METHODS FOR SEPARATING LARGE NUCLEIC ACIDS UNDER DENATURED CONDITIONS CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 62/748,768, filed October 22, 2018, the content of which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present disclosure provides methods for separating large nucleic acid molecules from large fragments thereof or impurities by separating the nucleic acids contained in the sample from each other by length using a capillary electrophoresis device with formamide as a denaturing agent and a non-aqueous separation matrix comprising a formamide-soluble polymer. Such methods can be used to determine the length of the nucleic acid(s) in the sample as well as the purity of the sample.

BACKGROUND OF THE INVENTION

Messenger RNA (mRNA)-based therapeutics are of great interest in the field of vaccination as a safe and efficient alternative to traditional live virus or protein-based vaccines. Unlike traditional vaccines, mRNA can be engineered to carry specific genetic information, which can be directly injected and delivered into cells where the antigen is generated in vivo. Promising results have shown that mRNA can stimulate immune response for influenza, HIV, OVA, etc. Currently, multiple mRNA vaccines are under evaluation in clinical trials.

RNA is not stable and can undergo degradation during preparation, process, formulation and storage. Characterization of RNA is crucial to quality assurance, understanding of their potency, and optimization of manufacture processes. Thus, reliable analytical methods that can measure the purity of RNA are required. One of the possible degradation pathways of RNA is hydrolytic degradation. Such degradation can be analyzed by the size difference of RNA.

Separation based on size difference is especially challenging for large RNA molecules because the size difference of, for example, large fragments compared to intact RNA is not as significant as for small RNAs ( e.g ., short interfering RNA or“siRNA”). Therefore, there is a need in the art to improve the efficiency of large RNA analysis.

BRIEF SUMMARY OF THE INVENTION

The present disclosure provides methods for separating one or more nucleic acids contained in a sample by length using a capillary electrophoresis device with formamide as a nucleic acid denaturing agent and a non-aqueous separation matrix comprising a formamide- soluble polymer. This method allows for separation of large nucleic acid molecules from other large nucleic acid molecules with good resolution and separation efficiency. Thus, also provided is a method for determining the purity of nucleic acid in the sample by separating the nucleic acids in the sample by length using capillary electrophoresis. The present disclosure also provides methods for determining the length of one or more nucleic acids contained in a sample using a capillary electrophoresis device with formamide as a nucleic acid denaturing agent and a non-aqueous separation matrix comprising a formamide-soluble polymer. The disclosure is based at least in part on the discovery that using 100% formamide as solvent for both the background electrolyte and gel, and preparing a formamide-containing gel containing a formamide-soluble polymer at low concentrations results in high resolution separations for large nucleic acid molecules.

In one aspect of the invention, provided is a method of separating nucleic acids by length, the method comprising: (a) loading a sample comprising one or more nucleic acids into a capillary containing a separation matrix comprising a formamide-soluble polymer and a formamide running buffer; and (b) applying a voltage to the capillary, thereby separating the one or more nucleic acids by length.

In one aspect of the invention, provided is a method of determining the length of one or more nucleic acids in a sample using a capillary electrophoresis device, the method comprising: (i) denaturing the sample containing the one or more nucleic acids in a formamide solution; (ii) loading the denatured sample from step (i) into a capillary containing a non-aqueous separation matrix; and (iii) applying a voltage to the loaded capillary of step (ii) so as to migrate the nucleic acid through the capillary, thereby determining the length of the nucleic acid in the sample, wherein the inner surface of the capillary is deactivated to block any silanol functional groups on the inner surface of the capillary and wherein the separation matrix comprises a formamide- soluble polymer.

In one aspect of the invention, provided is a method of determining the purity of a sample containing one or more nucleic acids, the method comprising separating the nucleic acids in the sample by length using a method described above, quantitating (or determining) the amount of nucleic acid of interest in the sample, and comparing the amount of nucleic acid of interest in the sample to a reference standard, thereby determining the purity of the sample.

In another aspect of the invention, provided is a non-aqueous separation matrix for capillary electrophoresis of denatured nucleic acids. In one aspect, the separation matrix comprises a formamide-soluble polymer dissolved in a non-aqueous buffer.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A and IB: Figures 1 A and 1B show CE separation of an RNA ladder (200, 500, 1000, 1500, 2000, 3000, 4000, and 6000 nt) using an aqueous BGE (0.16% w/v PEO (MW > 5 MDa), 100 mM MES, 1 mM EDTA, pH = 6) (Figure 1A) and formamide BGE (0.16% w/v PEO (MW > 5 MDa), 100 mM MES, 1 mM EDTA, pH = 6) (Figure 1B). PVA-coated capillary was used.

Figure 2 shows the correlation of the migration time and the number of nucleotides when 0.16% w/v PEO (MW > 5 MDa) formamide BGE (100 mM MES, 1 mM EDTA, pH = 6) is used. PVA-coated capillary was used. The number of nucleotides is on the Y axis and migration time is on the X axis.

Figures 3A - 3C are electropherograms of RNA ladder separation by CE (200, 500,

1000, 1500, 2000, 3000, 4000, and 6000 nt) using HEC (MW 1.3 MDa; Figure 3A), PAA (MW 5 MDa; Figure 3B), or PEO (MW > 5 MDa; Figure 3C) polymers (0.16%, w/v) in formamide BGE (100 mM MES, 1 mM EDTA, pH = 6). DMDCS-treated capillary was used.

Figure 4 shows efficiency (average plate number of the six RNAs with 200, 500, 1000, 1500, 2000, 3000, 4000 and 6000 nt) using different polymer gels prepared with HEC (MW 1.3 MDa), PAA (MW, 5 MDa) and PEO (MW > 5 MDa) polymers (0.16%, w/v) in formamide BGE (100 mM MES, 1 mM EDTA, pH = 6). DMDCS-treated capillary was used. Error bars represent standard deviation.

Figures 5A - 5C show electropherograms of RNA ladder separation by CE (200, 500, 1000, 1500, 2000, 3000, 4000 and 6000 nt) using PEO (0.25%, w/v) with molecular weight of 100 kE)a (Figure 5A), 600 kE)a (Figure 5B) and > 5 MDa (Figure 5C) in formamide BGE (100 mM MES, 1 mM EDTA, pH = 6). DMDCS-treated capillary was used.

Figure 6 shows separation factor (Y axis) of RNA ladder separated by CE (1500/2000 nt and 2000/3000 nt) using PEO (0.25%, w/v) of differing molecular weight (100 kDa, 600 kDa and > 5 MDa) in formamide BGE (100 mM MES, 1 mM EDTA, pH = 6). DMDCS-treated capillary was used. Error bars represent standard deviation.

Figures 7A - 7F are electropherograms of RNA ladder separated by CE (200, 500, 1000, 1500, 2000, 3000, 4000 and 6000 nt) using PEO (MW > 5 MDa) gels with different

concentration, 0.025% (Figure 7A), 0.05% (Figure 7B), 0.1% (Figure 7C), 0.16% (Figure 7D), 0.25% (Figure 7E) and 0.5% (Figure 7F), in formamide BGE (100 mM MES, 1 mM EDTA, pH = 6). DMDCS-treated capillary was used.

Figure 8 shows separation factor of RNA ladder (1500/2000 nt and 2000/3000 nt) using PEO (MW > 5 MDa) gels containing different concentrations of PEO, 0.025% w/v, 0.05% w/v, 0.1% w/v, 0.16% w/v, 0.25% w/v and 0.5% w/v, in formamide BGE (100 mM MES, 1 mM EDTA, pH = 6). DMDCS-treated capillary was used. Error bars represent standard deviation.

Figure 9 shows efficiency (plate number) of RNA ladder separated by CE (200, 500, 1000, 1500, 2000, 3000, 4000 and 6000 nt) using gels containing different concentrations of PEO (MW > 5 MDa), 0.025% w/v, 0.05% w/v, 0.1% w/v, 0.16% w/v, 0.25% w/v and 0.5% w/v, in formamide BGE (100 mM MES, 1 mM EDTA, pH = 6). DMDCS-treated capillary was used. Error bars represent standard deviation.

