Method for separating and detecting double-stranded and single-stranded ribonucleic acid (RNA)

Abstract

Provided herein are capillary electrophoresis methods for separating and detecting double-stranded ribonucleic acid (dsRNA) contaminants in a sample of single-stranded ribonucleic acid (ssRNA), such as an RNA therapeutic (e.g., an mRNA vaccine).

Description

[Technical field]

[0001] Claiming priority This application claims the benefit of U.S. Provisional Patent Application No. 63 / 314,936, filed February 28, 2022, which is incorporated by reference in its entirety. [Background technology]

[0002] Single-stranded RNA (ssRNA) is a key component in many RNA therapeutics, such as messenger (mRNA) vaccines. However, double-stranded RNA (dsRNA) is a by-product of the manufacture of ssRNA therapeutics, which is a major trigger of unwanted immune responses. Therefore, it is of interest to develop new assays to detect dsRNA contaminants in ssRNA therapeutics to ensure their safety and efficacy. Summary of the Invention

[0003] Provided herein is a method for separating and detecting dsRNA and ssRNA in a rapid and reproducible high-throughput manner, which comprises separating dsRNA and ssRNA by capillary electrophoresis, and then detecting the separated dsRNA and ssRNA via the signal generated by an intercalating agent bound to the dsRNA and ssRNA.

[0004] Accordingly, an aspect of the disclosure is a method for separating and detecting double-stranded ribonucleic acid (dsRNA) from single-stranded ribonucleic acid (ssRNA) by capillary electrophoresis, comprising the steps of: introducing a polymer solution and a sample comprising dsRNA and ssRNA into a first capillary channel, where the polymer solution is introduced before the sample, and where the polymer solution or the sample comprises an intercalating agent; and injecting a portion of the sample into a second capillary channel that intersects and is fluidly connected to the first capillary channel; The method includes applying a voltage gradient across the length of the second capillary channel to separate dsRNA into a first segment of the portion of the sample and ssRNA into a second segment of the portion of the sample, and detecting the amount of intercalating agent in the first segment and the second segment, wherein the amount of intercalating agent in the first segment indicates the amount of dsRNA in the sample and the amount of intercalating agent in the second segment indicates the amount of ssRNA in the sample.

[0005] In some embodiments, the intercalating agent is fluorescent and detecting the amount of the intercalating agent comprises detecting a fluorescent signal. In some embodiments, the concentration of the intercalating agent in the polymer solution or sample is 0.01 mM to 0.2 mM. In some embodiments, the intercalating agent is selected from the group consisting of SYBR dyes, ethidium bromide, acridine orange, propidium iodide, 7-aminoactinomycin D (7-AAD), 4',6-diamidino-2-phenylindole (DAPI), cyanine dyes, Alexa Fluor dyes, BODIPY dyes, and combinations thereof.

[0006] In some embodiments, the concentration of the polymer in the polymer solution is 0.01% to 20% (w / v). In some embodiments, the polymer has a molecular weight of 1 Kd to 5,000 Kd.

[0007] In some embodiments, the polymer is formed from monomers selected from the group consisting of acrylic acid, carboxylic acid, sulfonic acid, phosphate monomers, bisacrylamide acetic acid, 4,4-bis(4-hydroxyphenyl)pentanoic acid, 3-butene-1,2,3-tricarboxylic acid, 2-carboxyethyl acrylate, itaconic acid, methacrylic acid, and 4-vinylbenzoic acid.

[0008] In some embodiments, the polymer is an acrylic polymer, hi some embodiments, the acrylic polymer is selected from the group consisting of polyacrylamide polymers, polymethylacrylamide polymers, polydimethylacrylamide polymers, and polydimethylacrylamide-co-acrylic acid polymers.

[0009] In some embodiments, the polymer comprises between 0.01% and 2% net charge, which is the same charge as at least one surface of the capillary channel.

[0010] In some embodiments, the polymer comprises a negatively charged monomer subunit, and at least one surface of the capillary channel has a negative surface charge. In some embodiments, the negatively charged monomer subunit is selected from the group consisting of acrylic acid, bisacrylamide acetic acid, 4,4-bis(4-hydroxyphenyl)pentanoic acid, 3-butene-1,2,3-tricarboxylic acid, 2-carboxyethyl acrylate, itaconic acid, methacrylic acid, 4-vinylbenzoic acid, sulfonic acid, 2-acrylamido-2-methyl-1-propanesulfonic acid, 2-methyl-2-propene-1-sulfonic acid, 2-propene-1-sulfonic acid, 4-styrenesulfonic acid, 2-sulfoethyl methacrylate, 3-sulfopropyl dimethyl-3-methacrylamidopropyl ammonium inner salt, 3-sulfopropyl methacrylate, vinylsulfonic acid, bis(2-methacryloxyethyl)phosphate, and monoacryloxyethyl phosphate.

[0011] In some embodiments, the polymer comprises a positively charged monomer subunit, and at least one surface of the capillary channel has a positive surface charge. In some embodiments, the positively charged monomer subunit is selected from the group consisting of 2-acryloxyethyltrimethylammonium chloride, diallyldimethylammonium chloride, 2-methacryloxyethyltrimethylammonium chloride, and 3-methacryloxy-2-hydroxypropyltrimethylammonium chloride.

[0012] In some embodiments, the concentration of dsRNA in the sample is 1 pg / μL to 100 pg / μL. In some embodiments, the concentration of ssRNA in the sample is 1 pg / μL to 100 pg / μL.

[0013] In some embodiments, injecting a portion of the sample from the capillary channel into the second capillary channel comprises applying a voltage difference across the capillary channel to electrokinetically move a portion of the sample from the capillary channel to the second capillary channel.

[0014] In some embodiments, the methods described herein further include the steps of introducing an additional polymer solution and the sample into the first capillary channel, where the additional polymer solution is introduced before the sample, and the additional polymer solution or the sample contains an additional intercalating agent; injecting a portion of the sample into the second capillary channel that intersects and is fluidly connected to the first capillary channel; applying a second voltage gradient across the length of the second capillary channel to separate dsRNA into an additional first segment of the portion of the sample and ssRNA into an additional second segment of the portion of the sample; and detecting the amount of the additional intercalating agent in the additional first segment and the additional second segment, where the amount of the additional intercalating agent in the additional first segment indicates the amount of dsRNA in the sample and the amount of the additional intercalating agent in the additional second segment indicates the amount of ssRNA in the sample.

[0015] In some embodiments, one or more steps are performed in a microfluidic device.

[0016] An aspect of the disclosure is a method for separating and detecting double-stranded ribonucleic acid (dsRNA) from single-stranded ribonucleic acid (ssRNA) in a microfluidic device, comprising the steps of: providing a microfluidic device comprising: a substrate having a surface; an analytical channel disposed on the substrate; a sample loading channel disposed on the substrate and intersecting the analytical channel at an intersection; introducing a polymer solution and a sample comprising dsRNA and ssRNA into the loading channel, wherein the polymer solution is introduced before the sample, and wherein the polymer solution or the sample comprises an intercalating agent; The method includes the steps of injecting a portion of the sample into the analysis channel, applying a voltage gradient across the length of the analysis channel to separate dsRNA into a first segment of the portion of the sample and ssRNA into a second segment of the portion of the sample, and detecting the amount of intercalating agent in the first segment and the second segment, wherein the amount of intercalating agent in the first segment indicates the amount of dsRNA in the sample and the amount of intercalating agent in the second segment indicates the amount of ssRNA in the sample.

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Additionally, the materials, methods, and examples are merely illustrative and are not intended to be limiting.

[0018] Other features and advantages of the invention will be apparent from the following detailed description, and from the claims. [Brief description of the drawings]

