-toxic organic or inorganic base addition salt of any acidic compound. Acidic compounds that form a basic addition salt include, for example, compounds comprising a carboxylic acid group. Illustrative inorganic bases which form suitable salts include lithium, sodium, potassium, calcium, magnesium or barium hydroxide as well as ammonia. Illustrative organic bases which form suitable salts include aliphatic, alicyclic or aromatic organic amines such as isopropylamine, methylamine, trimethylamine, picoline, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2- diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, EGFRaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins, and the like. Exemplary organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. [See, for example, S. M. Berge, - 16 - 8660875

et aI., "Pharmaceutical Salts," J. Pharm. Sci. 1977, 66, 1-19]. The selection of the appropriate salt may be useful so that an ester functionality, if any, elsewhere in a compound is not hydrolyzed. The selection criteria for the appropriate salt will be known to one skilled in the art. [0071] Prodrugs of the compounds of the present application may be, for example, conventional esters formed with available hydroxy, thiol, amino or carboxyl groups. Some common esters which have been utilized as prodrugs are phenyl esters, aliphatic (C1-C24) esters, acyloxymethyl esters, carbamates and amino acid esters. [0072] The term “solvate” as used herein means a compound, or a salt or prodrug of a compound, wherein molecules of a suitable solvent are incorporated in the crystal lattice. A suitable solvent is physiologically tolerable at the dosage administered. Examples of suitable solvents are ethanol, water and the like. When water is the solvent, the molecule is referred to as a “hydrate”. [0073] The term “treating” or “treatment” as used herein and as is well understood in the art, means an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, and remission (whether partial or total), whether detectable or undetectable. “Treating” and “treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Treatment methods comprise administering to a subject a therapeutically effective amount of one or more of the compounds of the application and optionally consist of a single administration, or alternatively comprise a series of administrations. For example, in some embodiments, the compounds of the application may be administered at least once a week. In some embodiments, the compounds may be administered to the subject from about one time per three weeks, or about one time per week to about once daily for a given treatment. In another embodiment, the compounds are administered 2, 3, 4, 5 or 6 times daily. The length of the treatment period depends on a variety of factors, such as the severity of the disease, disorder or condition, the age of the subject, the concentration and/or the activity of the compounds of the application, and/or a combination thereof. It will also be - 17 - 8660875

appreciated that the effective dosage of the compound used for the treatment may increase or decrease over the course of a particular treatment regime. Changes in dosage may result and become apparent by standard diagnostic assays known in the art. In some instances, chronic administration may be required. For example, the compounds are administered to the subject in an amount and for duration sufficient to treat the patient. [0074] The term “preventing” or “prevention” as used herein includes prophylactic treatment. For example, a subject with early cancer can be administered a compound or composition of the application to prevent progression, or alternatively a subject in remission can be administered a compound or composition of the application to prevent recurrence. [0075] “Palliating” a disease or disorder means that the extent and/or undesirable clinical manifestations of a disorder or a disease state are lessened and/or time course of the progression is slowed or lengthened, as compared to not treating the disorder. [0076] As used herein, the term “effective amount” or “therapeutically effective amount” means an amount of a compound, or one or more compounds, of the application that is effective, at dosages and for periods of time necessary to achieve the desired result. For example, in the context of gene silencing or RNAi, an effective amount is an amount that, for example, decreases gene activity of the gene to be silenced, compared to the gene activity without administration of the one or more compounds. Effective amounts may vary according to factors such as the disease state, age, sex and/or weight of the subject. The amount of a given compound that will correspond to such an amount will vary depending upon various factors, such as the given drug or compound, the pharmaceutical formulation, the route of administration, the type of condition, disease or disorder, the identity of the subject being treated, and the like, but can nevertheless be routinely determined by one skilled in the art. The effective amount is one that following treatment therewith manifests as an improvement in or reduction of any disease symptom. When the disease is cancer, amounts that are effective can cause a reduction in the number, growth rate, size and/or distribution of tumours. [0077] The term “administered” as used herein means administration of a therapeutically effective amount of a compound, or one or more compounds, or a composition of the application to a cell either in cell culture or in a subject. - 18 - 8660875

[0078] The term “neoplastic disorder” as used herein refers to a disease, disorder or condition characterized by cells that have the capacity for autonomous growth or replication, e.g., an abnormal state or condition characterized by proliferative cell growth. The term “neoplasm” as used herein refers to a mass of tissue resulting from the abnormal growth and/or division of cells in a subject having a neoplastic disorder. Neoplasms can be benign (such as uterine fibroids and melanocytic nevi), potentially malignant (such as carcinoma in situ) or malignant (i.e. cancer). Exemplary neoplastic disorders include but are not limited to carcinoma, sarcoma, metastatic disorders (e.g., tumors arising from the prostate), hematopoietic neoplastic disorders, (e.g., leukemias, lymphomas, myeloma and other malignant plasma cell disorders), metastatic tumors and other cancers. Prevalent cancers include breast, prostate, colon, lung, liver, brain, ovarian and pancreatic cancers. [0079] The term “cancer” as used herein refers to cellular-proliferative disease states, including but not limited to: Acute Lymphoblastic Leukemia, Adult; Acute Lymphoblastic Leukemia, Childhood; Acute Myeloid Leukemia, Adult; Adrenocortical Carcinoma; Adrenocortical Carcinoma, Childhood; AIDS-Related Lymphoma; AIDS- Related Malignancies; Anal Cancer; Astrocytoma, Childhood Cerebellar; Astrocytoma, Childhood Cerebral; Bile Duct Cancer, Extrahepatic; Bladder Cancer; Bladder Cancer, Childhood; Bone Cancer, Osteosarcoma/Malignant Fibrous Histiocytoma; Brain Stem Glioma, Childhood; Brain Tumor, Adult; Brain Tumor, Brain Stem Glioma, Childhood; Brain Tumor, Cerebellar Astrocytoma, Childhood; Brain Tumor, Cerebral Astrocytoma/Malignant Glioma, Childhood; Brain Tumor, Ependymoma, Childhood; Brain Tumor, Medulloblastoma, Childhood; Brain Tumor, Supratentorial Primitive Neuroectodermal Tumors, Childhood; Brain Tumor, Visual Pathway and Hypothalamic Glioma, Childhood; Brain Tumor, Childhood (Other); Breast Cancer; Breast Cancer and Pregnancy; Breast Cancer, Childhood; Breast Cancer, Male; Bronchial Adenomas/Carcinoids, Childhood; Carcinoid Tumor, Childhood; Carcinoid Tumor, Gastrointestinal; Carcinoma, Adrenocortical; Carcinoma, Islet Cell; Carcinoma of Unknown Primary; Central Nervous System Lymphoma, Primary; Cerebellar Astrocytoma, Childhood; Cerebral Astrocytoma/Malignant Glioma, Childhood; Cervical Cancer; Childhood Cancers; Chronic Lymphocytic Leukemia; Chronic Myelogenous Leukemia; Chronic Myeloproliferative Disorders; Clear Cell Sarcoma of Tendon Sheaths; Colon Cancer; Colorectal Cancer, Childhood; Cutaneous T-CeIl Lymphoma; Endometrial - 19 - 8660875