Figure 10 is a double logarithmic plot of RNA length and mobility. RNA ladder includes RNAs with 200, 500, 1000, 1500, 2000, 3000, 4000, and 6000 nt. Experiment was performed using a PEO gel (MW > 5MDa) containing difference concentrations of PEO (0.025% w/v, 0.05% w/v, 0.1% w/v, 0.16% w/v, 0.25% w/v, and 0.5% w/v, in formamide BGE (100 mM MES, 1 mM EDTA, pH = 6)). DMDCS-treated capillary was used. DETAILED DESCRIPTION OF THE INVENTION

I. Definitions and Abbreviations

As used throughout the specification and appended claims, the following abbreviations apply:

BGE: Background electrolyte

CE: Capillary Electrophoresis

DTPA: Diethylenetriamine pentaacetic acid

DMDCS: Dimethyl dichlorosilane

EDTA: Ethylenediaminetetraaeetie acid

EGTA: Ethylene glycol-bis(P-ami noethyl ether)-N,N,N',N'-tetraacetic acid

HMDS : 1,1,1 ,3 ,3 ,3 -Hexamethyldisilazane

HEC: 2-Hydroxy ethyl cellulose

MES: 2-(N-Morpholino)ethanesulfonic acid

mRNA: Messenger RNA

nt: Nucleotide

NTA: Nitrilotriacetic acid

PAA: Polyacrylamide

PEO: Polyethylene oxide (also known as polyethylene glycol“PEG” or polyoxyethylene “POE”)

PIPES: Piperazine-N,N'-bis(2-ethanesulfonic acid)

TAPS: [Tris(hydroxymethyl)methylamino]propanesulfonic acid

TMCS: Trimethyl chlorosilane

TMS: Trimethyl silyl

So that the invention may be more readily understood, certain technical and scientific terms are specifically defined below. Unless specifically defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.

Reference to“or” indicates either or both possibilities unless the context clearly dictates one of the indicated possibilities. In some cases,“and/or” was employed to highlight either or both possibilities. As used herein, including the appended claims, the singular forms of words such as "a", "an", and "the" include their corresponding plural references unless the context clearly dictates otherwise.

The term "about", when modifying the quantity of a substance, the pH of a solution / formulation, or the value of a parameter characterizing a step in a method, or the like refers to variant in the numerical quantity that can occur, for example, through typical measuring, handling and sampling procedures involved in the preparation, characterization and/or use of the substance or composition; through inadvertent error in these procedures, through differences in the manufacture, source or purity of the ingredients employed to make or use the compositions or carryout the procedures and the like. In certain embodiments,“about” can mean a variation of greater or lesser than the value or range of values stated by 10 percent, e.g ., ± 0.2%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 10%. Each value or range of values preceded by the term "about" is also intended to encompass the embodiment of the stated absolute value or range of values.

“Comprising” or variations such as“comprise”,“comprises” or“comprised of’ are used throughout the specification and claims in an inclusive sense, i.e., to specify the presence of the stated features but not to preclude the presence or addition of further features that may materially enhance the operation or utility of any of the embodiments of the invention, unless the context requires otherwise due to express language or necessary implication.

"Consists essentially of and variations such as "consist essentially of or "consisting essentially of, as used throughout the specification and claims, indicate the inclusion of any recited elements or group of elements, and the optional inclusion of other elements, of similar or different nature than the recited elements, that do not materially change the basic or novel properties of the specified composition or method.

II. Aspects of the Invention

Provided herein is a method of separating large nucleic acid molecules, for example, mRNA, with high efficiency and resolution. Such method can be used, for example, to determine the purity of an mRNA sample. Separation of RNA based on size difference (i.e., separating RNAs of differing lengths from each other) can be challenging for large RNA molecules because the size difference of large fragments or impurities compared to intact or full- length RNA is not as significant as for small RNAs. Although methods such as ion pair chromatography and chip-based devices have been developed for RNA analysis, most of these methods have limited separation efficiencies for larger RNAs. Capillary electrophoresis (CE) provides high separation efficiency and can be a powerful tool for the analysis of RNA.

However, due to the size of large RNA molecules ( e.g ., mRNA or RNA ~ 1000 nt or larger), traditional analysis by CE is challenging. Large RNA molecules can form secondary structures, which causes size heterogeneity and peak broadening. In one aspect of the invention, this problem is solved by running CE under denaturing conditions. Linder ideal denaturing conditions, any secondary structures of RNA are disrupted and the separation is only dependent on the number of nucleotides. The peak shape thus can be improved, which also results in more accurate separation of large RNA fragments or impurities from the intact RNA and also provides a more accurate determination of purity. As described in the examples herein, the separation efficiency and resolution of large RNA molecules is improved using, for example, 100% formamide instead of aqueous buffer as a denaturing background electrolyte (BGE). Although formamide has previously been added to aqueous BGE to denature RNA, the percentage of formamide added was limited due to efficiency loss. It is believed that BGEs and gels with 100% formamide as solvent have never been used in CE for RNA analysis.

In another aspect of the invention, a non-aqueous gel comprising a low concentration of polymer is used as a separation matrix so as to achieve high separation efficiency. For large RNAs analyzed under denaturing conditions, broad peaks may still be observed. One possible reason is that the denaturing is incomplete since large RNAs require stronger denaturants compared to small RNA. Another possible reason is the high gel concentration that is usually used for large RNA CE analysis. Gels with high concentration have higher viscosity. The radial distribution of temperature can cause variation of viscosity of the gel, especially for high concentration gels. Because the electromobility of analytes is strongly dependent on viscosity of the matrix, heterogeneity of the viscosity in the gel can lead to larger distribution of

electromobilities for the same analyte and thus the separation efficiency is lowered. In addition to providing increased separation efficiency, using low concentration gel has other advantages, e.g., it is easy to degas the gel and remove particulates, and filling of the gel into longer and narrower-ID capillaries is easier. Thus, in another aspect of the invention, a low concentration non-aqueous gel is used, wherein the gel is comprised of a formamide-soluble polymer. As used herein, a formamide-soluble polymer is a polymer which, at the specified percentage of polymer, is soluble in formamide such that it can be dissolved to form a separation matrix (e.g, a gel).

The present disclosure provides methods for determining the length of one or more nucleic acids in a sample using a capillary electrophoresis device with formamide as a nucleic acid denaturing agent and a non-aqueous separation matrix comprising a formamide-soluble polymer. Such methods allow for high separation efficiency of, for example, large RNA molecules (e.g, RNA having 1000 nt or more) which differ from each other by, for example, as few as 10 nt.

Thus, in one embodiment, provided are methods of separating nucleic acids by length, the methods comprising: (a) loading a sample comprising one or more nucleic acids into a capillary containing a separation matrix comprising a formamide-soluble polymer and a formamide running buffer; and (b) applying a voltage to the capillary, thereby separating the one or more nucleic acids by length. Such methods can be used to determine the purity or quality of a nucleic acid in a sample, for example, by separating the nucleic acid from nucleic acids of different size (e.g., any smaller fragments of that nucleic acid). In this way, the methods disclosed herein can be used as a quality control for any nucleic acid production and/or purification process. For example, the methods disclosed herein can be used to determine the purity of an mRNA.

In another embodiment, provided is a method for determining the length of nucleic acids contained in a sample using a capillary electrophoresis device, the method comprising: (i) denaturing the nucleic acid in the sample in a formamide solution; (ii) loading the denatured sample from step (i) into a capillary containing a non-aqueous separation matrix; and (iii) applying a voltage to the loaded capillary of step (ii) so as to migrate the nucleic acid through the capillary, thereby separating the nucleic acids contained in the sample by length, wherein the inner surface of the capillary is treated to block any silanol functional groups on the inner surface of the capillary and wherein the separation matrix comprises a formamide-soluble polymer.