[0019] [Figure 1A]FIG. 1 is a schematic diagram illustrating sample loading and injection according to some embodiments of the technology described herein. [Figure 1B] FIG. 1 is a schematic diagram illustrating sample loading and injection according to some embodiments of the technology described herein. [Figure 1C] FIG. 1 is a schematic diagram illustrating sample loading and injection according to some embodiments of the technology described herein. [Figure 1D] FIG. 1 is a schematic diagram illustrating sample loading and injection according to some embodiments of the technology described herein. [Figure 1E] FIG. 1 is a schematic diagram illustrating sample loading and injection according to some embodiments of the technology described herein. [Figure 2A] FIG. 1 is a schematic diagram showing separation of dsRNA and ssRNA into portions of a sample and detection of the separated dsRNA and ssRNA according to some embodiments of the technology described herein. [Figure 2B] FIG. 1 is a schematic diagram showing separation of dsRNA and ssRNA into portions of a sample and detection of the separated dsRNA and ssRNA according to some embodiments of the technology described herein. [Figure 2C] FIG. 1 is a schematic diagram showing separation of dsRNA and ssRNA into portions of a sample and detection of the separated dsRNA and ssRNA according to some embodiments of the technology described herein. [Figure 2D] FIG. 1 is a schematic diagram showing separation of dsRNA and ssRNA into portions of a sample and detection of the separated dsRNA and ssRNA according to some embodiments of the technology described herein. [Figure 2E] FIG. 1 is a schematic diagram showing separation of dsRNA and ssRNA into portions of a sample and detection of the separated dsRNA and ssRNA according to some embodiments of the technology described herein. [Figure 2F] FIG. 1 is a schematic diagram showing separation of dsRNA and ssRNA into portions of a sample and detection of the separated dsRNA and ssRNA according to some embodiments of the technology described herein. [Diagram 3] FIG. 1 is a schematic diagram showing the arrangement of reagents on a chip. [Figure 4] 1 is a graph showing chromatographic separation of dsDNA standard samples according to some embodiments of the technology described herein, and the inset is an image showing separation of the dsDNA standard samples by agarose gel electrophoresis. [Diagram 5] 1 is a graph showing chromatographic separation of ssDNA standard samples according to some embodiments of the technology described herein, and the inset is an image showing separation of the ssDNA standard samples by agarose gel electrophoresis. [Figure 6] Chromatographic overlay of the chromatographic separation of dsDNA and ssDNA in FIG. 4 and FIG. 5, respectively. [Figure 7] 1 is a graph showing chromatographic separation of a dsRNA standard sample using standard pinch flow (top panel) and improved pinch flow according to some embodiments of the technology described herein (bottom panel). [Figure 8] FIG. 1 is a schematic diagram of a sample preparation and analysis workflow for detecting dsRNA contamination in an mRNA sample (e.g., an mRNA vaccine) according to some embodiments of the technology described herein. [Figure 9A] 9A-9C are graphs showing chromatographic separation of a dsRNA ladder (FIG. 9A) with (dashed line) and without (solid line) heating during sample preparation. The experiment was performed using a 6% gel matrix. [Figure 9B] 9B is a graph showing the chromatographic separation of a ssRNA ladder (FIG. 9B) with (dashed line) and without (solid line) heating during sample preparation. The experiment was performed using a 6% gel matrix. [Figure 10A] 10A-C are graphs showing fluorescent detection of dsRNA (FIG. 10A) using RNA dyes (triangles) and DNA dyes (squares) at the concentrations shown. [Figure 10B] 10B is a graph showing fluorescent detection of ssRNA (FIG. 10B) using RNA dye (triangles) and DNA dye (squares) at the concentrations shown. [Figure 11]Electropherogram showing an overlay of a ssRNA ladder analyzed using a 6% gel matrix (dashed line) followed by a 3% gel matrix (solid line). [Figure 12A] Graph showing the difference in migration between dsRNA (solid line) and ssRNA (dashed line) ladders using a DNA dye. Electropherograms showing the overlay of both ladders for each molecule type (FIG. 12A). Note that for dsRNA, the fragment size is in bp, whereas for ssRNA, the fragment size is in nt. [Figure 12B] 12B is a graph showing the difference in migration between dsRNA (solid line) and ssRNA (dashed line) ladders using DNA dyes. Migration time across fragment sizes for each molecule type (FIG. 12B). Note that for dsRNA, the fragment size is in bp, whereas for ssRNA, the fragment size is in nt. [Figure 12C] 12C is a graph showing the difference in migration between dsRNA (solid line) and ssRNA (dashed line) ladders using DNA dyes. Mobility across fragment sizes for each molecule type (FIG. 12C). Note that for dsRNA, the fragment size is in bp, whereas for ssRNA, the fragment size is in nt. [Figure 13A] 13A-13C are graphs showing data from an analysis of the difference in fluorescent labeling between dsRNA and ssRNA ladders using SYTO™ 61 and RiboRed. Electropherograms showing the overlay of dsRNA ladders labeled with both dye types (FIG. 13A). Note that for dsRNA, the fragment size is in bp, whereas for ssRNA, the fragment size is in nt. [Figure 13B] 13A-13C are graphs showing data from an analysis of the difference in fluorescent labeling between dsRNA and ssRNA ladders using SYTO™ 61 and RiboRed. Average area generated by each fragment of the dsRNA ladder (FIG. 13B). Note that for dsRNA, the fragment size is in bp, whereas for ssRNA, the fragment size is in nt. [Figure 13C]13C is a graph showing data from an analysis of the difference in fluorescent labeling between dsRNA and ssRNA ladders using SYTO™ 61 and RiboRed. Peak heights generated by each fragment of the dsRNA ladder (FIG. 13C). Note that for dsRNA, the fragment size is in bp, whereas for ssRNA, the fragment size is in nt. [Figure 13D] 13A-13D are graphs showing data from an analysis of the difference in fluorescent labeling between dsRNA and ssRNA ladders using SYTO™ 61 and RiboRed. Electropherograms showing the overlay of ssRNA ladders labeled with both dye types (FIG. 13D). Note that for dsRNA, the fragment size is in bp, whereas for ssRNA, the fragment size is in nt. [Figure 13E] 13A-13D are graphs showing data from an analysis of the difference in fluorescent labeling between dsRNA and ssRNA ladders using SYTO™ 61 and RiboRed. Average area generated by each fragment of the ssRNA ladder (FIG. 13E). Note that for dsRNA, the fragment size is in bp, whereas for ssRNA, the fragment size is in nt. [Figure 13F] 13A-13C are graphs showing data from an analysis of the difference in fluorescent labeling between dsRNA and ssRNA ladders using SYTO™ 61 and RiboRed. The peak heights generated by each fragment of the ssRNA ladder (FIG. 13F). Note that for dsRNA, the fragment size is in bp, whereas for ssRNA, the fragment size is in nt. [Figure 14A] Graphs showing data from analysis of samples containing custom-length dsRNA and long mRNA using SYTO™ 61. Electropherograms (FIG. 14A) of 4,001 bp and nt dsRNA and mRNA fragments, respectively, using SYTO™ 61. Run times summarized include data from three independent runs, each with two to three replicates. [Figure 14B]14A-14C are graphs showing data from the analysis of samples containing custom-length dsRNA and long mRNA using SYTO™ 61. Summarized runs of 4,001 bp and nt dsRNA and mRNA fragments, respectively, using SYTO™ 61 (FIG. 14B). Summarized run times include data from three independent runs, each with two to three replicates. [Figure 14C] Graph showing data from analysis of samples containing custom-length dsRNA and long mRNA using SYTO™ 61. Summary mobilities of 4,001 bp and nt dsRNA and mRNA fragments, respectively, using SYTO™ 61 (FIG. 14C). Summary migration times include data from three independent runs, each with two to three replicates. [Figure 15A] Graphs showing data from analysis of samples containing custom-length dsRNA and long mRNA using SYTO™ 61 and RiboRed. Custom dsRNA fragment electropherogram using SYTO™ 61 and RiboRed (FIG. 15A). Summarized data includes results from three independent runs, each with two to three replicates. [Figure 15B] 15A-15C are graphs showing data from the analysis of samples containing custom-length dsRNA and long mRNA using SYTO™ 61 and RiboRed. Custom dsRNA fragment area under the curve using SYTO™ 61 and RiboRed (FIG. 15B). Data summarized includes results from three independent runs, each with two to three replicates. [Figure 15C] Graph showing data from analysis of samples containing custom-length dsRNA and long mRNA using SYTO™ 61 and RiboRed. Custom dsRNA fragment peak heights using SYTO™ 61 and RiboRed (FIG. 15C). Summarized data includes results from three independent runs, each with two to three replicates. [Figure 15D]Graphs showing data from analysis of samples containing custom-length dsRNA and long mRNA using SYTO™ 61 and RiboRed. Custom mRNA fragment electropherogram using SYTO™ 61 and RiboRed (FIG. 15D). Summarized data includes results from three independent runs, each with two to three replicates. [Figure 15E] Graph showing data from analysis of samples containing custom-length dsRNA and long mRNA using SYTO™ 61 and RiboRed. Custom mRNA fragment area under the curve using SYTO™ 61 and RiboRed (FIG. 15E). Summarized data includes results from three independent runs, each with two to three replicates. [Figure 15F] Graph showing data from analysis of samples containing custom-length dsRNA and long mRNA using SYTO™ 61 and RiboRed. Custom mRNA fragment peak heights using SYTO™ 61 and RiboRed (FIG. 15F). Summarized data includes results from three independent runs, each with two to three replicates. [Figure 16A] Graphs showing data from analysis of mixed long dsRNA and mRNA samples. Electropherograms of mixed long dsRNA and mRNA samples (FIG. 16A) with SYTO™ 61 represented as a solid line and RiboRed represented as a dashed line. Summarized data includes results from three independent runs, each with two to three replicates. [Figure 16B] Graph showing data from analysis of mixed long dsRNA and mRNA samples. Area under the curve for dsRNA (FIG. 16B). Data summarized includes results from three independent runs, each with two to three replicates. [Figure 16C] Graph showing data from analysis of mixed long dsRNA and mRNA samples. dsRNA peak height (FIG. 16C). Summarized data includes results from three independent runs, each with two to three replicates. [Figure 16D]Graph showing data from analysis of mixed long dsRNA and mRNA samples. Area under the curve for mRNA (FIG. 16D). Data summarized includes results from three independent runs, each with two to three replicates. [Figure 16E] Graph showing data from analysis of mixed long dsRNA and mRNA samples. mRNA peak height (FIG. 16E). Data summarized includes results from three independent runs, each with two to three replicates. [Figure 17A] Graph showing the ratio between RNA and DNA dye (RiboRed / SYTO™ 61) for dsRNA (black) and mRNA (grey) when analyzed individually (FIG. 17A). These results include data from three independent runs, each with two to three replicates. [Figure 17B] Graph showing the ratio between RNA and DNA dye (RiboRed / SYTO™ 61) for dsRNA (black) and mRNA (grey) when analyzed mixed together (FIG. 17B). These results include data from three independent runs, each with two to three replicates. [Figure 18A] 18A-B are graphs showing an assessment of the linearity of dsRNA response with increasing concentrations of dsRNA (FIG. 18A) using SYTO™ 61 and RiboRed. In FIG. 18A, the line of best fit for dsRNA samples using SYTO™ 61 was y=79.05x+63.87 with R2=0.93, whereas the line for RiboRed was y=6.49x+39.21 with R2=0.75. [Figure 18B] 18B is a graph showing an assessment of the linearity of mRNA response with increasing concentrations of mRNA (FIG. 18B) using SYTO™ 61 and RiboRed. In FIG. 18B, the line of best fit for the mRNA samples using SYTO™ 61 was y=278.2x+1189 with R2=0.84, whereas the line for RiboRed was y=503.0x+1103 with R2=0.75. [Figure 19A]19A is a graph showing the evaluation of dye ratios (RiboRed / SYTO™ 61) at various concentrations of dsRNA. The dye ratios represented 5.0%, 7.5%, and 10.0% of the total mRNA concentration. FIG. 19B is a graph showing a comparison between the dsRNA (black) dye ratio and the mRNA (gray) dye ratio at various concentrations of dsRNA. [Figure 19B] 19B is a graph showing the evaluation of dye ratios (RiboRed / SYTO™ 61) at various concentrations of dsRNA. The dye ratios represent 5.0%, 7.5%, and 10.0% of the total mRNA concentration. FIG. 19B is a graph showing a comparison between the dye ratios of dsRNA and mRNA separately when the percentage of dsRNA relative to the percentage of mRNA is varied from 5.0% (black) to 7.5% (gray) to 10.0% (light gray). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] The details of one or more embodiments of the invention are set forth in the following description. Other features or advantages of the invention will be apparent from the following drawings and detailed description of certain embodiments, as well as from the appended claims.

