Method for purifying synthetic nucleic acid

Abstract

The present invention relates to a method for the separation and purification of synthetic nucleic acids. Unlike conventional nucleic acid purification methods using silica, the method of the present invention neither uses harmful substances nor employs the step of additionally binding cationic substances to silica, but enables the binding of nucleic acids to porous silica only by simply adding spermine, thus allowing for specific purification of nucleic acids. The method can selectively separate single-stranded nucleic acids without additional pre-processing after synthesis. Furthermore, by using a nucleic acid elution solution containing EDTA, the elution rate of single-stranded nucleic acids is significantly increased, making the method highly useful for purifying synthesized nucleic acids or nucleic acid production.

Description

Method for purifying synthetic nucleic acids

[0001] The present invention relates to a method for separating and purifying synthetic nucleic acids.

[0002] Advances in molecular biology have led to the development of numerous genetic technologies, enabling the isolation and identification of a growing number of disease-causing genes. Consequently, molecular biological techniques have been adopted in the medical field for diagnostic and testing purposes, making previously impossible diagnoses possible and dramatically reducing the time required for such testing. These advances are largely due to the practical application of nucleic acid amplification methods, particularly the polymerase chain reaction (PCR). PCR enables the sequence-specific amplification of nucleic acids in solution. Therefore, for example, the presence of a virus present in trace amounts in serum can be indirectly confirmed by amplifying and detecting the nucleic acid, the viral gene, using PCR. However, several challenges arise when using PCR for routine clinical testing. Among these challenges, the ability to purify nucleic acids, a preprocessing step for PCR-based evaluation, is crucial. Therefore, several methods for purifying nucleic acids have been proposed. Furthermore, the production of high-purity double-stranded plasmid DNA, single-stranded phage DNA, chromosomal DNA, and agarose gel-purified DNA fragments is crucial in molecular biology. Ideally, DNA purification methods should be simple, rapid, and require minimal, if any, additional sample handling steps. DNA obtained by these methods can be immediately used for transformation, restriction enzyme analysis, ligation, or sequencing.

[0003] Conventional methods for purifying nucleic acids using solid-phase materials have been known. For example, U.S. Patent No. 5,234,809 discloses a method for purifying nucleic acids using a solid-phase material that binds to nucleic acids. Specifically, the method comprises the steps of mixing a starting material, a chaotropic material, and a nucleic acid-binding solid-phase material, separating the solid-phase material containing the bound nucleic acids from a liquid, and washing the solid-phase nucleic acid complex. However, this method is complex due to its time-consuming nature and is unsuitable for lab-on-a-chip applications. Furthermore, the method suffers from the drawback of requiring the use of a chaotropic material.

[0004] Meanwhile, RNA, which contains repeating ribonucleotide units, plays a role in key cellular processes, including gene expression and protein synthesis. Therefore, RNA-based therapeutics are an attractive drug class for treating various diseases and an attractive target for modulating RNA vaccines that induce cellular immune responses. Significant advances have been made in RNA therapeutics, which modulate gene expression by delivering messenger RNA (mRNA) or non-coding RNAs, such as microRNA (miRNA), short interfering RNA (siRNA), and antisense oligonucleotides (ASOs), of the target gene. Furthermore, mRNA vaccines, starting with the coronavirus vaccine, have offered a new avenue for treating emerging infectious diseases and cancer. mRNA vaccines deliver mRNA synthesized through in vitro transcription (IVT) into human cells, where the delivered mRNA induces the cells to produce the viral spike protein, stimulating the humoral immune system. mRNA vaccines enable rapid production, stability, and personalized treatment, opening up the potential to address a variety of diseases. Accordingly, the demand for effective techniques for purifying non-immunogenic mRNA through in vitro transcription reactions has increased. mRNA synthesis uses DNA containing the T7 promoter sequence as a template, and T7 RNA polymerase synthesizes mRNA complementary to the template DNA. However, after the mRNA synthesis reaction is complete, the reaction solution retains not only single-stranded mRNA but also the nucleotides, enzyme proteins, and ions used as production materials. In particular, T7 RNA polymerase exhibits imprecise enzymatic activity, producing RNA corresponding to the synthesized RNA rather than the template DNA. Consequently, double-stranded RNA can be generated during the IVT reaction, which is an inducer of the innate immune response.Abnormal immune system activity and reduced translation efficiency caused by double-stranded RNA are being highlighted as causes of adverse effects of mRNA vaccines. Therefore, after IVT mRNA synthesis, the development of purification technologies that isolate double-stranded RNA, as well as purification of nucleotides, enzymes, proteins, and ions, is crucial.

[0005] Currently used mRNA purification methods include oligo-dT resin-based affinity chromatography, C18 resin-based reverse-phase liquid chromatography, and cellulose resin chromatography. The most widely used mRNA purification method is reverse-phase liquid chromatography using C18 resin. However, this purification method has the disadvantage of requiring the use of toxic substances such as triethyl ammonium acetate (TEAA) and acetonitrile as purification buffers.

[0006] An object of the present invention is to provide a composition for purifying synthetic nucleic acids.

[0007] In addition, it is an object of the present invention to provide a kit for purifying synthetic nucleic acids.

[0008] In addition, it is an object of the present invention to provide a method for purifying synthetic nucleic acids.

[0009] In addition, it is an object of the present invention to provide a method for producing nucleic acid.

[0010] To solve the above problem, the present invention provides a composition for purifying synthetic nucleic acids comprising porous silica and spermidine.

[0011] Additionally, the present invention provides a kit for purifying synthetic nucleic acids comprising the composition.