In another embodiment, provided is a method of determining the purity of a nucleic acid in a sample using a capillary electrophoresis device, the method comprising: (i) denaturing the nucleic acid in the sample in a formamide solution; (ii) loading the denatured sample from step (i) into a capillary containing a non-aqueous separation matrix; (iii) applying a voltage to the loaded capillary of step (ii) so as to migrate the nucleic acid through the capillary, thereby separating the nucleic acid molecules contained in the sample by length, wherein the inner surface of the capillary is treated to block any silanol functional groups on the inner surface of the capillary and wherein the separation matrix comprises a formamide-soluble polymer, and (iv) quantitating (or determining) the amount of nucleic acid in the sample compared to the amount of nucleic acid fragments in the sample. As a non-limiting example, the purity of a sample containing a nucleic acid of 2000 nt in length is determined by separating the nucleic acids in the sample by the methods described herein and determining the amount of (or detecting the presence of) the nucleic acid of interest ( e.g ., the nucleic acid of 2000 nt) as well as nucleic acid molecules that are longer or shorter than the nucleic acid of interest.

In one embodiment, the nucleic acid contained in the sample is DNA or RNA. In another aspect, the nucleic acid is RNA. In a further aspect, the RNA is mRNA. In one aspect, the nucleic acid sample comprises at least 200 nt. In another embodiment, the nucleic acid comprises at least 1500 nt. In another embodiment, the nucleic acid comprises at least 2000 nt.

In a further embodiment, the nucleic acid comprises at least 2500 nt.

In one embodiment, the length of the nucleic acid is at least 1500 nt. In another embodiment, the length of the nucleic acid is between 1000 nt and 6000 nt.

In one embodiment, the sample comprises a lipid nanoparticle containing a nucleic acid. In one embodiment, the nucleic acid is encapsulated in the lipid nanoparticle. In another embodiment, the lipid nanoparticle comprises a cationic lipid, cholesterol or a derivative thereof, a phospholipid, and a PEGylated lipid.

In one embodiment, the formamide solution of step (i) comprises 0-15% or 0-10% w/v of a surfactant. In another embodiment, the formamide solution of step (i) comprises 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% (w/v) of the surfactant. In a further embodiment, the formamide solution comprises 10% w/v of the surfactant. In another embodiment wherein the sample comprises a nucleic acid encapsulated in an LNP, the surfactant is present in an amount sufficient to disrupt the LNP encapsulating the nucleic acid. In another embodiment, the surfactant comprises a polyoxyethylene fatty ether derived from lauryl, cetyl, stearyl, or oleyl alcohols. In a further embodiment, the polyoxyethylene fatty ether is polyoxyethylene hexadecyl ether or polyoxyethylene lauryl ether. In some embodiments, the surfactant is a zwitterionic surfactant. In other embodiments, the surfactant is a non-ionic surfactant. Non-limiting examples of such non-ionic surfactants include BRIJ® 58 (polyoxyethylene cetyl ether) and BRIJ® 35 (polyoxyethylene lauryl ether), each available from Sigma Aldrich. In another embodiment, the formamide solution of step (i) comprises 10% w/v polyoxyethylene cetyl ether or polyoxyethylene lauryl ether.

In one embodiment of the method, the capillary is treated with dimethyldichlorosilane (“DMDCS”) in l,l,l,3,3,3-hexamethyldisilazane, trimethylchlorosilane, pyridine (3: 1 :9). In one embodiment, the capillary is coated with about 0.5 - 15 % v/v, 0.5 - 20% v/v, or 0.5 - 30% v/v DMDCS in l,l,l,3,3,3-hexamethyldisilazane, trimethylchlorosilane, pyridine (3:1 :9). In one embodiment, the capillary is coated with about 0.5 - 15 % v/v DMDCS in 1, 1,1, 3,3,3- hexamethyldisilazane, trimethylchlorosilane, pyridine (3:1 :9). In another embodiment, the capillary is coated with about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15% , 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% v/v DMDCS in l,l,l,3,3,3-hexamethyldisilazane, trimethylchlorosilane, pyridine (3:1 :9). In one embodiment, the capillary is coated with about 10% v/v DMDCS in 1, 1,1, 3,3,3- hexamethyldisilazane, trimethylchlorosilane, pyridine (3:1 :9). 1, 1,1, 3,3,3- hexamethyldisilazane, trimethylchlorosilane, pyridine (3: 1 :9), also known as SYLON® HTP, available from Sigma-Aldrich, contains l,l,l,3,3,3-hexamethyldisilazane, trimethylchlorosilane, and pyridine in a molar ratio of 3 : 1 :9.

In one embodiment, the separation matrix comprises about 0.1% to about 0.5% w/v of a formamide-soluble polymer dissolved in a non-aqueous formamide-containing buffer. In one embodiment of the method, the separation matrix comprises about 0.1% to about 0.25% w/v of a formamide-soluble polymer dissolved in a non-aqueous formamide-containing buffer. In one embodiment, the formamide-soluble polymer is present in an amount of about 0.16% w/v to about 0.25% w/v. In a further embodiment, the separation matrix comprises about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 0.10%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.20%, 0.21%, 0.22%, 0.23%, 0.24%, 0.25%, 0.26%, 0.27%,

0.28%, 0.29%, 0.30%, 0.31%, 0.32%, 0.33%, 0.34%, 0.35%, 0.36%, 0.37%, 0.38%, 0.39%,

0.40%, 0.41%, 0.42%, 0.43%, 0.44%, 0.45%, 0.46%, 0.47%, 0.48%, 0.49%, or 0.50% w/v of a formamide-soluble polymer. In one embodiment, the formamide-soluble polymer is 2- hydroxyethyl cellulose (HEC), polyacrylamide (PAA), or polyethylene oxide (PEO). In another embodiment, the PEO has a molecular weight of 600 kDa or greater. In a further embodiment, the PEO has a molecular weight of 600 kDa to > 5 MDa. In another embodiment, the separation matrix comprises from about 0.1% w/v to about 0.25 w/v% PAA having a molecular weight of 5 MDa as the formamide-soluble polymer. In another aspect, the separation matrix comprises from about 0.1% w/v to about 0.25% w/v PEO having a molecular weight of 600 kDa as the formamide-soluble polymer. In a further embodiment, the separation matrix comprises from about 0.1% w/v to about 0.25% w/v PEO having a molecular weight of > 5 MDa as the formamide-soluble polymer.

In another embodiment, the formamide-containing buffer comprises a buffering agent, a chelator, and a pH-adjusting agent dissolved in formamide. In one embodiment, the buffering agent is a zwitterionic buffering agent providing a final pH of 6.0 to 8.0. Non-limiting examples of buffering agents include, for example, MES, PIPES, and TAPS. In a further embodiment, the buffering agent is MES. In one embodiment, the chelator is EDTA, DTP A, or NTA. In a further embodiment, the chelator is EDTA. In one embodiment, the pH-adjusting agent is an acid or a base which is used to adjust the pH higher or lower to achieve the desired pH value. In another embodiment, the pH-adjusting agent is NaOH or HC1. In another embodiment, the pH-adjusting agent is NaOH. In a further embodiment, the buffering agent is MES, the chelator is EDTA, and the pH-adjusting agent is NaOH. In another embodiment, the pH of the formamide buffer is from about 6.0 to about 8.0. In one embodiment, the pH of the formamide buffer is about 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0. In another embodiment, the pH of the formamide buffer is from about 5.0 to about 7.0. In one embodiment, the pH of the formamide buffer is about: 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8,

5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0. In a further embodiment, the pH of the formamide buffer is about 6.0.

In one embodiment, the method further comprises use of an interference filter on the diode array detector to illuminate the capillary. In one embodiment, the interference filter illuminates the capillary with UV light within a range of 250 - 270 nm.