[0021] Currently available methods for detecting dsRNA contamination in ssRNA therapeutics utilize high performance liquid chromatography (HPLC). These methods are low throughput and time consuming (more than 30 minutes per sample). Therefore, it is of interest to develop new assays for detecting dsRNA contamination in ssRNA therapeutics and ensure their safety and efficacy.

[0022] The present disclosure is based at least in part on the development of a capillary electrophoresis method for separating and detecting dsRNA and ssRNA in a sample.The method described herein can be used to detect small amounts of dsRNA in a sample of ssRNA.Therefore, the method described herein is particularly useful in evaluating the purity of ssRNA therapy, such as mRNA therapy.

[0023] mRNA vaccines (e.g., COVID-19 vaccines) offer various advantages over traditional vaccines in preventing and reducing disease and shortening the time between pathogen discovery and vaccine creation. While the production of mRNA vaccines generates numerous nucleic acid and enzymatic by-products, most of which can be detected and removed, double-stranded RNA (dsRNA) contaminants pose particular challenges. Current purification and detection platforms for dsRNA vary in their effectiveness and are fraught with problems in scalability for mass production of mRNA vaccines. Effective detection of dsRNA is crucial to ensure vaccine safety and efficacy, as these strands can trigger length-symptom-dependent autoimmune responses and they can enhance mRNA degradation. Thus, the work described herein analyzed the differences in behavior in labeling mRNA and dsRNA molecules to develop a high-throughput microfluidic platform for dsRNA contaminant detection. The detection limits of the system for dsRNA were determined to be 17.7-76.6 pg / µL, whereas the detection limits for mRNA were 1.79-2.83 pg / µL, with a maximum loading capacity of 12.99 ng / µL. Based on these values, the proposed method should allow the detection of dsRNA contaminants present at percentages as low as 0.14-0.59% compared to the total mRNA concentration.

[0024] Vaccines have become a vital public health tool in preventing and reducing disease, morbidity, and mortality in millions of people each year [1]. The importance of vaccines has been highlighted by the COVID-19 pandemic and the widespread use of newly developed mRNA vaccines. While the potential of mRNA vaccines has been noted since the 1990s, many obstacles have prevented their mass production and distribution [2]. Nevertheless, much research has been going into nucleic acid-based vaccines, as they can provide precise targeting of the immune response and offer advantages in safety, efficiency, and specificity when compared to other vaccine platforms [3], [4]. Specifically, mRNA vaccines offer advantages including exonuclear activity, which confers very low risk for subsequent random genome integration and insertional mutagenesis, more controlled expression of the encoded antigen, no foreign genes, and the ability to be generated in a cell-free environment by in vitro transcription [3], [5]. If this technology is harnessed for widespread and large-scale use, the applications of mRNA vaccines could expand to cancer therapy, therapeutic protein replacement therapy, genetic diseases, and the treatment of a wide variety of infectious diseases [3], [5].

[0025] Challenges facing mRNA vaccine production and manufacturing include mRNA instability, in vivo delivery methods, and natural immunogenicity. mRNA instability has been mitigated by optimization of the non-translated region [5], [6]. After testing various delivery methods, the use of lipid nanoparticles (LPNs) has been found to be the most effective in protecting mRNA from rapid degradation by extracellular RNases and allowing mRNA to travel to target cells for cellular uptake and protein production [3], [7]. The body's recognition of exogenous mRNA can stimulate a robust immune response, which can be detrimental to the desired immune response [4]. This latter issue has been resolved by introducing modified nucleosides such as pseudouridine and 1-methylpseudouridine [4].

[0026] However, in addition to the above mentioned obstacles that have been largely addressed, due to the nature of the in vitro transcribed (IVT) mRNA development process, there exists the possibility of contaminants such as residual enzymes, free nucleotides, residual DNA, truncated RNA, and dsRNA [4]. These exogenous contaminants, specifically dsRNA, which has been identified as the major contaminant, are potent pathogen-associated molecular patterns (PAMPs). The creation of dsRNA in IVT production of mRNA results from the activity of T7 RNA polymerase and two potential outcome scenarios: 1) 3' extension of the runoff transcript annealing to a complementary sequence in the body of the runoff transcript, and 2) hybridization of antisense RNA to the runoff transcript [8]. Once recognized, PAMPs can trigger type I interferon activity, which leads to inhibition of translation and enhanced mRNA degradation. In turn, this can result in reduced production of the desired antigen protein. dsRNA contaminants can also activate proinflammatory cytokines that are associated with potential autoimmune responses, including effects on the central nervous system [4], [9]-

[11] . Furthermore, there appears to be a positive correlation between the length of the dsRNA fragments and the associated adverse effects

[12] , suggesting that both the presence and length of dsRNA fragments need to be carefully monitored to ensure vaccine safety.

[0027] Current mRNA vaccine purification platforms may include DNase digestion, precipitation, chromatography, or tangential flow filtration. However, an established manufacturing platform for mRNA is still lacking, so a number of mRNA synthesis and purification techniques can be combined [5]. The pharmaceutical industry often utilizes various forms of chromatography coupled with tangential flow filtration for purification due to its versatility, cost-effectiveness, selectivity, and importantly, its ability to be scaled up for large-scale production of mRNA vaccines [5]. However, various forms of this technique come with their own limitations. For example, ion-pair reversed-phase chromatography is an excellent purification method to remove dsRNA while maintaining high yields, but it is very costly to scale and uses toxic reagents such as acetonitrile [5],

[13] . Conversely, anion exchange chromatography can be used to purify mRNA cost-effectively on a large scale, but anion exchange chromatography requires denaturing conditions, strict control of temperature, and the use of potential chaotropic agents [5]. Finally, high-performance liquid chromatography has been shown to remove dsRNA and other contaminants, resulting in a 10- to 1000-fold increase in protein production levels

[14] , but there are issues with both the scale-up process and mRNA stability

[13] . New innovative purification techniques are under investigation, such as cellulose-based chromatography, which exploits the ability of dsRNA to bind to cellulose in the presence of ethanol. This process may provide a simple, efficient, high-throughput and cost-effective alternative for the removal of dsDNA

[15] .

[0028] However, even with these various purification platforms, only some have evidence of >90% dsRNA removal

[15] , and the variability in the quality and efficacy of current purification methods calls for a quality control system to detect residual and harmful dsRNA fragments in mRNA vaccines. Current preferred quality control detection assays for characterizing RNA transcripts include UV spectroscopy, fluorescence-based assays, electrophoresis on gels, Western blots for dsRNA, immunoassays (e.g., enzyme-linked immunosorbent assays), enzyme-linked immunosorbent assays, and chromatography. However, these are severely limited by factors such as resolution, precise handling, hazardous reagents, demanding labor, need for antibodies, long run times, or inability to be scalable

[13] ,

[16] -

[19] , leaving a critical need for a fast, high-throughput, low-cost quality control system. This can be achieved using microfluidics, whose versatility allows for the rapid development of novel analytical methodologies.

[0029] The development of microfluidics has revolutionized the way samples are analyzed due to its excellent automation compatibility and low volume requirements. Broadly speaking, microfluidics is the flow of microscale volumes (10 -9 ~10 -18 Microfluidics refers to a method or device for processing or manipulating fluids (L)

[20] . As a result of the miniaturization of traditional biomolecular analytical tools, the reduction in overall scale provides precise control of fluids, high-throughput capabilities, and rapid sample processing, allowing them to outperform traditional techniques and often leading to lower-cost alternatives

[20] ,

[21] . To obtain such fine control over the dynamics within a system, microfluidics is usually coupled with a driving force. The method described herein utilizes electrokinetics (EK) as a driving force, as EK allows for the exploitation of unique behaviors that are not easily achievable at large scale

[22] ,

[23] .

[0030] Thus, provided herein is a high-throughput microfluidic electrophoresis-based methodology that exploits the difference in labeling kinetics between dsRNA and mRNA to detect and characterize dsRNA contaminants in mRNA vaccines.

[0031] I. Assay Components The methods described herein include separating and detecting dsRNA and ssRNA via capillary electrophoresis in the presence of a polymer solution and an intercalating agent. In some examples, the methods described herein can be implemented in a microfluidic device.

[0032] Polymer Solutions The capillary electrophoresis method described herein includes the use of polymers to separate dsRNA and ssRNA. The polymers can be charged or uncharged. In some embodiments, when the polymer is charged, the charge on the polymer is the same as the charge on the inner surface of the capillary channel. For example, when the capillary channel contains negatively charged groups on the inner surface, the polymer contains negatively charged monomer subunits. In another example, when the capillary channel contains positively charged groups on the inner surface, the polymer contains positively charged monomer subunits.

[0033] Polymers of various charge percentages can be used in the methods described herein, in some embodiments, the polymer comprises a charge percentage of about 0.01% to about 2%, e.g., about 0.01% to about 1%, about 0.01% to about 0.5%, about 0.05% to about 0.5%, and about 0.05% to about 0.2%.

[0034] As used herein, the "charge percent" of a polymer refers to the molar percent of charged monomeric units relative to the total monomeric subunits used in the synthesis of the polymer. For example, if a polymer synthesis reaction is carried out by mixing 1 mmol of charged subunits and 99 mmol of uncharged monomeric subunits, then the polymer has a charge percent of 1%.