Cancer; Ependymoma, Childhood; Epithelial Cancer, Ovarian; Esophageal Cancer; Esophageal Cancer, Childhood; Ewing's Family of Tumors; Extracranial Germ Cell Tumor, Childhood; Extragonadal Germ Cell Tumor; Extrahepatic Bile Duct Cancer; Eye Cancer, Intraocular Melanoma; Eye Cancer, Retinoblastoma; Gallbladder Cancer; Gastric (Stomach) Cancer; Gastric (Stomach) Cancer, Childhood; Gastrointestinal Carcinoid Tumor; Germ Cell Tumor, Extracranial, Childhood; Germ Cell Tumor, Extragonadal; Germ Cell Tumor, Ovarian; Gestational Trophoblastic Tumor; Glioma, Childhood Brain Stem; Glioma, Childhood Visual Pathway and Hypothalamic; Hairy Cell Leukemia; Head and Neck Cancer; Hepatocellular (Liver) Cancer, Adult (Primary); Hepatocellular (Liver) Cancer, Childhood (Primary); Hodgkin's Lymphoma, Adult; Hodgkin's Lymphoma, Childhood; Hodgkin's Lymphoma During Pregnancy; Hypopharyngeal Cancer; Hypothalamic and Visual Pathway Glioma, Childhood; Intraocular Melanoma; Islet Cell Carcinoma (Endocrine Pancreas); Kaposi's Sarcoma; Kidney Cancer; Laryngeal Cancer; Laryngeal Cancer, Childhood; Leukemia, Acute Lymphoblastic, Adult; Leukemia, Acute Lymphoblastic, Childhood; Leukemia, Acute Myeloid, Adult; Leukemia, Acute Myeloid, Childhood; Leukemia, Chronic Lymphocytic; Leukemia, Chronic Myelogenous; Leukemia, Hairy Cell; Lip and Oral Cavity Cancer; Liver Cancer, Adult (Primary); Liver Cancer, Childhood (Primary); Lung Cancer, Non-Small Cell; Lung Cancer, Small Cell; Lymphoblastic Leukemia, Adult Acute; Lymphoblastic Leukemia, Childhood Acute; Lymphocytic Leukemia, Chronic; Lymphoma, AIDS-Related; Lymphoma, Central Nervous System (Primary); Lymphoma, Cutaneous T-CeIl; Lymphoma, Hodgkin's, Adult; Lymphoma, Hodgkin's, Childhood; Lymphoma, Hodgkin's During Pregnancy; Lymphoma, Non-Hodgkin's, Adult; Lymphoma, Non-Hodgkin's, Childhood; Lymphoma, Non- Hodgkin's During Pregnancy; Lymphoma, Primary Central Nervous System; Macroglobulinemia, Waldenstrom's; Male Breast Cancer; Malignant Mesothelioma, Adult; Malignant Mesothelioma, Childhood; Malignant Thymoma; Medulloblastoma, Childhood; Melanoma; Melanoma, Intraocular; Merkel Cell Carcinoma; Mesothelioma, Malignant; Metastatic Squamous Neck Cancer with Occult Primary; Multiple Endocrine Neoplasia Syndrome, Childhood; Multiple Myeloma/Plasma Cell Neoplasm; Mycosis Fungoides; Myelodysplastic Syndromes; Myelogenous Leukemia, Chronic; Myeloid Leukemia, Childhood Acute; Myeloma, Multiple; Myeloproliferative Disorders, Chronic; Nasal Cavity and Paranasal Sinus Cancer; Nasopharyngeal Cancer; Nasopharyngeal Cancer, Childhood; Neuroblastoma; Non-Hodgkin's Lymphoma, Adult; Non-Hodgkin's - 20 - 8660875

Lymphoma, Childhood; Non- Hodgkin's Lymphoma During Pregnancy; Non-Small Cell Lung Cancer; Oral Cancer, Childhood; Oral Cavity and Lip Cancer; Oropharyngeal Cancer; Osteosarcoma/Malignant Fibrous Histiocytoma of Bone; Ovarian Cancer, Childhood; Ovarian Epithelial Cancer; Ovarian Germ Cell Tumor; Ovarian Low Malignant Potential Tumor; Pancreatic Cancer; Pancreatic Cancer, Childhood; Pancreatic Cancer, Islet Cell; Paranasal Sinus and Nasal Cavity Cancer; Parathyroid Cancer; Penile Cancer; Pheochromocytoma; Pineal and Supratentorial Primitive Neuroectodermal Tumors, Childhood; Pituitary Tumor; Plasma Cell Neoplasm/Multiple Myeloma; Pleuropulmonary Blastoma; Pregnancy and Breast Cancer; Pregnancy and Hodgkin's Lymphoma; Pregnancy and Non-Hodgkin's Lymphoma; Primary Central Nervous System Lymphoma; Primary Liver Cancer, Adult; Primary Liver Cancer, Childhood; Prostate Cancer; Rectal Cancer; Renal Cell (Kidney) Cancer; Renal Cell Cancer, Childhood; Renal Pelvis and Ureter, Transitional Cell Cancer; Retinoblastoma; Rhabdomyosarcoma, Childhood; Salivary Gland Cancer; Salivary Gland Cancer, Childhood; Sarcoma, Ewing's Family of Tumors; Sarcoma, Kaposi's; Sarcoma (Osteosarcoma)/Malignant Fibrous Histiocytoma of Bone; Sarcoma, Rhabdomyosarcoma, Childhood; Sarcoma, Soft Tissue, Adult; Sarcoma, Soft Tissue, Childhood; Sezary Syndrome; Skin Cancer; Skin Cancer, Childhood; Skin Cancer (Melanoma); Skin Carcinoma, Merkel Cell; Small Cell Lung Cancer; Small Intestine Cancer; Soft Tissue Sarcoma, Adult; Soft Tissue Sarcoma, Childhood; Squamous Neck Cancer with Occult Primary, Metastatic; Stomach (Gastric) Cancer; Stomach (Gastric) Cancer, Childhood; Supratentorial Primitive Neuroectodermal Tumors, Childhood; T- CeIl Lymphoma, Cutaneous; Testicular Cancer; Thymoma, Childhood; Thymoma, Malignant; Thyroid Cancer; Thyroid Cancer, Childhood; Transitional Cell Cancer of the Renal Pelvis and Ureter; Trophoblastic Tumor, Gestational; Unknown Primary Site, Cancer of, Childhood; Unusual Cancers of Childhood; Ureter and Renal Pelvis, Transitional Cell Cancer; Urethral Cancer; Uterine Sarcoma; Vaginal Cancer; Visual Pathway and Hypothalamic Glioma, Childhood; Vulvar Cancer; Waldenstrom's Macro globulinemia; and Wilms' Tumor. Metastases of the aforementioned cancers can also be treated in accordance with the methods described herein. [0080] The compounds of the present application are suitably formulated in a conventional manner into compositions using one or more solvents or carriers. Accordingly, the present application also includes a composition comprising one or more - 21 - 8660875

compounds of the application and a carrier. The present disclosure also includes a composition comprising one or more compounds of the disclosure and a solvent. The compounds of the application are suitably formulated into pharmaceutical compositions for administration to subjects in a biologically compatible form suitable for administration in vivo. Accordingly, the present application further includes a pharmaceutical composition comprising one or more compounds of the application and a pharmaceutically acceptable carrier. [0081] The compounds of the application may be administered to a subject in a variety of forms depending on the selected route of administration, as will be understood by those skilled in the art. A compound of the application may be administered, for example, by oral, parenteral, buccal, sublingual, nasal, rectal, patch, pump or transdermal administration and the pharmaceutical compositions formulated accordingly. Administration can be by means of a pump for periodic or continuous delivery. [0082] Parenteral administration includes intravenous, intra-arterial, intraperitoneal, subcutaneous, intramuscular, transepithelial, nasal, intrapulmonary (for example, by use of an aerosol), intrathecal, rectal and topical (including the use of a patch or other transdermal delivery device) modes of administration. Parenteral administration may be by continuous infusion over a selected period of time. Conventional procedures and ingredients for the selection and preparation of suitable compositions are described, for example, in Remington’s Pharmaceutical Sciences (2000 - 2 ^0t h edition) and in The United States Pharmacopeia: The National Formulary (USP 24 NF19) published in 1999. [0083] A compound of the application may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsules, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the compound may be incorporated with excipient and used in the form of ingestible tablets, buccal tablets, troches, capsules, caplets, pellets, granules, lozenges, chewing gum, powders, syrups, elixirs, wafers, aqueous solutions and suspensions, and the like. In the case of tablets, carriers that are used include lactose, corn starch, sodium citrate and salts of phosphoric acid. Pharmaceutically acceptable excipients include binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium phosphate); lubricants (e.g., magnesium stearate, - 22 - 8660875

talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by methods well known in the art. In the case of tablets, capsules, caplets, pellets or granules for oral administration, pH sensitive enteric coatings, such as Eudragits™ designed to control the release of active ingredients are optionally used. Oral dosage forms also include modified release, for example immediate release and timed-release, formulations. Examples of modified-release formulations include, for example, sustained-release (SR), extended- release (ER, XR, or XL), time-release or timed-release, controlled-release (CR), or continuous-release (CR or Contin), employed, for example, in the form of a coated tablet, an osmotic delivery device, a coated capsule, a microencapsulated microsphere, an agglomerated particle, e.g., as of molecular sieving type particles, or, a fine hollow permeable fiber bundle, or chopped hollow permeable fibers, agglomerated or held in a fibrous packet. Timed-release compositions can be formulated, e.g. liposomes or those wherein the active compound is protected with differentially degradable coatings, such as by microencapsulation, multiple coatings, etc. Liposome delivery systems include, for example, small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines. For oral administration in a capsule form, useful carriers or diluents include lactose and dried corn starch. [0084] Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they are suitably presented as a dry product for constitution with water or other suitable vehicle before use. When aqueous suspensions and/or emulsions are administered orally, the compound of the application is suitably suspended or dissolved in an oily phase that is combined with emulsifying and/or suspending agents. If desired, certain sweetening and/or flavoring and/or coloring agents may be added. Such liquid preparations for oral administration may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methyl cellulose or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters or ethyl alcohol); and preservatives (e.g., methyl or propyl p-hydroxybenzoates or sorbic acid). Useful diluents include lactose and high molecular weight polyethylene glycols. [0085] It is also possible to freeze-dry the compounds of the application and use the lyophilizates obtained, for example, for the preparation of products for injection. - 23 - 8660875