In one embodiment, the method provides separation factor of 70 nt or less for nucleic acids comprising 1500 nt or more. Separation factor is calculated based on the resolution of the peaks. Separation factor can be calculated using the following equation Equation 1

where S is the separation factor, DN is the number of bases separating two peaks of interest, and R _s is the resolution of the two peaks. Separation factor estimates the length difference that can be separated by the method when the resolution equals to 1. In one embodiment, the separation factor is 100 nt or less. In one embodiment, the separation factor is 70 nt or less for nucleic acids comprising 1500 nt or more. In another embodiment, the separation factor is between 30 and 70 nt for nucleic acids comprising 1500 nt or more. In one embodiment, the separation factor is 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 60, 75, 80, 85, 90, 95, or 100 nt. In another

embodiment, the method provides a separation factor of about 70 nt for nucleic acids comprising 1500 nt or more. In another embodiment, the method provides a separation factor of about 30 nt for nucleic acids comprising 1500 nt or more. In another embodiment, the separation factor for nucleic acids comprising 2000 to 3000 nt is about 70 nt or less. In another embodiment, the separation factor for nucleic acids comprising 1500 to 2000 nt is about 30 nt or less.

The disclosure also provides a non-aqueous separation matrix for capillary

electrophoresis of denatured nucleic acids. In one aspect, the separation matrix comprises a formamide-soluble polymer dissolved in a non-aqueous buffer. In one embodiment, the separation matrix comprises about 0.1% to about 0.50% w/v of a formamide-soluble polymer dissolved in a formamide-containing buffer. In one embodiment, the separation matrix comprises about 0.1% to about 0.25% w/v of a formamide-soluble polymer dissolved in a formamide-containing buffer. In one embodiment, the formamide-soluble polymer is present in an amount of 0.16% w/v to 0.25% w/v. In a further embodiment, the separation matrix comprises about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 0.10%, 0.11%,

0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.20%, 0.21%, 0.22%, 0.23%, 0.24%, 0.25% , 0.26%, 0.27%, 0.28%, 0.29%, 0.30%, 0.31%, 0.32%, 0.33%, 0.34%, 0.35%, 0.36%, 0.37%, 0.38%, 0.39%, 0.40%, 0.41%, 0.42%, 0.43%, 0.44%, 0.45%, 0.46%, 0.47%, 0.48%, 0.49%, or 0.50% w/v of a formamide-soluble polymer. In one embodiment, the formamide-soluble polymer is 2-hydroxyethyl cellulose (HEC), polyacrylamide (PAA), or polyethylene oxide (PEO). In another embodiment, the PEO has a molecular weight of 600 kDa or greater. In a further embodiment, the PEO has a molecular weight of 600 kDa to > 5 MDa.

In another embodiment, the separation matrix comprises from about 0.1% w/v to about 0.25 w/v% PAA having a molecular weight of 5 MDa as the formamide-soluble polymer. In another aspect, the separation matrix comprises from about 0.1% w/v to about 0.25% w/v PEO having a molecular weight of 600 kDa as the formamide-soluble polymer. In a further embodiment, the separation matrix comprises from about 0.1% w/v to about 0.25% w/v PEO having a molecular weight of > 5 MDa as the formamide-soluble polymer.

In one aspect of the separation matrix, the non-aqueous formamide-containing buffer comprises a buffering agent, a chelator, and a pH adjusting agent dissolved in 100% formamide. In one embodiment, the buffering agent is a zwitterionic buffering agent between pH of 6.0 to 8.0. Non-limiting examples of buffering agents include, for example, MES, PIPES, and TAPS.

In a further embodiment, the buffering agent is MES. In one embodiment, the chelator is EDTA, DTPA and NTA. In a further embodiment, the chelator is EDTA. In one embodiment, the pH adjusting agent is an acid or a base. In one embodiment, the pH adjusting agent is NaOH or HC1. In another embodiment, the pH adjusting agent is NaOH. In a further embodiment, the buffering agent is MES, the chelator is EDTA, and the pH adjusting agent is NaOH. In another

embodiment, the pH of the formamide buffer is from about 6.0 to about 8.0. In one embodiment, the pH of the formamide buffer is about 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6., 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0. In a further embodiment, the pH of the formamide buffer is about 6.0.

In a further embodiment, the separation matrix is contained within a silica capillary coated with l,l,l,3,3,3-hexamethyldisilazane, trimethylchlorosilane, pyridine (3: 1 :9). In one embodiment, the capillary is coated with about 0.5 - 15 % v/v, 0.5 - 20% v/v, or 0.5 - 30% v/v DMDCS in l,l,l,3,3,3-hexamethyldisilazane, trimethylchlorosilane, pyridine (3:1 :9). In one embodiment, the capillary is coated with about 0.5 - 15 % v/v DMDCS in 1, 1,1, 3,3,3- hexamethyldisilazane, trimethylchlorosilane, pyridine (3:1 :9). In another embodiment, the capillary is coated with about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15% , 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% v/v DMDCS in l,l,l,3,3,3-hexamethyldisilazane, trimethylchlorosilane, pyridine (3:1 :9). In one embodiment, the capillary is coated with about 10% w/v DMDCS in 1, 1,1, 3,3,3- hexamethyldisilazane, trimethylchlorosilane, pyridine (3:1 :9). 1, 1,1, 3,3,3- Hexamethyldisilazane, trimethylchlorosilane, pyridine (3:1 :9), also known as SYLON® HTP, available from Sigma-Aldrich, contains l,l,l,3,3,3-hexamethyldisilazane, trimethylchlorosilane, and pyridine in a molar ratio of 3 : 1 :9.

III. Specific Embodiments of the Invention

Embodiment 1. A method of determining the length of one or more nucleic acids contained in a sample using a capillary electrophoresis device, the method comprising: (i) denaturing the sample containing the nucleic acids in a formamide solution; (ii) loading the denatured sample from step (i) into a capillary containing a non-aqueous separation matrix; and (iii) applying a voltage to the loaded capillary of step (ii) so as to migrate the denatured nucleic acids through the capillary, thereby determining the molecular weight of the nucleic acids in the sample, wherein the inner surface of the capillary is coated to block any silanol functional groups on the inner surface of the capillary and wherein the separation matrix comprises a formamide- soluble polymer.

Embodiment 2. The method of embodiment 1, wherein the nucleic acids comprise DNA, RNA, or both.

Embodiment 3. The method of embodiment 1 or 2, wherein the nucleic acids comprise

RNA.

Embodiment 4. The method of any one of embodiments 1-3, wherein the nucleic acids comprise mRNA.

Embodiment 5. The method of any one of embodiments 1-4, wherein at least one of the nucleic acids comprise at least 200 nucleotides.

Embodiment 6. The method of any one of embodiments 1-5, wherein at least one of the nucleic acids comprise at least 1500 nucleotides.

Embodiment 7. The method of any one of embodiments 1-6, wherein at least one of the nucleic acids comprise at least 2000 nucleotides.

Embodiment 8. The method of any one of embodiments 1-7, wherein at least one of the nucleic acids comprise at least 2500 nucleotides.

Embodiment 9. The method of any one of embodiments 1-8, wherein the sample containing the one or more nucleic acids further comprises a lipid nanoparticle. Embodiment 10. The method of embodiment 9, wherein the lipid nanoparticle comprises a cationic lipid, cholesterol or a derivative thereof, a phospholipid, a PEGylated lipid, or a combination thereof.

Embodiment 11. The method of any one of embodiments 1-10, wherein the formamide solution of step (i) comprises 0-10% v/v of a surfactant.

Embodiment 12. The method of embodiment 11, wherein the surfactant comprises a polyoxyethylene alkyl ether.

Embodiment 13. The method of embodiment 12, wherein the polyoxyethylene alkyl ether is polyoxyethylene cetyl ether or polyoxyethylene lauryl ether.

Embodiment 14. The method of any one of embodiments 1-10, wherein the formamide solution of step (i) comprises 10% v/v polyoxyethylene cetyl ether or polyoxyethylene lauryl ether.

Embodiment 15. The method of any one of embodiments 1-14, wherein the capillary is coated with 10% v/v dimethyldichlorosilane in l,l,l,3,3,3-hexamethyldisilazane,

trimethyl chlorosilane, pyridine (3 : 1 :9).

Embodiment 16. The method of any one of embodiments 1-15, wherein the separation matrix comprises about 0.1% w/v to about 0.25% w/v of a formamide-soluble polymer dissolved in a formamide buffer.

Embodiment 17. The method of embodiment 16, wherein the formamide buffer comprises a buffering agent dissolved in formamide, a chelator, and a pH-adjusting agent.