[0035] Polymers for use in the methods described herein can be formed from a variety of monomeric subunits, for example, negatively charged monomeric subunits, positively charged monomeric subunits, uncharged monomeric subunits, or combinations thereof.

[0036] Non-limiting examples of monomer subunits that may be included in a polymer for use in the methods described herein include acrylic acid, carboxylic acid, sulfonic acid, phosphate monomers, bisacrylamide acetic acid, 4,4-bis(4-hydroxyphenyl)pentanoic acid, 3-butene-1,2,3-tricarboxylic acid, 2-carboxyethyl acrylate, itaconic acid, methacrylic acid, and 4-vinylbenzoic acid. In some embodiments, the polymer is an acrylic polymer, such as polyacrylamide polymers, polymethylacrylamide polymers, polydimethylacrylamide polymers, and polydimethylacrylamide-co-acrylic acid polymers.

[0037] Non-limiting examples of negatively charged monomer subunits that can be included in the polymers for use in the methods described herein include acrylic acid, bisacrylamide acetic acid, 4,4-bis(4-hydroxyphenyl)pentanoic acid, 3-butene-1,2,3-tricarboxylic acid, 2-carboxyethyl acrylate, itaconic acid, methacrylic acid, 4-vinylbenzoic acid, sulfonic acid, 2-acrylamido-2-methyl-1-propanesulfonic acid, 2-methyl-2-propene-1-sulfonic acid, 2-propene-1-sulfonic acid, 4-styrenesulfonic acid, 2-sulfoethyl methacrylate, 3-sulfopropyldimethyl-3-methacrylamidopropylammonium inner salt, 3-sulfopropyl methacrylate, vinylsulfonic acid, bis(2-methacryloxyethyl)phosphate, and monoacryloxyethyl phosphate.

[0038] Non-limiting examples of positively charged monomer subunits that can be included in polymers for use in the methods described herein include 2-acryloxyethyltrimethylammonium chloride, diallyldimethylammonium chloride, 2-methacryloxyethyltrimethylammonium chloride, and 3-methacryloxy-2-hydroxypropyltrimethylammonium chloride.

[0039] Non-limiting examples of polymers that may be used as described herein are provided in U.S. Pat. Nos. 5,948,227, 6,042,710, 6,440,284, and 7,081,190, the relevant disclosures of each of which are incorporated herein by reference for the purposes and subject matter mentioned herein.

[0040] The concentration of the polymer in the polymer solution may vary depending on certain factors, such as the size of the capillary channel, the size and amount of RNA to be analyzed, and / or the desired resolution. In some embodiments, the concentration of the polymer in the polymer solution is about 0.01% to about 30% (w / v), e.g., about 0.01% to about 20% (w / v), about 0.01% to about 10% (w / v), about 0.01% to about 5% (w / v), about 0.1% to about 30% (w / v), about 1% to about 30% (w / v), about 10% to about 30% (w / v), or about 20% to about 30% (w / v).

[0041] The average molecular weight of the polymer in the polymer solution may vary depending on certain factors such as the size of the capillary channel, the size and amount of RNA to be analyzed, and / or the desired resolution. In some embodiments, the polymer has a molecular weight of 1 Kd to 5,000 Kd, e.g., 1 Kd to 2,500 Kd, 1 Kd to 1,000 Kd, 1 Kd to 500 Kd, 1 Kd to 100 Kd, 100 Kd to 5,000 Kd, 1,000 Kd to 5,000 Kd, or 2,500 Kd to 5,000 Kd.

[0042] The viscosity of the polymer solution may vary depending on certain factors, such as the size of the capillary channel, the size and amount of RNA to be analyzed, and / or the desired resolution. In some embodiments, the viscosity of the polymer solution is about 2 to about 1,000 centipoise, e.g., about 2 to about 750 centipoise, about 2 to about 500 centipoise, about 2 to about 250 centipoise, about 2 to about 100 centipoise, about 2 to about 50 centipoise, about 50 to about 1,000 centipoise, about 100 to about 1,000 centipoise, about 250 to about 1,000 centipoise, about 500 to about 1,000 centipoise, or about 750 to about 1,000 centipoise.

[0043] The synthesis of the polymers used in the methods of the present invention can be carried out by any number of methods well known in the art. Generally, synthesis conditions and protocols vary depending on the polymer to be synthesized and the nature and amount of charge to be incorporated. Examples of suitable polymer synthesis methods are described, for example, in Odian, Principles of Polymerization, 4th Edition (John Wiley, New York, 2004), and in U.S. Patent Nos. 5,264,101 and 5,567,292, the relevant disclosures of each of which are incorporated herein by reference for the purposes and subject matter mentioned herein.

[0044] Intercalating Agents "Intercalating agent" refers to a molecule that can reversibly bind to base pairs in nucleic acids such as DNA (e.g., dsDNA, ssDNA) and / or RNA (e.g., dsRNA, ssRNA). Generally, intercalating agents useful in the present method either insert between stacked bases in the center of a double-stranded nucleic acid (e.g., dsDNA, dsRNA) or they bind to adjacent bases along the backbone of a single-stranded nucleic acid (e.g., ssRNA). As a result, more molecules of intercalating agent are bound to double-stranded nucleic acids (e.g., dsRNA than ssRNA) than single-stranded nucleic acids at similar concentrations of double-stranded nucleic acids (e.g., dsRNA and ssRNA), which results in a stronger fluorescent signal being produced from double-stranded nucleic acids (e.g., dsRNA than ssRNA) than single-stranded nucleic acids at similar concentrations. The methods described herein take advantage of this difference in signal intensity to detect small amounts of dsRNA contaminants in ssRNA samples. Since the intercalating agent only fluoresces when bound to dsRNA or ssRNA, the fluorescent signal is specific and indicative of the amount of dsRNA or ssRNA in the sample.

[0045] The intercalating agent for use in the methods described herein can be any intercalating agent capable of binding to double-stranded and single-stranded nucleic acids (e.g., dsDNA, ssDNA, dsRNA and ssRNA). Non-limiting examples of intercalating agents for use in the methods described herein include SYTO™ dyes (e.g., SYTO™ 61), Ribo dyes (e.g., RiboRed, RiboGreen), SYBR dyes, ethidium bromide, acridine orange, propidium iodide, 7-aminoactinomycin D (7-AAD), 4',6-diamidino-2-phenylindole (DAPI), cyanine dyes (e.g., Cy3, Cy5), Alexa Fluor dyes, BODIPY dyes, and combinations thereof. In some embodiments, the intercalating agent is not ethidium bromide.

[0046] Various concentrations of intercalating agent can be used in the methods described herein to achieve desired results. In some embodiments, the concentration of intercalating agent in the sample is 0.01 mM to 0.2 mM, for example, 0.05 mM to 0.2 mM, 0.1 mM to 0.2 mM, 0.01 mM to 0.1 mM, or 0.01 mM to 0.05 mM.

[0047] The methods described herein include the use of intercalating agents that are intrinsically fluorescent (e.g., intercalating fluorescent dyes) or extrinsically fluorescent (e.g., intercalating agents labeled with fluorophores).

[0048] In some embodiments, the intercalating agent for use in the methods described herein preferentially binds a target nucleic acid. An intercalating agent is said to exhibit "preferential binding" if it reacts or binds to a particular target nucleic acid more frequently, more rapidly, for a longer duration, and / or with a higher affinity than with an alternative nucleic acid.

[0049] For example, an intercalating agent that preferentially binds to double-stranded nucleic acid is one that binds double-stranded nucleic acid with higher affinity, avidity, more readily, and / or longer duration than it binds to single-stranded nucleic acid. In another example, an intercalating agent that preferentially binds to single-stranded nucleic acid is one that binds single-stranded nucleic acid with higher affinity, avidity, more readily, and / or longer duration than it binds to double-stranded nucleic acid.

[0050] For example, an intercalating agent that preferentially binds to DNA is one that binds to DNA with higher affinity, avidity, more readily, and / or with longer duration than it binds to RNA. In another example, an intercalating agent that preferentially binds to RNA is one that binds to RNA with higher affinity, avidity, more readily, and / or with longer duration than it binds to DNA.

[0051] It will also be understood that an intercalating agent that preferentially binds to a first nucleic acid (e.g., DNA) may or may not preferentially bind to a second nucleic acid (e.g., RNA). Thus, preferential binding does not necessarily require (although it can include) exclusive binding.

[0052] The methods described herein include the use of one or more intercalating agents. For example, the methods described herein may include the use of an intercalating agent capable of binding to RNA and an intercalating agent capable of binding to DNA.

[0053] II. Methods for isolating and detecting dsRNA and ssRNA Also provided herein are methods for separating and detecting low amounts of dsRNA that commonly contaminate ssRNA preparations, including RNA therapeutics (e.g., mRNA therapeutics such as mRNA vaccines). The methods described herein involve capillary electrophoresis of samples of dsRNA and ssRNA in the presence of a polymer and an intercalating agent, each of which is disclosed herein.

[0054] To carry out the methods described herein, in some embodiments, a polymer solution is introduced into a capillary channel, followed by a sample containing dsRNA and ssRNA being introduced into the capillary channel. An intercalating agent for detecting dsRNA and ssRNA can be included in the polymer solution or in the sample. This introduction can be as simple as placing one end of the channel in contact with the polymer solution or sample and allowing the polymer solution or sample to be drawn into the channel. Alternatively, or in addition, a vacuum or pressure can be used to drive the polymer solution or sample into the capillary channel.

[0055] The capillary electrophoresis method described herein can be carried out using a single capillary channel or multiple capillary channels. In some embodiments, when using a single channel, a sample is introduced into the capillary channel (e.g., by hydrodynamic injection), and a voltage is applied across the capillary channel to separate dsRNA and ssRNA in the sample.

[0056] In some embodiments, when multiple channels are used, the sample is introduced into a first capillary channel (also referred to as the loading channel) and transported across the intersection of the first and second capillary channels (also referred to as the analysis channel). The volume or "plug" of sample located within the intersection of the two channels is then drawn into the second channel where it is subjected to the desired analysis.