[0086] A compound of the application may also be administered parenterally. Solutions of a compound of the application can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, DMSO and mixtures thereof with or without alcohol, and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. A person skilled in the art would know how to prepare suitable formulations. For parenteral administration, sterile solutions of the compounds of the application are usually prepared, and the pH of the solutions are suitably adjusted and buffered. For intravenous use, the total concentration of solutes should be controlled to render the preparation isotonic. For ocular administration, ointments or droppable liquids may be delivered by ocular delivery systems known to the art such as applicators or eye droppers. Such compositions can include mucomimetics such as hyaluronic acid, chondroitin sulfate, hydroxypropyl methylcellulose or polyvinyl alcohol, preservatives such as sorbic acid, EDTA or benzyl chromium chloride, and the usual quantities of diluents or carriers. For pulmonary administration, diluents or carriers will be selected to be appropriate to allow the formation of an aerosol. [0087] The compounds of the application may be formulated for parenteral administration by injection, including using conventional catheterization techniques or infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as sterile suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulating agents such as suspending, stabilizing and/or dispersing agents. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. Alternatively, the compounds of the application are suitably in a sterile powder form for reconstitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. [0088] Compositions for nasal administration may conveniently be formulated as aerosols, drops, gels and powders. [0089] For intranasal administration or administration by inhalation, the compounds of the application are conveniently delivered in the form of a solution, dry powder formulation or suspension from a pump spray container that is squeezed or pumped by the patient or as an aerosol spray presentation from a pressurized container or a - 24 - 8660875

nebulizer. Aerosol formulations typically comprise a solution or fine suspension of the active substance in a physiologically acceptable aqueous or non-aqueous solvent and are usually presented in single or multidose quantities in sterile form in a sealed container, which can take the form of a cartridge or refill for use with an atomising device. Alternatively, the sealed container may be a unitary dispensing device such as a single dose nasal inhaler or an aerosol dispenser fitted with a metering valve which is intended for disposal after use. Where the dosage form comprises an aerosol dispenser, it will contain a propellant which can be a compressed gas such as compressed air or an organic propellant such as fluorochlorohydrocarbon. Suitable propellants include but are not limited to dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, heptafluoroalkanes, carbon dioxide or another suitable gas. In the case of a pressurized aerosol, the dosage unit is suitably determined by providing a valve to deliver a metered amount. The pressurized container or nebulizer may contain a solution or suspension of the active compound. Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated containing a powder mix of a compound of the application and a suitable powder base such as lactose or starch. The aerosol dosage forms can also take the form of a pump-atomizer. [0090] Compositions suitable for buccal or sublingual administration include tablets, lozenges, and pastilles, wherein the active ingredient is formulated with a carrier such as sugar, acacia, tragacanth, or gelatin and glycerine. Compositions for rectal administration are conveniently in the form of suppositories containing a conventional suppository base such as cocoa butter. [0091] Suppository forms of the compounds of the application are useful for vaginal, urethral and rectal administrations. Such suppositories will generally be constructed of a mixture of substances that is solid at room temperature but melts at body temperature. The substances commonly used to create such vehicles include but are not limited to theobroma oil (also known as cocoa butter), glycerinated gelatin, other glycerides, hydrogenated vegetable oils, mixtures of polyethylene glycols of various molecular weights and fatty acid esters of polyethylene glycol. See, for example: Remington's Pharmaceutical Sciences, 1 ^6t h Ed., Mack Publishing, Easton, PA, 1980, pp. 1530-1533 for further discussion of suppository dosage forms. - 25 - 8660875

[0092] Compounds of the application may also be coupled with soluble polymers as targetable drug carriers. Such polymers can include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropylmethacrylamide-phenol, polyhydroxy-ethylaspartamide- phenol, or polyethyleneoxide-polylysine substituted with palmitoyl residues. Furthermore, compounds of the application may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and crosslinked or amphipathic block copolymers of hydrogels. [0093] The compounds of the application including pharmaceutically acceptable salts, solvates and prodrugs thereof are suitably used on their own but will generally be administered in the form of a pharmaceutical composition in which the one or more compounds of the application (the active ingredient) is in association with a pharmaceutically acceptable carrier. Depending on the mode of administration, the pharmaceutical composition will comprise from about 0.05 wt% to about 99 wt% or about 0.10 wt% to about 70 wt%, of the active ingredient (one or more compounds of the application), and from about 1 wt% to about 99.95 wt% or about 30 wt% to about 99.90 wt% of a pharmaceutically acceptable carrier, all percentages by weight being based on the total composition. [0094] Compounds of the application may be used alone or in combination with other known agents useful for gene silencing or for treating disease, disorder or conditions treatable with gene silencing or RNAi technology. The compounds of the disclosure can also be used alone or in combination with other anti-cancer therapy. When used in combination with other agents, it is an embodiment that the compounds of the application are administered contemporaneously with those agents. As used herein, “contemporaneous administration” of two substances to a subject means providing each of the two substances so that they are both biologically active in the individual at the same time. The exact details of the administration will depend on the pharmacokinetics of the two substances in the presence of each other, and can include administering the two substances within a few hours of each other, or even administering one substance within 24 hours of administration of the other, if the pharmacokinetics are suitable. Design of suitable dosing regimens is routine for one skilled in the art. In particular embodiments, two substances will be administered substantially simultaneously, i.e., within minutes of - 26 - 8660875

each other, or in a single composition that contains both substances. It is a further embodiment of the present application that a combination of agents is administered to a subject in a non-contemporaneous fashion. In an embodiment, a compound of the present application is administered with another therapeutic agent simultaneously or sequentially in separate unit dosage forms or together in a single unit dosage form. Accordingly, the present application provides a single unit dosage form comprising one or more compounds of the application (e.g. a compound of Formula I), an additional therapeutic agent, and a pharmaceutically acceptable carrier. [0095] The dosage of compounds of the application can vary depending on many factors such as the pharmacodynamic properties of the compound, the mode of administration, the age, health and weight of the recipient, the nature and extent of the symptoms, the frequency of the treatment and the type of concurrent treatment, if any, and the clearance rate of the compound in the subject to be treated. One of skill in the art can determine the appropriate dosage based on the above factors. Compounds of the application may be administered initially in a suitable dosage that may be adjusted as required, depending on the clinical response. Dosages will generally be selected to maintain a serum level of compounds of the application from about 0.01 µg/cc to about 1000 µg/cc, or about 0.1 µg/cc to about 100 µg/cc. As a representative example, oral dosages of one or more compounds of the application will range between about 1 mg per day to about 1000 mg per day for an adult, suitably about 1 mg per day to about 500 mg per day, more suitably about 1 mg per day to about 200 mg per day. For parenteral administration, a representative amount is from about 0.001 mg/kg to about 10 mg/kg, about 0.01 mg/kg to about 10 mg/kg, about 0.01 mg/kg to about 1 mg/kg or about 0.1 mg/kg to about 1 mg/kg will be administered. For oral administration, a representative amount is from about 0.001 mg/kg to about 10 mg/kg, about 0.1 mg/kg to about 10 mg/kg, about 0.01 mg/kg to about 1 mg/kg or about 0.1 mg/kg to about 1 mg/kg. For administration in suppository form, a representative amount is from about 0.1 mg/kg to about 10 mg/kg or about 0.1 mg/kg to about 1 mg/kg. In an embodiment of the application, compositions are formulated for oral administration and the compounds are suitably in the form of tablets containing 0.25, 0.5, 0.75, 1.0, 5.0, 10.0, 20.0, 25.0, 30.0, 40.0, 50.0, 60.0, 70.0, 75.0, 80.0, 90.0, 100.0, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 mg of active ingredient per tablet. Compounds of the - 27 - 8660875

application may be administered in a single daily, weekly or monthly dose or the total daily dose may be divided into two, three or four daily doses. [0096] To be clear, in the above, the term “a compound” also includes embodiments wherein one or more compounds are referenced. II. Compounds and Compositions [0097] Phosphoramidite derivatives have been synthesized using the compound of Formula III. Oligonucleotides have been synthesized using the phosphoramidite derivatives of the disclosure. [0098] In an aspect, the present disclosure includes a folic acid phoshoramidite derivative of Formula I I or a tautomer, derivative, or salt thereof, wherein Z is O, NH, or NHR ² ; L1 and L2 are each independently (CH2)2-6 or -CH2CH2OCH2CH2-; n is an integer of 2 to 6; m is an integer of 1 to 6; R ¹ is H or an acid-labile protective group; each R ² is independently H, -C(O)CH3, or -C(O)CF3; and R ³ is H, -C(O)CH3, -C(O)CF3, or C1-3alkyl. [0099] In another aspect, the present disclosure includes a phosphoramidite derivative of Formula II of a small molecule A, the small molecule A comprising at least one carboxylic acid, - 28 - 8660875