Embodiment 18. The method of embodiment 17, wherein the buffering agent is MES, the chelator is EDTA, and the pH-adjusting agent is NaOH.

Embodiment 19. The method of any one of embodiments 16-18, wherein the pH of the formamide buffer is from about 6.0 to about 8.0.

Embodiment 20. The method of any one of embodiments 16-18, wherein the pH of the formamide buffer is about 6.0. Embodiment 21. The method of any one of embodiments 16-20, wherein the formamide- soluble polymer is 2-hydroxyethyl cellulose (HEC), polyacrylamide (PAA), or polyethylene oxide (PEO).

Embodiment 22. The method of any one of embodiments 16-21, wherein the formamide- soluble polymer is PEO and has a molecular weight of 600 kDa or greater.

Embodiment 23. The method of any one of embodiments 16-22, wherein the formamide- soluble polymer is present in an amount of about 0.16% w/v to about 0.25% w/v.

Embodiment 24. The method of any one of embodiments 16-21, wherein the separation matrix comprises from about 0.1% w/v to about 0.25 w/v% PAA having a molecular weight of 5 MDa as the formamide-soluble polymer.

Embodiment 25. The method of any one of embodiments 16-21, wherein the separation matrix comprises from about 0.1% w/v to about 0.25% w/v PEO having a molecular weight of 600 kDa as the formamide-soluble polymer.

Embodiment 26. The method of any one of embodiments 16-21, wherein the separation matrix comprises from about 0.1% w/v to about 0.25% w/v PEO having a molecular weight of >

5 MDa as the formamide-soluble polymer.

Embodiment 27. The method of any one of embodiments 1-26, the method further comprising use of an interference filter on the diode array detector to illuminate the capillary.

Embodiment 28. The method of embodiment 27, wherein the interference filter illuminates the capillary with ETV light within a range of 250-270 nm.

Embodiment 29. The method of any one of embodiments 1-28, wherein the separation factor is 70 nucleotides or less for nucleic acids comprising 1500 nucleotides or more.

Embodiment 30. The method of embodiment 29, wherein the separation factor is from about 30 nucleotides to about 70 nucleotides for nucleic acids comprising 1500 nucleotides or more.

Embodiment 31. A non-aqueous separation matrix for capillary electrophoresis of denatured nucleic acids, the matrix comprising a formamide-soluble polymer dissolved in a non- aqueous buffer. Embodiment 32. The non-aqueous separation matrix of embodiment 31, wherein the formamide buffer comprises a buffering agent, a chelator, and a pH-adjusting agent dissolved in formamide.

Embodiment 33. The non-aqueous separation matrix of embodiment 32, wherein the buffering agent is MES, the chelator is EDTA, and the pH-adjusting agent is NaOH.

Embodiment 34. The non-aqueous separation matrix of embodiment 32 or 33, wherein the pH of the formamide buffer is from about 6.0 to about 8.0.

Embodiment 35. The non-aqueous separation matrix of embodiment 34, wherein the pH of the formamide buffer is about 6.0.

Embodiment 36. The non-aqueous separation matrix of any one of embodiments 31-35, wherein the formamide-soluble polymer is 2-hydroxyethyl cellulose (HEC), polyacrylamide (PAA), or polyethylene oxide (PEO), optionally, wherein the PEO has a molecular weight of 600 kDa or greater.

Embodiment 37. The non-aqueous separation matrix of any one of embodiments 31-36, wherein the formamide-soluble polymer is present in an amount of about 0.16% w/v to about 0.25% w/v.

Embodiment 38. The non-aqueous separation matrix of any one of embodiments 31-35, wherein the separation matrix comprises from about 0.1% w/v to about 0.25 w/v% PAA having a molecular weight of 5 MDa as the formamide-soluble polymer.

Embodiment 39. The non-aqueous separation matrix any one of embodiments 31-35, wherein the separation matrix comprises from about 0.1% w/v to about 0.25% w/v PEO having a molecular weight of 600 kDa as the formamide-soluble polymer.

Embodiment 40. The non-aqueous separation matrix any one of embodiments 31-35, wherein the separation matrix comprises from about 0.1% w/v to about 0.25% w/v PEO having a molecular weight of > 5 MDa as the formamide-soluble polymer.

Embodiment 41. The non-aqueous separation matrix of any one of embodiments 31-40, wherein the separation matrix is contained within a silica capillary coated with 1,1,1 ,3,3,3- hexamethyldisilazane, trimethylchlorosilane, pyridine (3: 1 :9) or 10% dimethyldichlorosilane in l,l,l,3,3,3-hexamethyldisilazane, trimethylchlorosilane, pyridine (3: 1 :9). Embodiment 42. A method of separating nucleic acids by length, the method comprising:

(a) loading a sample comprising one or more nucleic acids into a capillary containing a separation matrix comprising a formamide-soluble polymer and a formamide running buffer; and

(b) applying a voltage to the capillary, thereby separating the one or more nucleic acids by length.

Embodiment 43. The method of embodiment 42, wherein the one or more nucleic acids comprise DNA, RNA, or a combination thereof.

Embodiment 44. The method of embodiment 42 or 43, wherein the one or more nucleic acids comprise DNA.

Embodiment 45. The method of any one of embodiments 42-44, wherein the one or more nucleic acids comprise RNA.

Embodiment 46. The method of any one of embodiments 42-45, wherein the one or more nucleic acids comprise mRNA.

Embodiment 47. The method of any one of embodiments 42-46, wherein at least one of the nucleic acids is at least 100 nts, 200 nts, 300 nts, 400 nts, 500 nts, 750 nts, 1000 nts, 1500 nts, 2000 nts, 2500 nts, 3000 nts, 4000 nts, 5000 nts, 6000 nts, 7500 nts, or 10,000 nts in length.

Embodiment 48. The method of any one of embodiments 42-46, wherein at least one of the nucleic acids is at least 1000 nts in length.

Embodiment 49. The method of any one of embodiments 42-46, wherein at least one of the nucleic acids is at least 1500 nts in length.

Embodiment 50. The method of any one of embodiments 42-46, wherein at least one of the nucleic acids is at least 2000 nts in length.

Embodiment 51. The method of any one of embodiments 42-50, wherein the capillary is a silica capillary.

Embodiment 52. The method of any one of embodiments 42-51, wherein an inner surface of the capillary comprises a coating.

Embodiment 53. The method of embodiment 52, wherein the coating blocks silanol functional groups. Embodiment 54. The method of embodiment 52 or 53, wherein the coating is a dimethyldichlorosilane coating, a trimethylchlorosilane coating, or a combination thereof.

Embodiment 55. The method of any one of embodiments 52-54, wherein the coating is formed by contacting the capillary with l,l,l,3,3,3-hexamethyldisilazane, trimethylchlorosilane, pyridine (3: 1 :9).

Embodiment 56. The method of any one of embodiments 52-54, wherein the coating is formed by contacting the capillary with 10% v/v dimethyldichlorosilane in 1, 1,1, 3,3,3- hexamethyldisilazane, trimethylchlorosilane, pyridine (3: 1 :9).

Embodiment 57. The method of any one of embodiments 42-56, further comprising determining a length for at least one of the nucleic acids.

Embodiment 58. The method of embodiment 57, wherein the sample further comprises a nucleic acid ladder, and wherein determining the length for the at least one nucleic acid comprises comparing a migration time for the at least one nucleic acid to migration times for the nucleic acid ladder.

Embodiment 59. The method of any one of embodiments 42-58, further comprising denaturing the sample in a formamide solution prior to loading.

Embodiment 60. The method of embodiment 59, wherein the formamide solution comprises less than 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% water.

Embodiment 61. The method of embodiment 59, wherein the formamide solution comprises less than 0.1% water.

Embodiment 62. The method of any one of embodiments 59-61, wherein the formamide solution comprises a surfactant.

Embodiment 63. The method of embodiment 62, wherein the surfactant comprises a non ionic surfactant, a zwitterionic surfactant, or both.

Embodiment 64. The method of embodiment 62, wherein the surfactant comprises a non ionic surfactant.