[0057] 1A-1E show schematics of loading of sample into the loading channel (shown in shading) and injection of the sample into the analytical channel. As shown in FIG. 1A-1B, the sample is drawn into the loading channel across the intersection with the analytical channel. As shown in FIG. 1B-1C, transport of the sample across the intersection without diffusion into the analytical channel is achieved by applying a slight voltage gradient away from the flow path towards the top and bottom of the analytical channel. The result is a "pinching" of the sample flow at the intersection, thereby preventing diffusion of the sample into the analytical channel. The pinched volume of sample at the intersection can then be injected into the analytical channel by applying a voltage gradient across the length of the analytical channel, for example from the top to the bottom, as shown in FIG. 1D. To avoid any bleeding over of material from the sample loading channel during this injection, a low level flow is directed back into the side channel, resulting in a "pullback" of the material from the intersection, as shown in FIG. 1D-1E. The sample is electrophoresed through the analysis channel and flowed past the detection window (dashed box) as shown in Figure 1 E. When a fluorescent intercalating agent is used, a laser-activated fluorescence detection system can be used to monitor the detection window for the fluorescent signal generated by the intercalating agent bound to the dsRNA or ssRNA.

[0058] When a sample is loaded and injected as shown generally in Figures 1A-1E, dsRNA can be separated from ssRNA, and the separated dsRNA and ssRNA can then be sequentially detected as shown generally in Figures 2A-2F.

[0059] As shown in Figures 2A-2B, the sample is drawn into the loading channel across its intersection with the analysis channel, thereby aligning the dsRNA (indicated by crosses) and ssRNA (indicated by circles) in the applied electric field. Figures 2B-2C show "pinching" the sample at the intersection by applying a slight voltage gradient away from the flow path toward the top and bottom of the analysis channel. The pinched volume of sample at the intersection, including both dsRNA and ssRNA, is then injected into the analysis channel by applying a voltage gradient across the length of the analysis channel, e.g., from top to bottom, as shown in Figure 2D. As shown in Figures 2E-2F, the sample is electrophoresed through the analysis channel such that the faster migrating dsRNA is separated from the slower migrating ssRNA, allowing the dsRNA to flow separately past the detection window (dashed box) before the flow of the ssRNA past the detection window.

[0060] In some embodiments, the polymer solution may be introduced or "preloaded" into the capillary channel prior to introducing the dsRNA and ssRNA sample into the capillary channel. Thus, the sample is introduced into the capillary channel preloaded with the polymer solution. In such cases, the polymer solution and / or the sample may include an intercalating agent.

[0061] The sample may be present in an aqueous buffer having a similar or different ionic strength to the polymer solution. In some embodiments, the sample is present in an aqueous buffer having an ionic strength similar to that of the polymer solution. In some embodiments, the sample is present in an aqueous buffer having a lower ionic strength compared to that of the polymer solution.

[0062] The methods described herein encompass detecting dsRNA and ssRNA, or the absence thereof, in various samples. In some embodiments, the sample is an RNA-based therapeutic. In such a case, the methods provided herein can be used to evaluate the purity of the RNA-based therapeutic, for example, to detect the amount of dsRNA contamination in an mRNA therapeutic. Non-limiting examples of RNA-based therapeutics that can be analyzed as described herein include antisense oligonucleotides, aptamers, small interfering RNA, microRNA, and messenger RNA.

[0063] The methods described herein can be used to detect low concentrations of dsRNA and ssRNA in a sample. In some examples, the limit of detection (LOD) of the methods described herein can be 1 pg / μL to 100 pg / μL for dsRNA and ssRNA.

[0064] Thus, in some examples, the concentration of dsRNA in the sample is 10 pg / μL to 200 ng / μL, e.g., 50 pg / μL to 200 ng / μL, 100 pg / μL to 200 ng / μL, 150 pg / μL to 200 ng / μL, 10 pg / μL to 150 ng / μL, 10 pg / μL to 100 ng / μL, or 10 pg / μL to 50 ng / μL. In some examples, the concentration of dsRNA in the sample is 1 pg / μL to 100 pg / μL, for example, 10 pg / μL to 100 pg / μL, 25 pg / μL to 100 pg / μL, 50 pg / μL to 100 pg / μL, 75 pg / μL to 100 pg / μL, 1 pg / μL to 75 pg / μL, 1 pg / μL to 50 pg / μL, 1 pg / μL to 25 pg / μL, or 1 pg / μL to 10 pg / μL.

[0065] In some examples, the concentration of ssRNA in the sample is 50 pg / μL to 200 ng / μL, for example, 100 pg / μL to 200 ng / μL, 150 pg / μL to 200 ng / μL, 50 pg / μL to 150 ng / μL, or 50 pg / μL to 100 ng / μL. In some examples, the concentration of ssRNA in the sample is 1 pg / μL to 100 pg / μL, e.g., 10 pg / μL to 100 pg / μL, 25 pg / μL to 100 pg / μL, 50 pg / μL to 100 pg / μL, 75 pg / μL to 100 pg / μL, 1 pg / μL to 75 pg / μL, 1 pg / μL to 50 pg / μL, 1 pg / μL to 25 pg / μL, or 1 pg / μL to 10 pg / μL.

[0066] In some examples, the percentage of dsRNA contamination relative to the total amount of ssRNA in a sample is between 0.1% and 1%, e.g., between 0.25% and 1%, between 0.5% and 1%, between 0.75% and 1%, between 0.1% and 0.75%, between 0.1% and 0.5%, or between 0.1% and 0.25%.

[0067] The methods described herein can be performed using a variety of microfluidic devices and systems. A microfluidic device or system refers to a device or system incorporating at least two intersecting channels or fluid conduits, where at least one of the channels has at least one cross-sectional dimension in the range of about 0.1 to about 500 μm, e.g., about 1 to about 100 μm.

[0068] A microfluidic device for use in the methods described herein may include a central body structure on which various microfluidic elements are disposed. For example, the body structure of a microfluidic device for use in the methods described herein typically employs a solid or semi-solid substrate that is typically planar in structure, e.g., substantially flat or has at least one flat surface. A suitable substrate may be fabricated from any one of a variety of materials or combinations of materials. In many cases, the substrate is fabricated using solid substrates common in the field of microfabrication, e.g., silica, silicon, or gallium arsenide. For these substrates, common microfabrication techniques, such as photolithography techniques, wet chemical etching, micromachining, etc., may be readily applied in the fabrication of microfluidic devices and substrates. Alternatively, polymeric substrate materials, including, for example, polydimethylsiloxane (PDMS), polymethylmethacrylate (PMMA), polyurethane, polyvinyl chloride (PVC), polystyrene, polysulfone, polycarbonate, etc., may be used to fabricate devices for use in the methods described herein.

[0069] Generally, microfluidic devices for use in the methods described herein include an analysis channel and a loading channel in a cross-channel configuration, and a sample is loaded into the analysis channel by placing it in a reservoir at the end of the loading channel and applying a voltage to the loading channel until the sample is electrophoresed across the intersection of the loading and analysis channels. Typically, the voltage is applied via electrodes located in a reservoir or well at the end of a given channel (also referred to as a "port"). The plug of sample at the intersection is then electrophoresed down the analysis channel by applying a voltage to the analysis channel.

[0070] Examples of suitable microfluidic systems, methods, and devices are described, for example, in U.S. Pat. Nos. 5,976,336, 7,419,784, 7,276,330, 7,081,190, 5,948,227, 6,042,710, and 6,440,284, each of which is incorporated by reference herein in its entirety. EXAMPLES

[0071] In order that the invention described may be more fully understood, the following examples are set forth. The examples described in this application are provided to illustrate the methods and compositions provided herein and should not be construed in any way as limiting the scope thereof.

[0072] Example 1 Separation and detection of dsRNA in samples containing dsRNA and ssRNA Separation and detection of standard dsRNA and ssRNA samples in a microscale electrophoresis device (LabChip® GX / GXII Touch™ instrument) was performed as follows.

[0073] A. Preparation of the Reagent Chip The polymer-dye mixture was prepared by adding 550 μL of polymer to 7.5 μL of dye. The polymer-dye mixture was filtered using a spin filter and centrifuged at 9200 rcf for 7.5 minutes at room temperature. The reagent chip was then loaded by adding 75 μL of polymer-dye mixture to wells 3, 7, and 8, and 120 μL of polymer-dye mixture to well 10. The reagent chip was also loaded with 100 μL of marker solution (DNA High Sensitivity Reagent Kit, PN CLS760672) in well 4. See Figure 3 for the placement of the reagents on the chip.

[0074] B. Isolation of dsRNA ladder Separation was performed on a dsRNA ladder (New England Biolab, Cat# N0363S) containing a set of seven annealed double-stranded RNA molecules generated by in vitro transcription of 14 linear DNA templates, with ladder sizes of 500, 300, 150, 80, 50, 30 and 21 base pairs.

[0075] dsRNA samples were prepared by adding 6 μL of dsRNA ladder to 114 μL of DI water to form a 1× ladder solution. Then, 2 μL of 1× ladder and 8 μL of DI water were added to the wells of the sample plate. Samples were loaded into three wells and the analysis was performed in triplicate.

[0076] The samples and polymer-dye mixtures were mixed and flowed through the separation channel of the microfluidic device. Fluorescence was detected in the detection window. Representative graphs of fluorescence versus time for each sample are shown in Figure 4. Each dsRNA in the ladder (500, 300, 150, 80, 50, 30 and 21 base pairs) was separated and detected as shown.

[0077] C. Isolation of ssRNA ladder Separation was performed on ssRNA ladders (New England Biolab, Cat# N0364S) containing a set of six RNA molecules generated by in vitro transcription of a mixture of six linear DNA templates, with ladder sizes of 1000, 500, 300, 150, 80 and 50 bases.