II or a tautomer, derivative, or salt thereof, wherein L1 and L2 are each independently (CH2)2-6 or -CH2CH2OCH2CH2-; n is an integer of 2 to 6; m is an integer of 1 to 6; and R ¹ is H or an acid-labile protective group. [00100] In another aspect, the present disclosure includes compound of Formula III III or a tautomer, derivative, or salt thereof, wherein L1 and L2 are each independently (CH2)2-6 or -CH2CH2OCH2CH2-; n is an integer of 2 to 6; m is an integer of 1 to 6; R ¹ is H or an acid-labile protective group; R ³ is H or a protective group; and - 29 - 8660875

R ⁴ and R ⁵ are each independently H, or an amine-protective group. [00101] In another aspect, the present disclosure includes an oligonucleotide prepared by a method of the present disclosure. [00102] In another aspect, the present disclosure includes an oligonucleotide comprising a folic acid nucleotide analog having the following structure * H or a tautomer, derivative, or salt thereof, wherein Z is O or NH; L1 and L2 are each independently (CH2)2-6 or -CH2CH2OCH2CH2-; n is an integer of 2 to 6; m is an integer of 1 to 6; and R ⁴ is H or C1-3alkyl, optionally CH3; and wherein at least one of the * is a point of attachment to an adjacent nucleotide and the remaining * if present is a terminus of the oligonucleotide. [00103] In another aspect, the present disclosure includes an oligonucleotide comprising a small molecule nucleotide analog having the following structure - 30 - 8660875

or a tautomer, derivative, or salt thereof, wherein A is a small molecule as defined herein; L1 and L2 are each independently (CH2)2-6 or -CH2CH2OCH2CH2-; n is an integer of 2 to 6; and m is an integer of 1 to 6; and wherein at least one of the * is a point of attachment to an adjacent nucleotide and the remaining * if present is a terminus of the oligonucleotide. [00104] In another aspect, the present disclosure includes a kit for oligonucleotide synthesis comprising a folic acid phosphoramidite derivative of the present disclosure in a container. [00105] In another aspect, the present disclosure includes a kit for oligonucleotide synthesis comprising a small molecule phosphoramidite derivative of the present disclosure in a container. [00106] In some embodiments, n is an integer of 2 to 4, and m is an integer of 1 to 3. In some embodiments, n is 2, and m is 1. [00107] In some embodiments, the acid-labile protective group is selected from DMT, trityl, and MMT, optionally the acid-labile protective group is DMT. [00108] In some embodiments, R ² is -C(O)CF3. [00109] In some embodiments, L1 and L2 are each independently (CH2)2-6. [00110] In some embodiments, Z is O, and R ³ is H, -C(O)CH3, or -C(O)CF3. [00111] In some embodiments, Z is NH and R ³ is C1-3 alkyl. In some embodiments, R ³ is CH3 [00112] In some embodiments, the folic acid phosphoramidite derivative is - 31 - 8660875

alt th . [00113] In some embodiments, the small molecule A is selected from folic acid, retinoic acid, niacin, biotin, pyruvic acid, ibuprofen, arachidonic acid, amino acid, and peptides, optionally dipeptides and tripeptides. [00114] For example, the small molecule can be retinoic acid and the small molecule phosphoramidite can have the structure as shown below: alt the eo, ee , , , a , ae as e e ee . [00115] Accordingly, the oligonucleotide comprising a retinoic acid nucleotide analog can have the structure or a tautomer, a derivative or a salt thereof, wherein L ¹ , L ² , n, m, and * are as defined herein. [00116] For example, the small molecule can be niacin, and the small molecule phosphoramidite can have the structure as shown below: - 32 - 8660875

tautomer, a derivative or a salt thereof, wherein L ¹ , L , n, and m, are as defined herein. [00117] Accordingly, the oligonucleotide comprising a niacin nucleotide analog can have the structure tautomer, a derivative or a salt thereof, wherein L ¹ , L ² , n, m, and * are as defined herein. [00118] In some embodiments, the small molecule A is folic acid. [00119] In some embodiments, R ⁵ is selected from H, TBS, and acetyl, optionally R ⁵ is H. In some embodiments, R ⁶ and R ⁶ are H. [00120] It can be appreciated that the compounds described herein can exist in tautomeric forms. As such, it is contemplated that the present application also includes tautomeric forms of the compounds. [00121] For example, when Z is NH, the compound of Formula I can exist in the forms below and - 33 - 8660875

. It is contemplated that both keto and enol tautomeric forms are included in the present application. It can be appreciated that when Z is NH2, the nitrogen of Z can be protected with a protective group such as R ² . Accordingly, the following structure is also included in the compounds of Formula I . [00122] In some embodiments, the amine-protective group is selected from Boc, Fmoc, Cbz, and benzyl. [00123] In some embodiments, the compound of Formula III is or a derivative or salt thereof. [00124] In some embodiments, the oligonucleotide is about 2 to about 500 nucleotides in length, about 2 to about 400 nucleotide in length, about 2 to about 300 nucleotide in length, about 2 to about 200 nucleotide in length, about 2 to about 150 nucleotide in length, about 2 to about 100 nucleotide in length, about 2 to about 70 nucleotide in length, about 2 to about 50 nucleotide in length, about 2 to about 30 nucleotide in length, about 5 to about 30 nucleotide in length, about 10 to about 30 nucleotide in length, about 15 to about 30 nucleotide in length, or about 15 to about 25 nucleotide in length. - 34 - 8660875

[00125] In some embodiments, the oligonucleotide of the present application has a sequence selected from: CUUACGCUGAGUACUUCGAF (SEQ ID No.1); CUUACGCUGAGUACUFCGATT (SEQ ID No.2); CUUACGCUGFGUACUUCGATT (SEQ ID No.3); and CUUACGCUFAGUACUUCGATT (SEQ ID No.4), and wherein F represents a folic acid nucleotide analog as defined herein. [00126] In some embodiments, the oligonucleotide has one of the following sequences, wherein at least one nucleotide of the sequence has been replaced by the folic acid nucleotide analog. III. Methods, Uses and Compositions for Use [00127] It has been shown that the compound of Formula III can be used to generate small molecule phosphoramidite derivatives such as folic acid phosphoramidite derivatives. Further, it has been demonstrated that the phosphoramidite derivatives can be used in a standard solid-phase oligonucleotide synthesis. It is demonstrated that exemplary oligonucleotides disclosed herein results in enhanced gene silencing in cells. [00128] In another aspect, the present disclosure includes a method of preparing a compound of Formula I of the present disclosure comprising ^ reacting a compound of Formula IIIa IIIa or a tautomer, derivative, or salt thereof, wherein L1 and L2 are each independently (CH2)2-6 or -CH2CH2OCH2CH2-; N is an integer of 2 to 6; - 35 - 8660875

m is an integer of 1 to 6; and R ¹ is H or an acid-labile protective group; with a compound of Formula IV IV, or a tautomer, derivative, or salt thereof, wherein Z, each R ² and R ³ are each as defined herein, to obtain a compound of Formula V V or a tautomer, derivative, or salt thereof; and ^ reacting the compound of Formula V or a tautomer, derivative, or salt thereof, with a phosphoramadite forming reagent, optionally 2-cyanoethyl- N,N-diisopropylchlorophosphoramidite or 2-cyanoethyl N,N,N,N- tetraisopropylphosphoramidite. [00129] In some embodiments, the method of preparing a compound of Formula I further comprises reacting folic acid with trifluoroacetic anhydride (TFAA) or acetic anhydride to obtain the compound of Formula IV, wherein R ² is -C(O)CF3, or -C(O)CH3 respectively. - 36 - 8660875