Embodiment 65. The method of any one of embodiments 62-64, wherein the surfactant comprises a polyoxyethylene alkyl ether. Embodiment 66. The method of embodiment 65, wherein the polyoxyethylene alkyl ether is polyoxyethylene cetyl ether or polyoxyethylene lauryl ether.

Embodiment 67. The method of any one of embodiments 62-66, wherein the surfactant is present in the formamide solution at up to about 15% w/v.

Embodiment 68. The method of any one of embodiments 62-66, wherein the surfactant is present in the formamide solution at up to about 10% w/v.

Embodiment 69. The method of any one of embodiments 62-66, wherein the surfactant is present in the formamide solution at about: 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% w/v.

Embodiment 70. The method of any one of embodiments 62-66, wherein the surfactant is present in the formamide solution at about 10% w/v.

Embodiment 71. The method of any one of embodiments 62-66, wherein the surfactant comprises polyoxyethylene cetyl ether or polyoxyethylene lauryl ether, and wherein the surfactant is present at 10% w/v in the formamide solution.

Embodiment 72. The method of any one of embodiments 62-71, wherein the sample further comprises one or more lipid nanoparticles (LNPs), and wherein the surfactant is present in the formamide solution in an amount sufficient to disrupt the LNPs.

Embodiment 73. The method of embodiment 72, wherein the LNPs comprise a cationic lipid, cholesterol or a derivative thereof, a phospholipid, a PEGylated lipid, or a combination thereof.

Embodiment 74. The method of any one of embodiments 42-73, wherein the formamide- soluble polymer comprises 2-hydroxyethyl cellulose (ELEC), polyacrylamide (PAA),

polyethylene oxide (PEO), or a combination thereof.

Embodiment 75. The method of any one of embodiments 42-73, wherein the formamide- soluble polymer comprises 2-hydroxyethyl cellulose (ELEC).

Embodiment 76. The method of any one of embodiments 42-73, wherein the formamide- soluble polymer comprises polyacrylamide (PAA). Embodiment 77. The method of any one of embodiments 42-73, wherein the formamide- soluble polymer comprises polyethylene oxide (PEO).

Embodiment 78. The method of any one of embodiments 42-77, wherein the formamide- soluble polymer has a molecular weight of 600 kDa or greater.

Embodiment 79. The method of any one of embodiments 42-77, wherein the formamide- soluble polymer has a molecular weight of 5 MDa or greater.

Embodiment 80. The method of any one of embodiments 42-79, wherein the formamide- soluble polymer is present in the separation matrix at about 0.1% w/v to about 0.25% w/v.

Embodiment 81. The method of any one of embodiments 42-79, wherein the formamide- soluble polymer is present in the separation matrix at about 0.16% w/v to about 0.25% w/v.

Embodiment 82. The method of any one of embodiments 42-81, wherein the formamide running buffer comprises less than 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% water.

Embodiment 83. The method of any one of embodiments 42-81, wherein the formamide running buffer comprises less than 0.1% water.

Embodiment 84. The method of any one of embodiments 42-83, wherein the formamide running buffer comprises a buffering agent.

Embodiment 85. The method of embodiment 84, wherein the buffering agent is a zwitterionic buffering agent.

Embodiment 86. The method of embodiment 84, wherein the buffering agent comprises 2-(N-Morpholino)ethanesulfonic acid (MES); Piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES); [Tris(hydroxymethyl)methylamino]propanesulfonic acid (TAPS); or a combination thereof.

Embodiment 87. The method of embodiment 84, wherein the buffering agent comprises 2-(N-Morpholino)ethanesulfonic acid (MES).

Embodiment 88. The method of any one of embodiments 84-87, wherein the buffering agent is present in the formamide running buffer at a concentration of about: 1 mM, 5 mM, 10 mM, 25 mM, 50 mM, 75 mM, 100 mM, 150 mM, 200 mM, 250 mM, 500 mM, or 1 mM. Embodiment 89. The method of any one of embodiments 84-87, wherein the buffering agent is present in the formamide running buffer at a concentration of about 100 mM.

Embodiment 90. The method of any one of embodiments 42-89, wherein the formamide running buffer comprises a chelator.

Embodiment 91. The method of embodiment 90, wherein the chelator comprises EDTA, EGTA, DTP A, NT A, or a combination thereof.

Embodiment 92. The method of embodiment 90, wherein the chelator is EDTA.

Embodiment 93. The method of any one of embodiments 90-92, wherein the chelator is present in the formamide running buffer at a concentration of about: 0.01 mM, 0.05 mM, 0.1 mM, 0.25 mM, 0.5 mM, 0.75 mM, 1 mM, 1.25 mM, 1.5 mM, 2 mM, 2.5 mM, 5 mM, 7.5 mM, or 10 mM.

Embodiment 94. The method of any one of embodiments 90-92, wherein the chelator is present in the formamide running buffer at a concentration of about 1 mM.

Embodiment 95. The method of any one of embodiments 42-94, wherein the formamide running buffer has a pH of from 5 to 7.

Embodiment 96. The method of any one of embodiments 42-94, wherein the formamide running buffer has a pH of about: 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.

Embodiment 97. The method of any one of embodiments 42-94, wherein the formamide running buffer has a pH of about 6.

Embodiment 98. The method of any one of embodiments 42-97, wherein the method is capable of achieving a separation factor for nucleic acids of about: 100, 95, 90, 85, 80, 75, 70,

65, 60, 55, 50, 45, 40, 35, or 30 or less for nucleic acids from 2000 to 3000 nts in length.

Embodiment 99. The method of any one of embodiments 42-97, wherein the method is capable of achieving a separation factor for nucleic acids of about 70 or less for nucleic acids from 2000 to 3000 nts in length. Embodiment 100. The method of any one of embodiments 42-99, wherein the method is capable of achieving a separation factor for nucleic acids of about: 50, 45, 40, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 20, 15 or less for nucleic acids from 1500 to 2000 nts in length.

Embodiment 101. The method of any one of embodiments 42-99, wherein the method is capable of achieving a separation factor of about 31 or less for nucleic acids of from 1500 to 2000 nts in length.

The following examples are intended to promote a further understanding of the present invention.

EXAMPLES

The examples below demonstrate that RNA with different sizes can be separated using PEO gel in formamide, resulting in a high separation efficiency, especially for large RNA, by methods of the present invention. When compared to using aqueous BGE, the high separation efficiency obtained with formamide BGE is ascribed to maintaining denaturing conditions for large RNA in formamide and the low gel concentration of polymer used in the separation matrix. The heterogeneity of the size of RNA is improved when using formamide as a denaturant so as to minimize band broadening. A separation matrix comprising a low concentration of polymer in formamide provides good separation for large RNA. This is highly beneficial because a more dilute polymer gel can increase the RNA separation efficiency and is easier to operate. In addition, the resulting low gel viscosity enables the use of a longer capillary that can provide higher separation resolution. The experimental conditions, including types of polymers, molecular weight of the polymers and polymer concentration, were optimized for the analysis of RNA with approximately 2000 nt, which is the target size of mRNA. The results based on the mechanism of the separation also shows reptation regime offers better separation compared to the reptation with orientation regime.