[0078] The ssRNA samples were prepared as described for the dsRNA ladder. Specifically, 6 μL of ssRNA ladder was added to 114 μL of DI water to form a 1× ladder solution. Then, 2 μL of 1× ladder and 8 μL of DI water were added to the wells of the sample plate. Samples were loaded into three wells and the analysis was performed in triplicate.

[0079] The sample and polymer-dye mixture were mixed and flowed through the separation channel of the microfluidic device. Fluorescence was detected in the detection window. Representative graphs of fluorescence versus time for each sample are shown in Figure 5. Each dsRNA in the ladder (1000, 500, 300, 150, 80 and 50 bases) was separated and detected as shown.

[0080] D. Separation of dsRNA and ssRNA mixtures Separation is performed on a mixture of dsRNA ladder (New England Biolab, Cat# N0363S) and ssRNA ladder (New England Biolab, Cat# N0364S). The sample and polymer-dye mixture are mixed and run through the separation channel of the microfluidic device. Fluorescence is detected in the detection window. Representative graphs of fluorescence versus time for dsRNA ladder and ssRNA ladder are shown in FIG. 6. As shown, at the same molecular weight, dsRNA migrates faster than ssRNA, e.g., 300 nt (150 bp) dsRNA migrates faster than 300 nt ssRNA (approximately 34 seconds compared to approximately 45 seconds). Also shown is that at similar concentrations, ssRNA (7.14 ng / μL) showed a lower fluorescence signal than dsRNA (6.25 ng / μL) (0-400 fluorescence units compared to 500-1000 fluorescence units).

[0081] Example 2 Increasing the sensitivity of dsRNA detection by improving pinch flow Low amounts of dsRNA contamination in ssRNA samples make dsRNA detection difficult. Therefore, an improved pinch flow technique was developed to improve the sensitivity of dsRNA detection. Separation and detection of standard dsRNA samples in a microscale electrophoretic device (LabChip® GX / GXII Touch™ instrument) were performed using standard pinch flow conditions and improved conditions. Standard pinch flow conditions and improved pinch flow conditions used the same voltage to drive the samples in the loading channel and the separation channel. The improved pinch flow conditions utilized longer loading times and higher currents to allow injection of larger plugs of sample into the separation channel. As shown in Figure 7, the fluorescence signal increased about 6-fold in the chromatographic separation of dsRNA standard samples using improved pinch flow (lower panel) compared to the fluorescence signal using standard pinch flow (upper panel).

[0082] Example 3 Development of an assay to detect dsRNA contamination in ssRNA samples This example describes the development of an assay for the detection and characterization of harmful dsRNA contaminants in mRNA vaccines, using a dsRNA ladder to simulate dsRNA contaminants and a ssRNA ladder to simulate mRNA.Ladders were used because long dsRNA and mRNA molecules are not readily available, especially in highly purified form, and limited data has been collected to characterize them.The findings from these experiments were then used as a reference or starting point for the analysis of custom long (4,001 bp or nt) dsRNA and mRNA samples.

[0083] The ssRNA (catalog #N0364S) and dsRNA ladders (catalog #N0363S) used in the experiments described herein were purchased from New England Biolabs (New England Biolabs, Ipswich, MA). The ssRNA ladders contain fragments of 50nt, 80nt, 150nt, 300nt, 500nt and 1,000nt, whereas the dsRNA ladders contain fragments of 21bp, 30bp, 50bp, 80bp, 150bp, 300bp and 500bp. We note that the fragment concentrations in the ladders, especially the dsRNA ladders, are not fully characterized by the supplier, as difficulties in synthesis may lead to significant differences between batches.

[0084] The assays were performed using a high-throughput LabChip GX Touch II platform (PerkinElmer, Waltham, MA). As seen in FIG. 8, samples were analyzed in a chip loaded with a DNA dye gel matrix containing polydimethysiloxane (PDMS) and SYTO™ 61, and once complete, the same samples were analyzed using the same chip loaded with PDMS and an RNA dye gel matrix containing RiboRed. Once both assays were performed, the results were analyzed as described herein. The assays were performed using a variety of fluorescent dyes (SYTO™ 61 and RiboRed, PerkinElmer) and various concentrations of fluorescent dyes and gel polymers. Similar results were obtained using the DNA dye gel matrix and the RNA dye gel matrix, regardless of the order of sample analysis.

[0085] Single-stranded RNA molecules are generally heated before analysis to prevent their tertiary structure (e.g., hairpins) from interfering with accurate measurement of their mobility. However, it has been observed that the use of heat denatures the smaller strands of dsRNA, which causes the creation of dsRNA peaks (Figure 9A). Heating produces clear peaks for ssRNA samples (Figure 9B). Thus, the assay described herein excludes heating from sample preparation, since the denaturation caused by heating the sample makes it more difficult to characterize dsRNA.

[0086] Dye concentrations were varied and DMSO was used to keep the dye to gel matrix volume ratio constant for a given dye type. DNA dyes (SYTO™ 61, PerkinElmer) were diluted to concentrations of 0.58%, 1.17%, 1.75%, 2.34%, and 2.95%, and RNA dyes (RiboRed, PerkinElmer) were diluted to concentrations of 11.67%, 13.33%, 15.00%, 16.67%, and 18.33%. These percentages are based on the concentrations provided for the dye stock vials, and therefore the percentages do not represent the molar concentration of the solutions. Experiments were performed using 50 bp or nt fragments for each ladder to serve as representative fragment sizes and 6% gel matrix. At the concentrations tested, an increase in fluorescence was observed for dsRNA using the DNA dye (Figure 10A) and for ssRNA using the RNA dye (Figure 10B).

[0087] The ssRNA ladder was analyzed using a 6% gel followed by a 3% gel matrix mixture. The ladder showed significantly greater peak broadening in the 6% gel than in the 3% gel (Figure 11), which may be partially attributed to the significantly slower migration using the 6% gel. The dsRNA ladder was also evaluated using both gel percentages and similar results were obtained, although the change in separation was less evident for the dsRNA ladder due to the absence of larger fragments such as 1,000 nt present in the ssRNA ladder (data not shown).

[0088] The dsRNA and ssRNA ladders were analyzed using a microfluidic electrophoresis chip platform to obtain electropherograms (Figure 12A). To determine whether any differences exist between the two electropherograms, we evaluated whether there were significant differences between the migration time (Figure 12B) and mobility (Figure 12C) between the two types of molecules (Table 1). However, no significant differences in migration time and mobility were observed (Figures 12B-C).

[0089] [Table 1]

[0090] Example 4 Use of DNA and RNA dyes to label dsRNA and ssRNA fragments Due to the structure between dsRNA and dsDNA, and the overlap in bases between dsRNA and ssRNA, we investigated both DNA dyes and RNA dyes for visualization of our molecules. The DNA dye and RNA dye used in the experiment were SYTO™ 61 and RiboRed, respectively. To do so, we performed a new experiment in which we analyzed dsRNA and ssRNA ladders in a chip loaded with a gel matrix containing DNA dye, and then analyzed the same samples in the same chip loaded with a gel matrix containing RNA dye. Similar to the previous results, we found that the overall fluorescence of the ssRNA peaks was higher than that of the dsRNA peaks, but more interestingly, we found that the ladders responded to the dyes in the opposite way. While dsRNA ladder fragments generally yielded lower fluorescence than ssRNA ladder fragments when labeled with both dyes, we noticed the following trend: dsRNA exposed to DNA dyes was better labeled than dsRNA exposed to RNA dyes, whereas ssRNA was better labeled with RNA dyes (Figures 13A-F, Tables 2-3). As discussed below, this difference in response may be due to differences in the overall structure of both types of nucleic acid molecules and how each fluorophore may fit within their conformation.

[0091] [Table 2]

[0092] [Table 3]

[0093] When comparing the areas obtained using DNA dyes with those obtained using RNA dyes, on average, a 56% decrease in area was observed for the dsRNA ladders, whereas a 118% increase in area was observed for the ssRNA ladders. Interestingly, while the same trends were observed in both peak area and peak height for each fragment size, it was noted that for three out of five ssRNA fragments analyzed, the peak heights showed no statistical difference. While the standard deviation was higher in the case of peak heights, the change in peak heights when comparing the peak heights obtained with DNA dyes with those obtained with RNA dyes showed similar values; a 57% decrease for the dsRNA ladders and a 96% increase for the ssRNA ladders. This suggests that while both parameters are consistent, the peak area, which shows smaller error bars on average, can be used as an indicator when evaluating the impact of different dyes and conditions.

[0094] Example 5 Assay performance using long dsRNA and mRNA fragments To assess whether the findings made using shorter dsRNA (less than 500 bp) and ssRNA fragments (less than 1,000 nt) are applicable to longer fragments (4,001 bp or nt), we analyzed long dsRNA and long mRNA molecules using DNA and RNA dyes, SYTO™ 61 and RiboRed, respectively. These molecules were custom made to have the same sequence to reduce any effects that may be caused by differences in sequence (e.g., charge, flexibility). Interestingly, while FIG. 12C suggests that there is no significant difference in the mobility of the shorter ssRNA and dsRNA fragments, FIG. 14A-C suggests that there is a significant difference in mobility between the long dsRNA and mRNA molecules. This difference may be due to the length of the molecules or the use of mRNA instead of ssRNA. It was also found that, despite dsRNA and mRNA having similar concentrations, they show significant differences in the area under the curve, similar to the experiments carried out using the shorter fragments. Here, dsRNA was analyzed at a concentration of 2.60ng / μL, whereas mRNA was used at a concentration of 5.90 / μL. However, instead of showing a 2.3-fold increase in area, which corresponds to the change in concentration between the two, on average, the area of ​​the mRNA sample was 9.6 times larger than that of the dsRNA sample. Therefore, to better understand the mechanism behind this disproportionate change, we evaluated how each molecule type responded to a separate type of dye.