[00130] In another aspect, the present disclosure includes a method of preparing a compound of Formula II of the present disclosure comprising ^ reacting a compound of Formula III of the present disclosure with the small molecule A comprising at least one COOH under conditions to form an amide bond between the small molecule A and the compound of Formula III to obtain a compound of Formula VI or a tautomer, derivative, or salt thereof, wherein L1 and L2 are each independently (CH2)2-6 or - CH2CH2OCH2CH2-; n is an integer of 2 to 6; m is an integer of 1 to 6; and R ¹ is H or an acid-labile protective group; and ^ reacting the compound of Formula IV or a tautomer or salt thereof with a phosphoramidite forming reagent, optionally 2-cyanoethyl-N,N- diisopropylchlorophosphoramidite or 2-cyanoethyl N,N,N,N- tetraisopropylphosphane. [00131] In some embodiments, the forming of the amide bond is carried out with a peptide coupling reagent, optionally selected from HATU, HBTU, HCTU, BOP, PyBOP, EDC, DCC, DIC, and PyAOP. [00132] In another aspect, the present disclosure includes a method of preparing an oligonucleotide comprising a folic acid nucleotide analog, the method comprising - 37 - 8660875

reacting a folic acid phosphoramidite or a tautomer or salt thereof of the present disclosure with a nucleotide having a free 3’ hydroxyl group or an oligonucleotide chain having a free 3’ hydroxyl group. [00133] In another aspect, the present disclosure includes a method of preparing an oligonucleotide comprising a small molecule nucleotide analog, the method comprising reacting a small molecule phosphoramidite of the present disclosure with a nucleotide having a free 3’ hydroxyl group or an oligonucleotide chain having a free 3’ hydroxyl group. [00134] In some embodiments, the method of preparing an oligonucleotide further comprises deprotecting any protective group in the oligonucleotide if present. [00135] In some embodiments, the reacting of the method of preparing an oligonucleotide is carried out on a solid support, optionally the solid support is a silica solid support. [00136] In another aspect, the present disclosure includes a method of gene silencing in a cell comprising administrating an oligonucleotide comprising a folic acid nucleotide analog of the present disclosure. [00137] In another aspect, the present disclosure includes the oligonucleotide of the present disclosure for use in gene silencing. [00138] In another aspect, the present disclosure includes the oligonucleotide of the present disclosure for use as an siRNA. [00139] In some embodiments, the cell expresses folic acid receptor. In some embodiments, the cell is selected from a breast cell, ovarian cell, endometrial cell, lung cell, brain cell, kidney cell, cervical cell, colon cell, and combinations thereof. In some embodiments, the cell is selected from a breast cancer cell, ovarian cancer cell, endometrial cancer cell, lung cancer cell, brain cancer cell, kidney cancer cell, cervical cancer cell, colon cancer cell, and combinations thereof. [00140] In another aspect, the present disclosure includes a method of treating or preventing cancer comprising administering an oligonucleotide comprising a folic acid nucleotide analogue of the present disclosure to a subject in need thereof. - 38 - 8660875

[00141] In another aspect, the present disclosure includes a use of an oligonucleotide comprising a folic acid nucleotide analogue of the present disclosure for treating or preventing cancer. [00142] In another aspect, the present disclosure includes a use of an oligonucleotide comprising a folic acid nucleotide analogue of the present disclosure in the preparation of a medication for treating or preventing cancer. [00143] In another aspect, the present disclosure includes an oligonucleotide comprising a folic acid nucleotide analogue of the present disclosure for use in the treatment or prevention of cancer. [00144] In some embodiments, the cancer comprises cells that express folic acid receptor. [00145] In some embodiments, the cancer is selected from breast cancer, ovarian cancer, cervical cancer, colon cancer, and combinations thereof. [00146] In another aspect, the present disclosure includes a use of a compound of Formula I of the present disclosure in the synthesis of an oligonucleotide. [00147] In another aspect, the present disclosure includes a use of a compound of Formula II of the present disclosure in the synthesis of an oligonucleotide. [00148] In another aspect, the present disclosure includes a use of a compound of Formula III of the present disclosure in the synthesis of a phosphoramidite derivative. [00149] An aspect of the present disclosure relates to using the oligonucleotides disclosed herein to silence expression of a target gene in a cell expressing folate receptors (FR). To allow an siRNA to translocate across the cell membrane, a carrier, such as for example liposome, is typically used. It is demonstrated herein that the folate-siRNAs of the present disclosure allows carrier-free delivery into a cell that expresses FR. [00150] The oligonucleotides of the present disclosure may also be used with a carrier if delivery into non-FR expressing cells is desired. [00151] Various methods are known in the art to design siRNA sequences for specific gene targets. For example, low G/C content and a bias towards low internal stability at the sense strand 3’-terminus are some of the known characteristics associated with siRNA functionality. Algorithms have been developed to optimize siRNA sequences - 39 - 8660875

(See e.g. Fakhr, E., Zare, F. and Teimoori-Toolabi, L., 2016. Precise and efficient siRNA design: a key point in competent gene silencing. Cancer gene therapy, 23(4), pp.73-82, the content of which is incorporated herein in its entirety). [00152] In some embodiments, the cancer is a cancer that over-expresses FR. Examples of cancer that over-expresses FR include but not limited to breast cancer, ovarian cancer, endometrial cancer, cervical cancer, colon cancer, lung cancer, brain cancer, and kidney cancers. [00153] The oligonucleotides of the present application can be used to suppress expression of a target gene to provide therapeutic benefits in a subject in need thereof. For example, the target gene can be an oncogene, such as BCL-2, EGFR, VEGF or PLK1. [00154] In some embodiments, the oligonucleotides of the present disclosure are used to silence an oncogene in a subject with cancer. In some embodiments, the oncogene is BCL-2. [00155] Any suitable methods to enhance the stability and/or functionality of a siRNA therapy known in the art can be used with the oligonucleotides of the present disclosure. [00156] The oligonucleotides may be modified. For example, once administered to a subject in need thereof, the siRNA may be subjected to degradation by nucleases. It is known in the art that modifications of the RNA backbone can improve stability of the RNA. Examples of such modifications include but are not limited to 2’-O-methyl, 2’-fluoro, locked nucleic acid (LNA)and phosphorothioate linkages. [00157] Therefore, in some embodiments, the backbone of the oligonucleotides comprises one or more modifications. [00158] In an embodiment, the disease, disorder or condition is a neoplastic disorder. Accordingly, the present application also includes a method of treating or preventing a neoplastic disorder comprising administering a therapeutically effective amount of one or more oligonucleotides of the application to a subject in need thereof. The present application also includes a use of one or more oligonucleotides of the application for treatment or prevention of a neoplastic disorder as well as a use of one or more oligonucleotides of the application for the preparation of a medicament for treatment of a neoplastic disorder. The application further includes one or more oligonucleotides of the application for use in treating or preventing a neoplastic disorder. In an embodiment, - 40 - 8660875

the treatment is in an amount effective to ameliorate at least one symptom of the neoplastic disorder, for example, reduced cell proliferation or reduced tumor mass, among others, in a subject in need of such treatment. In one embodiment, the neoplastic disorder is a cancer. [00159] In an embodiment, the cancer is selected from a cancer of the skin, blood, prostate, colorectum, pancreas, kidney, ovary, breast, for example mammary, liver, tongue and lung. In another embodiment, the cancer is selected from leukaemia, lymphoma, non-Hodgkin’s lymphoma and multiple myeloma. In a further embodiment of the present application, the cancer is selected from leukemia, melanoma, lung cancer, colon cancer, brain cancer, ovarian cancer, breast cancer, prostate cancer and kidney cancer. [00160] In an embodiment, the disease, disorder or condition that is treated by gene silencing using the oligonucleotides of the present disclosure relates to uncontrolled and/or abnormal gene activity. In another embodiment, the uncontrolled and/or abnormal gene activity is related to proliferative activity in a cell. Accordingly, the application also includes a method of inhibiting proliferative activity in a cell, comprising administering an effective amount of one or more oligonucleotides of the application to the cell. The present application also includes a use of one or more oligonucleotides of the application for inhibition of proliferative activity in a cell as well as a use of one or more oligonucleotides of the application for the preparation of a medicament for inhibition of proliferative activity in a cell. The application further includes one or more oligonucleotides of the application for use in inhibiting proliferative activity in a cell. IV. Preparation of Compounds of the Application [00161] Compounds of the present application can be prepared by various synthetic processes. The choice of particular structural features and/or substituents may influence the selection of one process over another. The selection of a particular process to prepare a given compound of the present disclosure is within the purview of the person of skill in the art. Some starting materials for preparing compounds of the present application are available from commercial chemical sources. Other starting materials, for example as described below, are readily prepared from available precursors using straightforward transformations that are well known in the art. In the Schemes below showing the - 41 - 8660875