General Conditions

The following are the basic materials and methods for the experimental details that follow this section: Material: 2-Hydroxyethyl cellulose (HEC) (MW -1.3 MDa), MES (2-(N-morpholino) ethanesulfonic acid) hydrate, Polyacrylamide (PAA, MW - 5 MDa), polyvinyl alcohol (PVA, MW 146 kDa-l86 kDa), polyethylene oxide (PEO, MW lOOkDa, 600kDa), NaOH (10N), formamide, HMDS+TMCS+Pyridine (l,l,l,3,3,3-hexamethyldisilazane, trimethylchlorosilane, pyridine (3: 1 :9)) and dimethyldichlorosilane were purchased from Sigma- Aldrich (St. Louis,

MO, ETSA). Polyethylene oxide (PEO, MW > 5 MDa), EDTA (0.5 M), Brij® 58 (synonyms: polyethylene glycol hexadecyl ether, Polyoxyethylene (20) cetyl ether), Brij®35 (synonyms: polyoxyethylene lauryl ether, polyethyleneglycol lauryl ether) and RNA ladder (200, 500, 1000, 1500, 2000, 3000, 4000 and 6000 nt) were purchased from Fisher Scientific (Norristown, PA, ETSA). Bare silica and PVA-coated capillaries were purchased from Agilent (Extended Light Path, 64.5 cm total, 56 cm effective, 50-pm ID, Agilent Technologies, Santa Clara, CA, USA). Gel and sample preparation. MES (2-(N-morpholino)ethanesulfonic acid; 0.9759 g) was dissolved in 100% formamide (50 mL). EDTA (0.5 M, 0.1 mL) and NaOH (10 N, 0.164 mL) were added. The pH of the formamide buffer was adjusted to approximately 6.0. Because the pH of an organic solution cannot be measured directly, 1 mL of the formamide buffer was mixed with 9 mL water and the pH of the formamide/water solution was then measured. Polymer gel was prepared by dissolving polymer powder in the formamide buffer at different concentrations. The mixture was mixed overnight. The polymer gel was degassed using sonication and centrifuged before use. RNA ladder was diluted lO-fold in 10% w/v polyoxyethylene alkyl ether /formamide.

Capillary coating. l,l,l,3,3,3-hexamethyldisilazane, trimethylchlorosilane, pyridine (3: 1 :9) or 10% dimethyldichlorosilane in l,l,l,3,3,3-hexamethyldisilazane, trimethylchlorosilane, pyridine (3: 1 :9) was infused in to a bare silica capillary (Extended Light path, 64.5 cm total, 56 cm effective, 50-pm ID, Agilent Technologies, Santa Clara, CA, USA) for two hours. The capillary was then rinsed with ethanol and water and dried with air flow before use.

Capillary electrophoresis: Agilent 7100 CE system (Agilent Technologies, Santa Clara, CA, USA) was used. All the experiments were run at -29 kV (reverse polarity), 25 °C. Samples were injected using electrokinetic injection mode (20 kV, 10 s). RNA was detected by UV absorption at 260 nm. Before running samples, the capillary was preconditioned by running two injections of RNA ladder. The capillary was infused with fresh gel after each injection was completed. Data were processed using OpenLab software (Agilent Technologies, Santa Clara, CA, USA) for efficiency (plate number) and resolution calculation.

Efficiency (plate number) N = Equation 2

Where t _r is retention time and w is peak width.

Resolution Equation 3

Where t ₂ and ti are retention time of two peaks and wi and w ₂ are peak width of the two peaks.

EXAMPLE 1

NON-AQUEOUS DENATURATION

Compared to traditional gel electrophoresis for RNA analysis, capillary electrophoresis provides high separation efficiency, reproducibility, and automation which enables high throughput analysis. In addition, it allows for the use of small sample volume and high sensitivity can be achieved. CE analysis of RNA has been done using aqueous gel, or a mixture of aqueous and organic solvent gel. Although non-aqueous gels have been used for analysis of synthetic polymers, enantiomers, benzodiazepines, etc., they have never been used for RNA analysis.

In this example, formamide was used to denature the RNA ladder and demonstrates improved separation efficiency. Briefly, mRNA was subject to capillary electrophoresis using either (A) aqueous BGE or (B) formamide BGE prepared as described supra. Aqueous BGE has the same composition of the formamide BGE except the solvent is water instead of formamide. RNA in aqueous BGE was not denatured whereas RNA in the formamide BGE was denatured. The separation matrix was a 0.16% PEO gel, in the indicated run buffer. The separation matrix was infused into a PVA coated capillary. The conditions for CE were as described supra. The results are set forth in Figures 1 A and 1B, which show a comparison of CE separation of the RNA ladder using aqueous (A) and formamide (B) BGE. The RNA ladder comprises high molecular weight RNA standards ranging from 200 nt to 6000 nt (200, 500, 1000, 1500, 2000, 3000, 4000, and 6000 nt).

When formamide was used to denature RNAs, the current used during the CE was much lower compared to when aqueous buffer was used (data not shown). This is beneficial for the separation efficiency because joule heating was reduced in the formamide BGE. As shown in Figures 1A and IB, the use of formamide buffer to denature the RNA resulted in higher resolution. The separation resolution is related to efficiency, which is determined by multiple parameters, including the heterogeneity of samples or gel matrices. The heterogeneity of size in the native state of RNA can impact their peak shape resulting in peak broadening. Formamide BGE, as a strong denaturant of nucleic acids, can better denature of RNA molecules. Linder the denatured condition, RNA is free of intramolecular interaction, and the size homogeneity is significantly improved, which is highly desired for efficient CE separation.

In addition, determining the size (length) of nucleic acids in terms of the number of nucleotides by CE analysis can be used to characterize RNA integrity. Linder CE conditions, the migration time of RNA in the polymer gel is dependent on the size of the RNA. Polymer long chains form a sieving matrix in the gel. Charged RNA passes through the gel following a sieving mechanism. Under denatured condition, determination of the length of RNA(s) {i.e., the number of nucleotides) is more accurate because the RNA is free of intramolecular interaction. Thus, the migration time is primarily dependent on the number of nucleotides. Figure 2 shows the correlation of the migration time and the length of RNA(s) in the sample when formamide BGE is used. It can be fitted using a fourth order polynomial equation with R ² of 0.9999. In this exemplary run, the size (i.e., the length) of RNA can be determined by the equation of y = 0.0352x ⁴ - 3.88l3x ³ + 146.95x ² - 1916.3x + 2195.2, where y is the length and x is the migration time of an RNA.

EXAMPLE 2

CAPILLARY COATING

Suppression of electroosmotic flow (EOF) can increase efficiency and reduce band broadening in capillary gel electrophoresis. Silanol functional groups on the surface of bare silica capillary are the major cause of EOF, which needs to be deactivated or blocked. Several coating materials, such as PEO, PVA and polyacryloylaminoethanol (PAAE), have been reported. Initially, a commercially-available PVA-coated capillary was used. However, it showed poor reproducibility and conditioning the capillary was time consuming when using formamide BGE. Therefore, deactivated capillaries were prepared in house. Capillary coatings were prepared as described above, using either l,l,l,3,3,3-hexamethyldisilazane, trimethylchlorosilane, pyridine (3: 1 :9) or 10% v/v dimethyldichlorosilane (DMDCS) in l,l,l,3,3,3-Hexamethyldisilazane, trimethylchlorosilane, pyridine (3:1 :9). 1,1, 1,3, 3, 3- hexamethyldisilazane, trimethylchlorosilane, pyridine (3:1 :9), also known as SYLON® HTP, available from Sigma-Aldrich, was used without further modification to functionalize and deactivate the inner surface of a bare silica capillary. SYLON® HTP ostensibly adds the trimethyl silyl (TMS) group to free silanols on the surface of silica, thereby eliminating EOF.

The results indicate that bare silica capillaries treated with SYLON® HTP showed improved reproducibility and minimum pre-conditioning of the capillary was required. However, the stability of SYLON® HTP -treated capillaries was poor as they only performed well for about one week. Broad peaks were observed after that time, which was attributed to degradation of the coating and the gradual reappearance of EOF in the capillaries. Additionally, the reproducibility of migration time was not good using capillaries treated with SYLON® HTP alone.

To resolve the stability issue, 10% v/v dimethyldichlorosilane (DMDCS) in 1, 1,1, 3,3,3- hexamethyldisilazane, trimethylchlorosilane, pyridine (3:1 :9) was used to functionalize and deactivate bare silica capillaries. DMDCS serves as a cross-linking agent by virtue of the dichlorosilane functional group. In addition, the reaction between DMDCS and surface silanol functional groups is more efficient, resulting in formation of a surface layer on bare silica capillary with better coverage and improved stability. DMDCS-treated capillaries show similar RNA separation compared to capillaries treated with SYLON® HTP alone. However, the stability of DMDCS-treated capillaries is much improved, and they can be used for at least one month with more than two hundred injections without losing resolution.