[0095] When custom-length dsRNA and mRNA were analyzed using both dye types (Figures 15A-C, Tables 4-5), we observed differences in how dsRNA and mRNA responded. There was an overall trend that dsRNA was more effectively labeled with DNA dyes, whereas mRNA was more effectively labeled with RNA dyes. For long dsRNA fragments, in all three independent replicates (each with 2-3 replicates), the area under the curve (Figure 15B) and peak heights (Figure 15C) were greater using DNA dyes than using RNA dyes. However, while 3 / 3 of the differences in area were statistically different, only 2 / 3 of the peak heights were statistically different. For long mRNA fragments, the averages for each replicate showed a greater area under the curve (Figure 15E) and peak heights (Figure 5F) when using RNA dyes, but only 2 / 3 of the area differences were statistically different and only 1 / 3 of the peak heights were statistically different. This suggests, as before, that the area under the curve appears to be a better indicator of the difference in response, in part due to the presence of narrower error bars. For long dsRNA fragments, when comparing values ​​obtained with the DNA dye to those obtained with the RNA dye, a 64% decrease in area was observed, while a 76% decrease in peak height was observed. For long mRNA fragments, a 74% increase in area was observed, while a 150% increase in peak height was observed.

[0096] [Table 4]

[0097] [Table 5]

[0098] Next, to better simulate a realistic scenario for detecting dsRNA in mRNA vaccines, we mixed dsRNA and mRNA samples and evaluated their individual responses in the presence of both dye types, as seen in Figures 16A-16E and Tables 6-7. Based on the triplicate nanodrop readings, dsRNA was used at a concentration of 2.60 ng / μL, whereas mRNA was used at a concentration of 5.90 / μL. Similar to the experiments with short nucleic acid fragments, dsRNA was better labeled with DNA dyes, whereas mRNA was better labeled with RNA dyes. When the samples were analyzed together, as opposed to when they were analyzed individually, the dsRNA area was reduced by 84% and the peak height was reduced by 87%, when comparing the values ​​obtained with the DNA dye to those obtained with the RNA dye. For the mRNA samples, the area showed a 74% increase, while a 54% increase was observed in the peak height. Interestingly, the changes in dsRNA samples are more pronounced when samples are mixed, whereas the changes in mRNA samples are less clear.However, regardless of the difference in changes when samples are mixed compared to when samples are analyzed individually, the statistical difference between the two dyes is greater when samples are mixed.In the case of dsRNA, 3 / 3 of the area and 2 / 3 of the peak height are now statistically different, while in the case of mRNA samples, 3 / 3 of the area and 3 / 3 of the peak height are statistically different, whereas only 2 / 3 of the area and 1 / 3 of the peak height were statistically different before.

[0099] [Table 6]

[0100] [Table 7]

[0101] Although the exact binding method of the DNA and RNA dyes used in this study is unknown, our data suggests that DNA and RNA dyes may exhibit some selectivity when staining nucleic acids. Without wishing to be bound by theory, the observed selectivity of the dyes for different nucleic acids may be due to the different sizes of the dyes and nucleic acids. For example, RNA dyes may have a larger size, making it unable to effectively fit into the grooves or spaces between dsRNA. Evidence of the lower preference of RNA dyes for dsRNA samples can be seen by the dramatic reduction in the labeling area of ​​dsRNA samples when compared to the preference of DNA dyes. Similarly, DNA dyes may exhibit properties that make them too small to effectively fit and label ssRNA and mRNA molecules. For example, dsRNA is known to have an A-form helix, with an increase in length per base pair of approximately 2.8 Å and a radius of approximately 1.2 nm, effectively making it approximately 20% shorter and wider than dsDNA, which has a B-form helix

[25] . The shorter and wider spacing of dsRNA base pairs and grooves compared to that of DNA may explain the observation that most fragments in ssRNA ladders fluoresced brighter than dsRNA fragments in the early stages of experiments with DNA dyes. If DNA dyes were created to bind to dsDNA through groove binding or intercalation, the DNA dyes may be too large to effectively bind dsRNA, but rather the DNA dyes may fit into the larger spaces in ssRNA.

[0102] Example 6 Detecting dsRNA Contaminants in mRNA Samples Using Peak Area This example describes the identification of the type of molecule using the peak areas generated in the presence of RNA and DNA dyes, RiboRed and SYTO™ 61, respectively. Given that in some experiments there was no statistical difference between the peak heights or even the peak areas, the inventors developed a classification index to determine the type of molecule resulting in the peak. Based on the observed behavior of mRNA, ssRNA and dsRNA when exposed to various dye molecules, the inventors determined that the type of molecule can be identified based on the following relationship:

[0103]

number

[0104] If it is, then the molecule giving rise to the peak is dsRNA.

[0105]

number

[0106] If so, then the molecule giving rise to the peak is an mRNA (or ssRNA).

[0107] [Table 8]

[0108] Across all three independent runs (Table 8), the dsRNA peak ratio was less than 1, whereas the mRNA peak ratio was greater than 1. In this case, for each molecule type, 3 / 3 of the samples analyzed in either duplicates or triplicates met the identification criteria. Interestingly, the ratios obtained by the mRNA peak in both pure and mixed forms were close in value (1.68 for the pure sample and 1.77 for the mixed sample), whereas the ratios obtained by the dsRNA were quite different (0.40 in its pure form and 0.15 when mixed). This difference is likely due in part to degradation of the sample overlapping with the dsRNA peak. For example, in Figure 16A, an increase in baseline is seen to the left of the mRNA peak, which is likely due to sample degradation overlapping with the dsRNA peak. Although the dsRNA and mRNA peaks overlap, they are still distinguishable. For example, when averaging three independent runs, a statistical difference was found between the values ​​obtained for the dsRNA and mRNA samples (Figures 17A-B), and thus the difference between the ratios of the two types of molecules could be determined when they were mixed in a single sample. Furthermore, the dsRNA fragments used in these experiments had the same length as the mRNA fragments. This means that the shorter dsRNA fragments that may be found in the mRNA vaccine samples will migrate faster and overlap less with the mRNA peaks.

[0109] Example 7 Detection of dsRNA contamination in mRNA samples This example describes the detection of dsRNA contaminants in mRNA samples using the methods described herein. The linearity of area versus sample concentration was assessed for both types of molecules using DNA and RNA dyes, SYTO™ 61 and RiboRed, respectively (FIGS. 8A-B). While all samples showed a nearly linear relationship between area and concentration for both dsRNA and mRNA, the RNA dye had the lowest R of 0.75 for both molecules. 2It was found that the best-fit lines yielded the highest values ​​with values ​​of 0.93 and 0.84, respectively, with the DNA dye yielding the highest values. Also, for both molecule types, the slope of the best-fit line was found to be highest depending on the dye, specifically the DNA dye for dsRNA and the RNA dye for mRNA. Based on the equations for the lines of best fit obtained for each molecule with both dyes (Figures 8A-B), the lines for dsRNA converge when the dsRNA concentration is -0.34 ng / μL, whereas the lines for mRNA converge when the mRNA concentration is 0.38 ng / μL.

[0110] The limits of detection (LOD) were calculated by determining the minimum concentration that could be observed using the areas obtained by each sample in Figures 8A-8B and 9A-9B. This was done by dividing the concentration used by the area obtained. For dsRNA, the area used was the area using the RNA dye since they were smaller, and for mRNA, the area used was the area using the DNA dye since they were smaller. The results suggested that the LOD for dsRNA was 17.7-76.6 pg / μL, while the LOD for mRNA was 1.79-2.83 pg / μL. The difference in LOD is consistent with previous experiments showing that both ssRNA molecules and mRNA yielded larger areas, regardless of dye.

[0111] Furthermore, it is important to note that while the area tends to become larger as the concentration increases, the maximum concentration of mRNA that can be used without clogging or affecting the chip was determined to be 12.99 ng / μL. Considering that we can detect dsRNA concentrations as low as 17.7-76.6 pg / μL, if we load 12.99 ng / μL of mRNA, we should be able to detect contaminants that are present at percentages as low as 0.14-0.59% compared to the total mRNA concentration. Since the actual percentage of dsRNA present in the vaccine has not been fully quantified and characterized, it is very important to have a system with a low LOD that can detect low levels of contaminants.

[0112] During the synthesis and purification of mRNA vaccines, the mRNA concentration is routinely controlled, but manufacturers have little control over the exact percentage of dsRNA contaminants present in the sample. Therefore, we evaluated the ability of the method described herein to detect dsRNA contaminants at various concentrations relative to the total mRNA concentration. As shown in Figure 19A, there was an equal statistically significant difference between the dye ratios obtained by dsRNA peaks and the dye ratios of mRNA peaks, regardless of dsRNA concentration. Furthermore, as shown in Figure 19B, there was no statistical difference between the ratios obtained by dsRNA peaks at various concentrations, regardless of concentration, and by mRNA peaks at equal concentrations when dsRNA concentration is changed. These results suggest that although the areas obtained by each peak are diverse, the method described herein is robust and may be useful for detecting dsRNA contaminants regardless of the concentration of dsRNA relative to the concentration of mRNA in a sample.

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[0114] Other embodiments While the invention has been described in conjunction with its detailed description, it will be understood that the foregoing description is intended to be illustrative, and not limiting, of the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims.

Claims

1. 1. A method for separating and detecting double-stranded ribonucleic acid (dsRNA) from single-stranded ribonucleic acid (ssRNA) by capillary electrophoresis, comprising: introducing a polymer solution and a sample comprising dsRNA and ssRNA into a first capillary channel, the polymer solution being introduced before the sample, and the polymer solution or the sample comprising an intercalating agent; injecting a portion of the sample into a second capillary channel that intersects and is fluidly connected to the first capillary channel; applying a voltage gradient across the length of the second capillary channel to separate dsRNA into a first segment of the portion of the sample and ssRNA into a second segment of the portion of the sample; detecting the amount of intercalating agent in the first segment and the second segment, wherein the amount of intercalating agent in the first segment indicates the amount of dsRNA in the sample and the amount of intercalating agent in the second segment indicates the amount of ssRNA in the sample. and

2. 2. The method of claim 1, wherein the intercalating agent is fluorescent and detecting the amount of the intercalating agent comprises detecting a fluorescent signal.