preparation of compounds of the application, all variables are as defined herein, unless otherwise stated. [00162] In some embodiments, the compounds of Formula I are prepared as shown in Scheme A. For example, folic acid can be reacted with an anhydride R ^a C(O)O(O)CR ^a to obtain the compound A-1. For example, trifluoroacetic anhydride can be used. It can be appreciated that other nucleophilic nitrogens in folic acid may be acylated. It can also be appreciated that acylated nitrogens such as trifluoroacetamides can be hydrolyzed to reveal the corresponding amines using methods known in the art. Compounds A-1 can be reacted with compounds A-2 to form an amide bond to obtain compounds A-3. Compounds A-3 can undergo optional, selective deprotection of the hydroxyl group OR ⁵ (if R ⁵ is a protective group) and the resulting OH group can react with a phosphoramidite reagent such as 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite to provide compounds of Formula I. It can be appreciated that where R ¹ and R ⁵ are both protective groups, one can be selectively deprotected in the presence of the other. Scheme A – Preparation of Compounds of Formula I [00163] In some embodiments, the compounds of Formula II can be prepared as shown in Scheme B. Therefore, in some embodiments, a small molecule A comprising at least one carboxylic acid group can undergo amide bond formation with the free amine group in compounds B-1 using peptide coupling agents to obtain compounds B-2. For example, suitable peptide coupling agents include but are not limited to HATU, HBTU, HCTU, EDC, DCC, DIC, BOP, PyAOP, and PyBOP. Compounds B-2 can undergo - 42 - 8660875

selective deprotection of the hydroxyl group OR ⁵ in the case where R ⁵ is a protective group and the resulting OH group can react with a phosphoramidite reagent such as 2- cyanoethyl-N,N-diisopropylchlorophosphoramidite to provide compounds of Formula II. It can be appreciated that where R ¹ and R ⁵ are both protective groups, one can be selectively deprotected in the presence of the other. L ¹ OR ¹ L ¹ OR ¹ Sm I ph op II Scheme B – Preparation of compounds of Formula II [00164] In some embodiments, the compounds of Formula III can be prepared as shown in Scheme C. Therefore, for example, alkyne compounds C-1 can undergo Cu- catalysed cyclization with azide compounds C-2 to generate compounds of Formula III. Scheme C – Preparation of compounds of Formula III [00165] Salts of the compounds of the application are generally formed by dissolving the neutral compound in an inert organic solvent and adding either the desired acid or base and isolating the resulting salt by either filtration or other known means. [00166] The formation of solvates of the compounds of the application will vary depending on the compound and the solvate. In general, solvates are formed by dissolving the compound in the appropriate solvent and isolating the solvate by cooling or - 43 - 8660875

using an antisolvent. The solvate is typically dried or azeotroped under ambient conditions. The selection of suitable conditions to form a particular solvate can be made by a person skilled in the art. [00167] Prodrugs of the compounds of the present application may be, for example, conventional esters formed with available hydroxy, thiol, amino or carboxyl groups. For example, available hydroxy or amino groups may be acylated using an activated acid in the presence of a base, and optionally, in inert solvent (e.g. an acid chloride in pyridine). [00168] Throughout the processes described herein it is to be understood that, where appropriate, suitable protecting groups will be added to, and subsequently removed from, the various reactants and intermediates in a manner that will be readily understood by one skilled in the art. Conventional procedures for using such protecting groups as well as examples of suitable protecting groups are described, for example, in “Protective Groups in Organic Synthesis”, T.W. Green, P.G.M. Wuts, Wiley-Interscience, New York, (1999). It is also to be understood that a transformation of a group or substituent into another group or substituent by chemical manipulation can be conducted on any intermediate or final product on the synthetic path toward the final product, in which the possible type of transformation is limited only by inherent incompatibility of other functionalities carried by the molecule at that stage to the conditions or reagents employed in the transformation. Such inherent incompatibilities, and ways to circumvent them by carrying out appropriate transformations and synthetic steps in a suitable order, will be readily understood to one skilled in the art. Examples of transformations are given herein, and it is to be understood that the described transformations are not limited only to the generic groups or substituents for which the transformations are exemplified. References and descriptions of other suitable transformations are given in “Comprehensive Organic Transformations – A Guide to Functional Group Preparations” R.C. Larock, VHC Publishers, Inc. (1989). References and descriptions of other suitable reactions are described in textbooks of organic chemistry, for example, “Advanced Organic Chemistry”, March, ^4t h ed. McGraw Hill (1992) or, “Organic Synthesis”, Smith, McGraw Hill, (1994). Techniques for purification of intermediates and final products include, for example, straight and reversed phase chromatography on column or rotating plate, recrystallisation, distillation and liquid-liquid or solid-liquid extraction, which will be readily understood by one skilled in the art. - 44 - 8660875

[00169] The following non-limiting examples are illustrative of the present application: EXAMPLES Example 1 Methods and Materials [00170] General Methods [00171] All starting reagents and solvents were obtained from commercial sources and used without additional purification, unless otherwise stated. Compounds A and B were synthesized as previously described [36]. Standard flash chromatography was performed using Silicycle Siliaflash 60 (230-400 mesh). ¹ H, ¹³ C and ³¹ P NMRs were recorded in DMSO-d6, CDCl3 or CD3OD using a Bruker Avance ^TM III NMR spectrometer. Synthesis of folate phosphoramidite Synthesis of Compound C [00172] Compound A (0.7 g, 1.57 mmol) was first dissolved in anhydrous ACN and triethylamine (0.5 eq, 0.785 mmol, 0.11 mL) under an Argon atmosphere. Compound B (1.5 eq., 2.36 mmol, 0.2 g) was then added and the mixture was stirred for 5 minutes at room temperature. CuI (0.3 eq., 0.471 mmol, 0.090 g) and sodium ascorbate (0.5 eq., 0.785 mmol, 0.16 g) were subsequently added, and the reaction was stirred vigorously for 3 hours at room temperature. After the reaction reached completion, the mixture was dried under vacuum. The resulting residue was purified using silica gel column chromatography (5 to 40% MeOH/CH2Cl2 with 2% triethylamine) to afford compound C as a crystalline foam (0.63 g, 76%). ¹ H NMR (400 MHz, CD3OD) δ ppm 2.66 (t, 2 H) 2.78 (t, 2 H) 3.11 (s, 2 H), 3.23 (t, 2 H), 3.61 (t, 2 H), 3.78 (s, 6 H), 3.84 (s, 2 H), 4.44 (t, 2 H), 6.79 - 6.89 (m, 4 H), 7.16 – 7.44 (m, 9 H), 7.85 (s, 1 H) ^{. 13} C NMR (101 MHz, CD3OD) δ ppm 158.64, 145.24, 136.17, 129.86, 127.92, 127.39, 126.38, 124.02, 112.69, 86.24, 61.83, 59.33, 56.00, 54.37, 53.71, 49.31. ESI HRMS (ES+) m/z calculated for C30H37N- 5O4: 531.2846, found: 531.2100 [M+H] ⁺ - 45 - 8660875

Scheme 1 Synthesis of triazole-linker C. Reagents and conditions: (i) CuI, sodium ascorbate, Et3N/ACN, rt, 3 h, 76%. Synthesis of Compound 1 [00173] Folic acid (2 g, 4.5 mmol) was suspended in anhydrous THF (20 mL) and stirred at 0 °C in the dark, under an argon atmosphere. Trifluoroacetic anhydride (8 eq., 36 mmol, 5 mL) was added over a 30 min period, and the reaction mixture was then allowed to equilibrate to room temperature. As the reaction proceeded, the mixture turned into a dark brown homogenous phase. After 12 hours, the solvent was evaporated under vacuum and the product was precipitated in ether (~100 mL). The dark brown crystals were collected by filtration and washed with ether (30 mL x 3). ¹ H NMR (400 MHz, DMSO- d6) δ ppm 1.99 - 2.07 (m, 1 H), 2.41-2.60 (overlap, 3H), 4.73 (dd, 1 H), 5.76 (s, 2 H), 7.64 (s, 4 H), 8.68 (s, 1 H). ¹⁹ F NMR (377 MHz, DMSO-d6) δ ppm -74.30, -66.09. ESI HRMS (ES+) m/z calculated for C23H15F6N7O7: 615.0937, found: 615.0987 [M+H] ⁺ . Synthesis of Compound 2 [00174] Compound C (0.5 g, 0.94 mmol) was dissolved in anhydrous DMF (20 mL) under argon. Compound 1 (0.87 g, 1.41 mmol) was added, and the reaction was allowed to stir in the dark under an argon atmosphere for 36 hours. DMF was removed using a miVac Quattro concentrator. The resulting residue was purified using silica gel chromatography, eluting from 5 to 60% MeOH/CH2Cl2 with 5% triethylamine to yield compound 2 as brown crystals (0.70 g, 74%). ¹⁹ F NMR (377 MHz, DMSO-d6) δ ppm - 73.46, -66.09. ESI HRMS (ES+) m/z calculated for C48H45F6N11O8: 1017.3357, found: 1016.4338 [M+H] ⁺ . Synthesis of Compound 3 [00175] To a flame-dried round-bottomed flask was added a solution of compound 2 (0.2 g, 0.196 mmol) in anhydrous 1:1 DCM and THF (10 mL) and triethylamine (0.16 mL, 1.18 mmol), under an argon atmosphere. This was followed by the dropwise addition - 46 - 8660875