EXAMPLE 3

SEPARATION MATRIX

Different polymers have been used as a separation matrix for RNA or DNA analysis, including HEC, PAA, and PEO. Polymers with different physical properties could impact the CE separation. In this example, three polymers, HEC, PAA, and PEO were tested for the CE separation of RNA. Previous results showed high molecular weight HEC is effective for separation of larger RNA in aqueous BGE. Although use of HEC gel in formamide BGE showed good separation (Figure 3A), high molecular weight HEC is very difficult to dissolve in formamide. Compared to HEC, PEO, and PAA are more soluble in formamide. Three separation matrices were prepared by dissolving polymer powder in the formamide buffer at a concentration of 0.16% w/v polymer. Specifically, either HEC (MW 1.3 MDa), PAA (MW 5 MDa), or PEO (MW > 5 MDa) polymer (0.16% w/v) was dissolved in 100% formamide buffer (100 mM MES, 1 mM EDTA, pH = 6.0) as described in Example 1. The separation matrix was infused into a DMDCS-treated capillary prepared as described in Example 2. The conditions for CE were as described supra. Figures 3B - 3C show the separation of the RNA ladder using PEO or PAA gel. The electropherograms show the peak shape for large RNA with 6000 nt became slightly broader, but still retains well defined peak shape compared to aqueous gel, which show very broad peaks for large RNA. High separation efficiency of large RNA enables the use of longer capillaries without significant broadened peaks, which is desirable because separation factor increases with the length of capillary. Although the molecular weight and gel concentration of PEO and PAA that were tested are similar, the electropherograms show sharper peaks when using PEO gels. The average separation efficiency of the six RNA standards for different polymers is shown in Figure 4. Although both polymers work to separate large RNA, PEO shows approximately two times higher average separation efficiency compared to PAA (Figure 4).

EXAMPLE 4

POLYMER MOLECULAR WEIGHT

The impact of molecular weight of the polymer used in the separation matrix was evaluated using PEO as the polymer. An RNA ladder was separated using PEO gels having a different molecular weight of PEO (100 kDa, 600 kDa and > 5 MDa) at the same polymer concentration of 0.25% w/v. Gels were prepared as described in Example 2. As shown in

Figures 5A - 5C, PEO with low molecular weight, 100 kDa, showed poor separation, especially for the large RNA. The separation was improved when higher molecular weight PEO was used, between 600 kDa and > 5 MDa. This trend is consistent with previous results. To further evaluate the separation of RNA, separation factor was calculated based on the resolution of the peaks. Separation factor can be calculated using Equation 1 as described supra. Separation factor estimates the length difference that can be separated by the method when the resolution equals to 1. The separation of RNA with approximately 2000 nt is important because it is the target size of mRNA. The separation factor of an RNA ladder (1500 to 2000 nt and 2000 to 3000 nt) was calculated using PEO (0.25% w/v) gels with different molecular weights (100 kDa and between 600 kDa and > 5 MDa) in formamide buffer (100 mM MES, 1 mM EDTA, pH = 6). The results are set forth in Figure 6. PEO with MW of between 600 kDa and > 5 MDa provides better separation of the RNA ladder around 2000 nt. From 1500 nt to 2000 nt, the average separation factor is as low as 31 nt, which indicates that RNAs having length difference of 31 could be separated within the range. Similarly, from 2000 nt to 3000 nt the average separation factor is about 70, which indicates that RNAs having length difference of 70 nt could be separated within the range. Based on previous results, there appears to be a trend that the separation of large RNA requires larger polymer. Therefore, PEO with MW > 5 MDa is selected for potential applications to separated larger RNA ( e.g ., mRNA, nt > 6000).

EXAMPLE 5

POLYMER CONCENTRATION

The concentration of polymer in the gel is also an important factor to the separation of RNA. When the polymer concentration in the gel is too low, there are not enough polymer chains to form a sieving matrix for separation. On the other hand, when the concentration of the polymer in the gel is too high, the gel becomes too viscous and the migration of the RNA molecule is very slow and the peaks are broad. While the polymer in the gel has to be concentrated enough to provide effective separation, low polymer concentration in the gel is desirable. One reason for this is that analytes migrate faster when the polymer concentration in the gel is lower, which improves speed of analysis. Additionally, a low polymer gel

concentration has lower viscosity, which enables accurate and reproducible filling of longer and narrower-ID capillaries.

Polymer gels with different concentrations of polymer ranging from 0.025% to 0.5% w/v were tested to optimize the resolution of RNA separation. Gels having 0.5% w/v of polymer were prepared by dissolving PEO powder in the prepared formamide BGE to result in a gel containing 0.5% w/v PEO with different molecular weight. The gels with other polymer concentrations were prepared by dilution of 0.5% w/v gel using the prepared formamide BGE. The electropherograms are shown in Figures 7A-7F. The gel containing 0.025% w/v PEO showed poor separation since the polymer gel concentration was too low to form the sieving matrix and therefore it is not able to provide effective separation. As the polymer concentration increases, the migration of RNA becomes slower as expected. A comparison of the separation factor between RNAs of 1500 nt to 2000 nt and 2000 nt to 3000 nt when using gels having differing concentrations of polymer is shown in Figure 8. The best separation of RNA around 2000 nt was obtained when 0.1% to 0.25% w/v polymer was used. This concentration is much lower than the reported optimal polymer gel concentration for RNA separation in aqueous media, which is approximately 0.4%. Because PEO with molecular weight of 600 kDa showed similar separation compared to PEO with molecular weight of > 5MDa and HEC molecular weight of l.3MDa, the low polymer concentration in formamide BGE does not appears to be related to only the molecular weight of the polymers. The reason for the lower optimal polymer gel concentration in formamide BGE is still not yet clear. However, it is clear that gels with low polymer concentration and low viscosity are preferred insofar as they are easier to prepare and easier to fill into longer and narrower-ID capillaries. Low polymer concentration could contribute to the improved efficiency as well. The separation efficiency using different polymer concentrations is shown in Figure 9. As the polymer concentration of the gel increases from 0.025% to 0.5% w/v, the separation efficiency decreased for small RNA molecules, 200 nt, 500 nt and 1000 nt, as expected. It is interesting that the larger RNA molecules, 1500 nt to 6000 nt, show similar or slightly lower separation efficiency. Moreover, the electropherogram of the RNA ladder using 0.5% w/v polymer gel show different separation selectivity, better separation for smaller RNA but less separation for larger RNA.

In order to understand the root cause for the fact that the separation using 0.5% w/v polymer gel does not follow the trend in terms of efficiency and selectivity, the mechanism of the CE separation was investigated. There are different mechanisms for the separation of RNA in polymer gel, depending on the size of RNA and pore size of gel matrix. When the RNA molecules are smaller than the pore size of the gel, RNA molecules can move through pores without altering their shape, which is called Ogston regime. When RNA molecules are larger than the pore size of the gel, the molecules need to deform in order to migrate through pores, which is called reptation model. Figure 10 shows the double logarithmic plot of RNA length and mobility. There are clearly three regimes in the plot, Ogston (section I), reptation regime (section II), and reptation with orientation (section III). The plot shows that when using 0.5% w/v polymer gel, the migration of RNA molecules with 1500 or more nucleotides falls in a different regime, reptation with orientation, (section III), while the smaller ones with 500 and 1000 nt are still in reptation regime (section II). It is likely that because of the different separation mechanism, large RNA molecules in 0.5% w/v polymer gel show poorer separation and the separation efficiency is less dependent on the gel concentration. The separation factor of 2000 and 3000 nt RNA is also significantly higher in 0.5% w/v polymer gel compared to the gels with lower polymer concentrations (Figure 8). RNA with 2000 and 3000 nt appear in the reptation regime (section II) when gel with lower polymer concentrations were used and the separation in those gels was much improved. Similarly, in 0.25% w/v polymer gel, RNA standards with 3000 and 4000 nt fall in section III (reptation with orientation) in Figure 10 and the electropherogram shows less separation of the two peaks. Large RNA separation usually happens in reptation or reptation with orientation regime. The result indicates that for large RNA the separation in reptation regime is better compared to reptation with orientation regime.

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