3. 3. The method according to claim 1 or 2, wherein the concentration of the intercalating agent in the polymer solution or in the sample is between 0.01 mM and 0.2 mM.

4. 4. The method of any one of claims 1 to 3, wherein the intercalating agent is selected from the group consisting of SYTO™ dyes, Ribo dyes, SYBR dyes, ethidium bromide, acridine orange, propidium iodide, 7-aminoactinomycin D (7-AAD), 4',6-diamidino-2-phenylindole (DAPI), cyanine dyes, Alexa Fluor dyes, BODIPY dyes, and combinations thereof.

5. The method according to any one of claims 1 to 4, wherein the concentration of the polymer in the polymer solution is from 0.01% to 20% (w / v).

6. 6. The method of any one of claims 1 to 5, wherein the polymer has a molecular weight of from 1 Kd to 5,000 Kd.

7. 7. The method of any one of claims 1 to 6, wherein the polymer is formed from monomers selected from the group consisting of acrylic acid, carboxylic acid, sulfonic acid, phosphate monomers, bisacrylamide acetic acid, 4,4-bis(4-hydroxyphenyl)pentanoic acid, 3-butene-1,2,3-tricarboxylic acid, 2-carboxyethyl acrylate, itaconic acid, methacrylic acid, and 4-vinylbenzoic acid.

8. 8. The method of claim 1, wherein the polymer is an acrylic polymer.

9. 9. The method of claim 8, wherein the acrylic polymer is selected from the group consisting of polyacrylamide polymers, polymethylacrylamide polymers, polydimethylacrylamide polymers, and polydimethylacrylamide-co-acrylic acid polymers.

10. 10. The method of claim 1, wherein the polymer comprises a net charge of 0.01% to 2%, the net charge being the same charge as at least one surface of the capillary channel.

11. The method of claim 10, wherein the polymer comprises negatively charged monomer subunits and the at least one surface of the capillary channel has a negative surface charge.

12. 12. The method of claim 11, wherein the negatively charged monomer subunit is selected from the group consisting of acrylic acid, bisacrylamide acetic acid, 4,4-bis(4-hydroxyphenyl)pentanoic acid, 3-butene-1,2,3-tricarboxylic acid, 2-carboxyethyl acrylate, itaconic acid, methacrylic acid, 4-vinylbenzoic acid, sulfonic acid, 2-acrylamido-2-methyl-1-propanesulfonic acid, 2-methyl-2-propene-1-sulfonic acid, 2-propene-1-sulfonic acid, 4-styrenesulfonic acid, 2-sulfoethyl methacrylate, 3-sulfopropyldimethyl-3-methacrylamidopropylammonium inner salt, 3-sulfopropyl methacrylate, vinylsulfonic acid, bis(2-methacryloxyethyl)phosphate, and monoacryloxyethyl phosphate.

13. The method of claim 10, wherein the polymer comprises positively charged monomer subunits and the at least one surface of the capillary channel has a positive surface charge.

14. 14. The method of claim 13, wherein the positively charged monomer subunit is selected from the group consisting of 2-acryloxyethyltrimethylammonium chloride, diallyldimethylammonium chloride, 2-methacryloxyethyltrimethylammonium chloride, and 3-methacryloxy-2-hydroxypropyltrimethylammonium chloride.

15. The method according to any one of claims 1 to 14, wherein the concentration of dsRNA in the sample is between 1 pg / μL and 100 pg / μL.

16. The method according to any one of claims 1 to 15, wherein the concentration of ssRNA in the sample is between 1 pg / μL and 100 pg / μL.

17. 17. The method of claim 1, wherein the step of injecting the portion of the sample from the capillary channel into the second capillary channel comprises applying a voltage difference across the capillary channel to electrokinetically move the portion of the sample from the capillary channel to the second capillary channel.

18. introducing a further polymer solution and the sample into the first capillary channel, the further polymer solution being introduced before the sample, and the further polymer solution or the sample comprising a further intercalating agent; injecting a portion of the sample into the second capillary channel that intersects and is fluidly connected to the first capillary channel; applying a second voltage gradient across the length of the second capillary channel to separate dsRNA into a further first segment of the portion of the sample and ssRNA into a further second segment of the portion of the sample; detecting the amount of the further intercalating agent in the further first segment and the further second segment, wherein the amount of the further intercalating agent in the further first segment indicates the amount of dsRNA in the sample and the amount of the further intercalating agent in the further second segment indicates the amount of ssRNA in the sample. The method of any one of claims 1 to 17, further comprising:

19. 19. The method of any one of claims 1 to 18, wherein one or more steps are carried out in a microfluidic device.

20. 1. A method for separating and detecting double-stranded ribonucleic acid (dsRNA) from single-stranded ribonucleic acid (ssRNA) in a microfluidic device, comprising: a substrate having a surface; an analytical channel disposed in said substrate; a sample loading channel disposed on the substrate and intersecting the analytical channel at an intersection; Providing a microfluidic device comprising: introducing a polymer solution and a sample comprising dsRNA and ssRNA into the loading channel, the polymer solution being introduced before the sample, and the polymer solution or the sample comprising an intercalating agent; injecting a portion of the sample into the analytical channel; applying a voltage gradient across the length of the analysis channel to separate dsRNA into a first segment of the portion of the sample and ssRNA into a second segment of the portion of the sample; detecting the amount of intercalating agent in the first segment and the second segment, wherein the amount of intercalating agent in the first segment indicates the amount of dsRNA in the sample and the amount of intercalating agent in the second segment indicates the amount of ssRNA in the sample. and

21. 21. The method of claim 20, wherein the intercalating agent is fluorescent and the detecting step comprises detecting a fluorescent signal.

22. 22. The method according to claim 20 or 21, wherein the concentration of the intercalating agent in the sample is between 0.01 mM and 0.2 mM.

23. 23. The method of any one of claims 20 to 22, wherein the intercalating agent is selected from the group consisting of SYTO™ dyes, Ribo dyes, SYBR dyes, ethidium bromide, acridine orange, propidium iodide, 7-aminoactinomycin D (7-AAD), 4',6-diamidino-2-phenylindole (DAPI), cyanine dyes, Alexa Fluor dyes, BODIPY dyes, and combinations thereof.

24. 24. The method of any one of claims 20 to 23, wherein the concentration of the polymer in the sample is between 0.01% and 20% (w / v).

25. 25. The method of any one of claims 20 to 24, wherein the polymer has a molecular weight of from 1 Kd to 5,000 Kd.

26. 26. The method of any one of claims 20 to 25, wherein the polymer is formed from monomers selected from the group consisting of acrylic acid, carboxylic acid, sulfonic acid, phosphate monomers, bisacrylamide acetic acid, 4,4-bis(4-hydroxyphenyl)pentanoic acid, 3-butene-1,2,3-tricarboxylic acid, 2-carboxyethyl acrylate, itaconic acid, methacrylic acid, and 4-vinylbenzoic acid.

27. 27. The method of any one of claims 20 to 26, wherein the polymer is an acrylic polymer.

28. 28. The method of claim 27, wherein the acrylic polymer is selected from the group consisting of polyacrylamide polymers, polymethylacrylamide polymers, polydimethylacrylamide polymers, and polydimethylacrylamide-co-acrylic acid polymers.

29. 29. The method of any one of claims 20 to 28, wherein the polymer comprises a net charge of between 0.01% and 2%, the net charge being the same charge as at least one surface of a separation channel.

30. 30. The method of claim 29, wherein the polymer comprises negatively charged monomeric subunits and the at least one surface of the separation channel has a negative surface charge.

31. 31. The method of claim 30, wherein the negatively charged monomer subunit is selected from the group consisting of acrylic acid, bisacrylamide acetic acid, 4,4-bis(4-hydroxyphenyl)pentanoic acid, 3-butene-1,2,3-tricarboxylic acid, 2-carboxyethyl acrylate, itaconic acid, methacrylic acid, 4-vinylbenzoic acid, sulfonic acid, 2-acrylamido-2-methyl-1-propanesulfonic acid, 2-methyl-2-propene-1-sulfonic acid, 2-propene-1-sulfonic acid, 4-styrenesulfonic acid, 2-sulfoethyl methacrylate, 3-sulfopropyldimethyl-3-methacrylamidopropylammonium inner salt, 3-sulfopropyl methacrylate, vinylsulfonic acid, bis(2-methacryloxyethyl)phosphate, and monoacryloxyethyl phosphate.

32. 30. The method of claim 29, wherein the polymer comprises positively charged monomeric subunits and the at least one surface of the separation channel has a positive surface charge.

33. 33. The method of claim 32, wherein the positively charged monomeric subunit is selected from the group consisting of 2-acryloxyethyltrimethylammonium chloride, diallyldimethylammonium chloride, 2-methacryloxyethyltrimethylammonium chloride, and 3-methacryloxy-2-hydroxypropyltrimethylammonium chloride.

34. The method of any one of claims 20 to 33, wherein the concentration of dsRNA in the sample is between 1 pg / μL and 100 pg / μL.

35. The method of any one of claims 20 to 34, wherein the concentration of ssRNA in the sample is between 1 pg / μL and 100 pg / μL.

36. introducing a further polymer solution and the sample into the first capillary channel, the further polymer solution being introduced before the sample, and the further polymer solution or the sample comprising a further intercalating agent; injecting a portion of the sample into the second capillary channel that intersects and is fluidly connected to the first capillary channel; applying a second voltage gradient across the length of the second capillary channel to separate dsRNA into a further first segment of the portion of the sample and ssRNA into a further second segment of the portion of the sample; detecting the amount of the further intercalating agent in the further first segment and the further second segment, wherein the amount of the further intercalating agent in the further first segment indicates the amount of dsRNA in the sample and the amount of the further intercalating agent in the further second segment indicates the amount of ssRNA in the sample. The method of any one of claims 1 to 18, further comprising:

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