of 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (0.13 mL, 0.59 mmol). The reaction was stirred at room temperature, and in the dark, for 3 hours. After removing the solvent in vacuo, the crude product was taken up in dichloromethane, washed with water and dried with anhydrous sodium sulphate. The organic layer was then concentrated in vacuo. The extracted product was dissolved in 1 mL dichloromethane and crashed out by adding 50 mL of n-hexanes. The resulting crystals were collected by filtration and dried in vacuo to yield compound 3, which was immediately used for solid-phase oligonucleotide synthesis (0.19 g, 79%). ¹⁹ F NMR (377 MHz, CDCl3) δ ppm -75.48, -67.01. ³¹ P NMR (162 MHz, CDCl3) ^^ ppm 149.43, 149.37. ESI HRMS (ES+) m/z calculated for C48H45F6N11O8: 1217.4435, found: 1134.1402 [M+H] ⁺ (hydrolyzed product). Scheme 2. Synthesis of folate phosphoramidite 3. Reagents and conditions: (i) TFAA, THF, 0 °C, 30 min, 0 °C → rt, 12 h (91%), (ii) C, DMF, rt, 36 h (74%), (iii) 2-cyanoethyl- N,N-diisopropylchlorophosphoramidite, THF/DCM (1:1), Et3N, rt, 3 h (79%). Oligonucleotide synthesis [00176] Oligonucleotides were prepared following standard solid-phase oligonucleotide synthetic procedures, using a 1.0 μM controlled-pore glass (CPG) support on an Applied Biosystems 394 DNA/RNA synthesizer. Immediately before use, phosphoramidites were resuspended to a final concentration of 0.1 M. Commercial phosphoramidites were resuspended in anhydrous acetonitrile whereas phosphoramidite 3 was resuspended in a 1:1 mixture of anhydrous THF and DCM. To cleave the oligonucleotides from the solid support, each CPG column was flushed with 1 mL EMAM solution (1:1 methylamine 33 wt% in ethanol/methylamine 40 wt% in water) for 1 hour at room temperature and then incubated overnight in EMAM to deprotect the bases. - 47 - 8660875

Oligonucleotides were concentrated in a miVac Quattro Concentrator before being resuspended in DMSO (100 μL). To remove the remaining protecting groups, each oligonucleotide was treated with 3HF-Et3N (125 μL) and incubated for 3 hours at 65°C. After drying the DMSO in a miVac Quattro Concentrator, oligonucleotides were precipitated in ethanol and desalted using Millipore Amicon Ultra 3000 MW cellulose centrifugal filters. Strands were purified using reverse-phase HPLC eluting from 5% to 95% ACN in 0.1 M TEAA buffer (pH 7.0). Biophysical characterization [00177] CD and thermal denaturation studies were performed on a Jasco J-815 Circular Dichroism (CD) Spectropolarimeter equipped with a temperature controller. Duplexes were formed by combining equimolar amounts of complementary sense and antisense strands in 300 μL pH 7 sodium phosphate buffer (90.0 mM NaCl, 10.0 mM Na2HPO4, 1.00 mM EDTA), heating them to at 90 °C for 2 min and allowing them to equilibrate to room temperature. Circular dichroism spectra were recorded at 25°C, scanning from 200 to 350 nm with a screening rate of 100 nm/min and a 0.20 nm data pitch. The melting temperature (Tm) of each duplex was determined by measuring the change in absorbance at 260 nm against a temperature gradient from 15 to 90 °C at a rate of 1 °C per minute. Data were analysed using Meltwin ^TM v3.5 software. Cell culture and transfection [00178] HeLa and HT-29 cells were maintained in Dulbecco′s modified Eagle′s medium (DMEM) and Roswell Park Memorial Institute (RPMI) 1640 Medium, respectively, at 37 °C in a humidified atmosphere with 5% CO2. Media were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin (Sigma). HeLa and HT-29 cells were transfected as previously described for both the standard and carrier-free assays [36]. Dual-luciferase® reporter assay [00179] Cells were lysed with 1X passive lysis buffer for 30 min at room temperature. Cell lysates were transferred to microcentrifuge tubes and were immediately used to assess the gene-silencing activity of siRNAs using the Dual-Luciferase® Reporter Assay (Promega) following the manufacturer′s protocol. Cell lysates (10 μL) were transferred to Costar 96-well plates in triplicate. Luminescence measurements were taken on a Synergy - 48 - 8660875

HT (Bio-Tek) plate luminometer. Results are expressed as the ratio of firefly/Renilla luminescence taken as a percentage of an untreated control. Example 2 Preparation and characterization of folate oligonucleotides [00180] Folate phosphoramidite 3 was prepared and purified as described in Example 1. The few previous reports of folate phosphoramidites, such as the one commercialized by Berry and Associates, do not employ silica gel chromatography as a purification method [39]. These phosphoramidite derivatives have poor solubility profiles and can be very labile, so an alternative approach involves precipitating them out of solution, using an appropriate solvent, and then immediately using them for solid-phase oligonucleotide synthesis. Once the oligonucleotides were cleaved from the solid support and deprotected, they were purified using reverse-phase HPLC and characterized by mass spectrometry (Table 1). Table 1. Oligonucleotide sequences and mass spectrometry data Code Sequence Mass (predicted) Mass (found) Fo 27 Fo 11 Fo 23 Fo 08 All strands code for firefly luciferase and correspond to the sense strand. F indicates t position of the folate modification. [00181] Synthesized oligonucleotides were annealed to their complementary antisense sequences prior to CD and thermal denaturation studies. As seen in Figure 2, all siRNAs adopted the desired A-form helical conformation which is recognized by the RISC [40]. Consistent with previous findings, higher thermal destabilization was observed when a folate modification is placed within the central region of the sense strand, compared to the 3′ end. Tm values are summarized in Table 2. In siRNA Fol1, the folate modification replaces the 3′ dTdT overhang and imparts a small destabilizing effect - 49 - 8660875

(ΔTm = −6), likely due to the loss of stacking interactions in this region [41]. A similar effect was observed when replacing the nucleotide at position 6 from the sense strand 3′ end (ΔTm = −6). Notably, this novel modification was more destabilizing than the one reported previously[36], particularly when placed at position 9 (Fol4) or 10 (Fol3) from the sense strand 5′ end (ΔTm = −25 and −24 °C, respectively). This trend is consistent with with literature reports examining the effect of central modifications on the thermal stability of the siRNA duplex [42,43]. Table 2. Sequences and Tm data of anti-luciferase wild-type (wt) and folate-siRNAs. Code Sequence Tm Δ Tm IC50 ± SE (°C) (°C) (nM) wt ^{5′ CUU ACG CUG AGU ACU UCG AdTdT 3′ (SEQ ID No 4)} 76 - NA Fo Fo Fo Fo The top strand corresponds to the sense strand; the bottom strand corresponds to the antisense strand. F indicates the location of the folate modification. IC50 values were calculated after carrier-free siRNA transfections in HeLa cells. Example 3 Biological activity of folate siRNAs Gene-silencing activity in HeLa cells [00182] The biological activity of the folate siRNAs in HeLa cells after transfection with Lipofectamine 2000™ was assessed using the methods described in Example 1. As seen in Figure 3, all siRNAs displayed excellent dose-dependent activity. At the highest concentration tested (800 pM), all siRNAs achieved potent knockdown of exogenous firefly luciferase mRNA. [00183] The ability of the folate modification to mediate siRNA uptake into these folate receptor-expressing cells was investigated. To achieve this, siRNAs were transfected into HeLa cells at concentrations ranging from 0.5 to 3000 nM, without the - 50 - 8660875

use of a transfection reagent. As expected, the unmodified (wt) siRNA was unable to cross the cellular membrane and thus did not result in gene silencing. On the other hand, all folate siRNAs achieved potent gene-silencing activity in the absence of a transfection carrier (Figure 4). With the previous folate modification reported in Salim et al (2020) [36], enhanced potency was observed when placing it within the central region, as opposed to the 3′ end, of the siRNA. The novel folate modification presented herein did not impart a position-dependent effect on siRNA potency although it was placed in the same regions that we previously investigated. Overall, siRNAs Fol1-Fol4 displayed potent and comparable activity, with IC50 values ranging from 23.2 to 98.5 nM. Gene-silencing activity in HT-29 cells [00184] To assess the selectivity of our folate siRNAs for FR-expressing cells, the dual-luciferase® reporter assays were repeated in the FR-negative cell line HT-29. We first transfected siRNAs into HT-29 cells at concentrations ranging from 8 to 800 pM, using Lipofectamine® LTX. As seen in Figure 5, when siRNAs are internalized by the transfection carrier, all siRNAs show dose-dependent knockdown. The gene-silencing trend displayed by the siRNAs in HT-29 cells was comparable to that observed in HeLa cells. Next, the ability of the folate modification to mediate uptake in the absence of a transfection carrier was assessed. None of the siRNAs displayed gene-silencing activity in HT-29 cells when tested without a transfection reagent (Figure 6). This was expected since the internalization of these molecules is mediated by cell-surface folate receptors. Cell viability after siRNA treatment [00185] HeLa cell viability after carrier-free treatment with unmodified (wt) and folate siRNAs at five concentrations (5, 25, 100, 750 and 3000 nM) was assessed using a colorimetric MTT assay, as described in Example 1. As seen in Figure 7, treatment with high concentrations of wild-type siRNA (750 and 3000 nM) led to ~24% decrease in HeLa cell viability. This is consistent with previous findings [36]. On the other hand, the novel folate-modified siRNAs disclosed herein did not impart any cytotoxic effects, resulting in over 90% cell viability across all concentrations tested. The previously reported folate siRNA formulation led to 80-90% cell viability after siRNA treatment, indicating that low cytotoxic effects were present with some of the concentrations tested [36]. [00186] Overall, it is shown herein a straightforward and cost-effective approach to prepare a folic acid phosphoramidite derivative that is compatible with solid-phase - 51 - 8660875

oligonucleotide synthesis. Incorporation of this folate modification at different positions within the siRNA sense strand resulted in a new generation of self-delivering folate siRNAs that display enhanced gene-silencing activity. This work is very significant as folate phosphoramidites are not commercially available and there are not many reliable protocols to synthesize them. This has limited the application of folate-based gene- silencing therapies, as most current approaches rely on post-column Cu(I)-catalyzed azide/alkyne cycloaddition which can pose challenges when it comes to cytotoxicity and scalability. The method of the present disclosure will allow for streamlined synthesis of folate-conjugated oligonucleotides and provide a means to better investigate these molecules for therapeutic applications. Example 4 Synthesis of Compounds of Formula VI [00187] Exemplary compounds of Formula VI were prepared by reacting different small molecules having a carboxylic acid group with Compound C as precursors to phosphoramidite compounds of Formula II. Synthesis of Compound 4 [00188] Firstly, benzoic acid was conjugated to compound C to afford compound 4 in 33% yield via amide-bond coupling. The reaction is shown in Scheme 3. [00189] Benzoic Acid (2eq., 0.38 mmol, 0.046g) was first dissolved in anhydrous DMF (2.5mL) under argon. EDC (1.6eq., 0.30mmol, 0.058g) and HOBt (1.6eq., 0.30mmol, 0.041g) were added subsequently and left to stir at room temperature for 15 minutes. Anhydrous triethylamine (8eq., 1.51mmol, 0.21mL) was then added to adjust the reaction pH to 7. Compound C (0.10g, 0.19 mmol) was dissolved with anhydrous DMF (2.5mL), added to the reaction mixture, and stirred for 24hr hours at room temperature. A liquid- liquid extraction (3X) was then performed for the crude product using DCM, and brine. The organic layer was then dried down using the miVac Quattro ^TM concentrator and purified using silica gel column chromatography (1 to 10% MeOH/CH2Cl2) to afford an orange oil (33% yield). ¹ H NMR (400 MHz, (CD3)2CO) δ ppm 2.75(t, 2H), 3.18 (t, 2H), 3.54 (t, 2H), 3.79 (s, 6H), 3.83 (s, 2H), 3.86-3.90 (m, 2H), 4.65 (t, 2H), 6.87-8.09 (m, 19H). ¹³ C NMR (101 MHz, (CD3)2CO) δ ppm 166.87, 158.61, 145.59, 144.33, 136.43, 134.57, 131.20, 130.02, 128.26, 127.65, 127.18, 126.53, 123.46, 112.95, 85.96, 62.20, 59.39, - 52 - 8660875

56.10, 54.63, 53.57, 49.25, 48.95, 46.04, 39.91. ESI (ES+) m/z calculated for C37H41N5O5: 635.76, found 635.31 [M+H] Scheme 3. Synthesis of Compound 4 Synthesis of Compound 5 [00190] Retinoic acid was used next as an example small molecule. The reaction is shown in Scheme 4. [00191] Retinoic Acid (1.6eq., 0.15 mmol, 0.045g) was first dissolved in anhydrous DMF (2.5 mL) under argon. EDC (1.2eq., 0.11 mmol, 0.022g) and HOBt (1.2eq., 0.30mmol, 0.015g) were added subsequently and left to stir at room temperature for 15 minutes. Anhydrous triethylamine (1.2 mL) was then added to adjust the reaction pH to 7. Compound C (0.05g, 0.094 mmol) was dissolved with anhydrous DMF (2.5mL) and added to the reaction mixture, stirred for 24hr hours at room temperature. A liquid-liquid extraction (3X) was then performed for the crude product using DCM, and brine. The organic layer was then dried down using the miVac Quattro concentrator and purified using silica gel column chromatography (1 to 10% MeOH/CH2Cl2) to afford a dark yellow oil (39% yield). ¹ H NMR (400 MHz, CD3OD) δ ppm 1.05 (s, 6H), 1.50-1.52 (m, 2H), 1.63- 1.68 (m, 2H), 1.72 (s, 3H), 1.98 (s, 3H), 2.05 (t, 2H), 2.26 (s, 3H), 2.64 (t, 2H), 2.75 (t, 2H), 3.22 (t, 2H), 3.60 (t, 2H), 3.69 (t, 2H), 3.78 (s, 6H), 3.83 (s, 2H), 4.53 (s, 2H), 5.73 (s, 1H), 6.08-6.31 (m, 4H), 6.84-6.86 (m, 4H), 6.93-6.99 (m, 1H), 7.18-7.22 (m, 1H), 7.26-7.32 (m, 6H), 7.42-7.44 (m, 2H), 7.80 (s, 1H). ¹³ C NMR (101MHz, CD3OD) δ ppm 168.46, 158.65, 148.65, 145.24, 144.36, 138.24, 137.61, 136.15, 135.46, 129.85, 127.92, 127.36, 126.34, 124.08, 120.73, 112.67, 86.24, 61.84, 59.33, 55.79, 54.32, 53.51, 52.21, 39.37, 38.94, 33.86, 32.59, 28.04, 20.57, 18.93, 12.54, 11.48, 8.98, 6.24. ESI (ES+) m/z calculated for C50H63N5O5: 813.48, found 812.42 [M+H] ⁺ - 53 - 8660875

Scheme 4. Synthesis of Compound 5. Synthesis of Phosphoramidite [00192] It can be appreciated that compounds of Formula II such as Compounds 4 and 5 can be phosphotitylated to obtain the corresponding phosphoramidite that can be used in oligonucleotide synthesis as described herein. The preparation of the phosphoramidite can be done for example according to procedures shown in Example 1. [00193] As shown herein, small molecules including bioactive small molecules having a carboxylic acid functionality can be used in the methods of the present disclosure to prepare phosphoramidites that can be used to incorporate the small molecule into an oligonucleotide. Synthesis of Oligonucleotide [00194] The synthesis of oligonucleotides using the compounds and phosphoramidites of this Example can be done by methods known in the field such as shown in Example 1. [00195] While the present application has been described with reference to examples, it is to be understood that the scope of the claims should not be limited by the embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole. [00196] All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety. Where a term in the present application is found to be defined differently in a document incorporated herein by reference, the definition provided herein is to serve as the definition for the term. - 54 - 8660875

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