[0012] The present invention also provides a method for purifying a synthetic nucleic acid.

[0013] In addition, the present invention provides a method for producing a nucleic acid.

[0014] Unlike conventional nucleic acid purification methods using silica, the present invention can specifically purify nucleic acids by binding them to porous silica only by adding spermidine without using hazardous substances and without a separate process of additionally binding a positively charged substance to silica, and can selectively separate only single-stranded nucleic acids without a separate pretreatment process after the synthesis reaction, and has the effect of significantly increasing the elution rate of single-stranded nucleic acids by using a nucleic acid elution solution containing EDTA.

[0015] Figure 1 is a schematic diagram showing an IVT RNA purification method using porous silica and spermidine of the present invention.

[0016] Figure 2 is a diagram confirming the induction of binding of porous silica and IVT RNA by spermidine:

[0017] A: Schematic diagram showing the process of conjugating IVT RNA to porous silica using spermidine in a microtube (1.5 mL);

[0018] B: IVT RNA bound to porous silica according to IVT buffer solution composition;

[0019] C: IVT RNA bound to porous silica by spermidine contained in IVT buffer; and

[0020] D: IVT RNA bound to porous silica according to the RNA binding effect according to the N / P ratio of spermidine and IVT RNA (N: amino groups of spermidine and phosphates of RNA).

[0021] Figure 3 compares the IVT RNA binding efficiency when spermidine is present in the stationary or mobile phase:

[0022] A: Schematic diagram of experimental design using spin columns; and

[0023] B: Amount of IVT RNA bound to porous silica when spermidine is present in the mobile phase (top) or stationary phase (bottom).

[0024] Figure 4 is a diagram comparing IVT RNA bound / adsorbed to silica according to the form of silica:

[0025] A: IVT RNA binding and elution of porous silica or spherical silica in the presence of spermidine at increasing N / P ratios; and

[0026] B: Relative amounts of RNA present in the flowthrough (unbound RNA) and eluant (silica-bound RNA).

[0027] Figure 5 is a diagram comparing the binding and desorption effects of IVT RNA according to three polyamines (ethylenediamine, spermidine, and spermine).

[0028] Figure 6 is a diagram comparing the desorption efficiency of IVT RNA according to the buffer solution (elution solution) used to desorb IVT RNA.

[0029] Figure 7 is a diagram showing the derivation of EDTA as the optimal dissolution solution and its effectiveness:

[0030] A: RNA elution amount according to stepwise increase in concentration of salt (NaCl) elution solution and chelating agent (EDTA) elution solution;

[0031] B: RNA recovery rate of each elution solution;

[0032] C: Concentration of EDTA elution solution required depending on the amount of RNA; and

[0033] D: Integrity of eluted RNA.

[0034] Figure 8 is a diagram confirming the removal effect of dsRNA from IVT RNA using the method of the present invention:

[0035] A: The ratio of double-stranded RNA content in the eluate obtained by gradually increasing the EDTA concentration;

[0036] B: The amount of double-stranded RNA contained in RNA eluted using EDTA; and

[0037] C: Recovery rate of purified single-stranded RNA.

[0038] Hereinafter, the present invention will be described in detail with reference to the attached drawings and embodiments thereof. However, the following embodiments are provided as examples of the present invention. If a detailed description of a technology or configuration well known to those skilled in the art is judged to unnecessarily obscure the gist of the present invention, such detailed description may be omitted, and the present invention is not limited thereby. The present invention is capable of various modifications and applications within the scope of the following claims and equivalents interpreted therefrom.

[0039] Additionally, the terminology used in this specification is intended to appropriately express preferred embodiments of the present invention, and may vary depending on the intent of the user or operator, or the customs of the field to which the present invention pertains. Therefore, the definitions of these terms should be determined based on the contents throughout this specification. Throughout this specification, when a part is said to "include" a certain component, unless specifically stated otherwise, this does not mean that other components are excluded, but rather that other components may be included.

[0040] Unless otherwise defined, all technical terms used in this invention have the same meaning as commonly understood by those skilled in the art. While preferred methods and samples are described herein, similar or equivalent methods are also included within the scope of the present invention. The contents of all publications cited herein as references are incorporated herein by reference.

[0041] In one aspect, the present invention relates to a composition for purifying synthetic nucleic acids comprising porous silica and spermidine.

[0042] In one embodiment, the nucleic acid may be DNA or RNA.

[0043] In one embodiment, the particle size of the porous silica may be 100 to 200 μm, and the surface area may be 800 to 1500 m 2 / g may be.

[0044] In one embodiment, the synthetic nucleic acid may be a nucleic acid synthesized in vitro using a polymerase, may be single-stranded DNA or RNA, may be in vitro transcribed (IVT) RNA synthesized in vitro using an RNA polymerase, and may be single-stranded RNA.

[0045] In one embodiment, the composition of the present invention may further comprise a nucleic acid elution solution containing EDTA (Ethylenediaminetetraacetic acid) for desorbing / eluting synthetic nucleic acids bound / adsorbed to porous silica by spermidine.

[0046] In the present invention, the synthesized nucleic acid comprises single-stranded DNA or RNA, and may comprise coding RNA or non-coding RNA. In one example, the coding RNA may comprise messenger RNA (mRNA). In another example, the non-coding RNA may comprise guide RNA (gRNA), small interfering RNA (siRNA), microRNA (miRNA), short hairpin (shRNA), piwi-interacting RNA (piRNA), RNA aptamer, transfer RNA (tRNA), or antisense oligonucleotide (ASO).

[0047] In one aspect, the present invention relates to a kit for purifying synthetic nucleic acids comprising the composition of the present invention.

[0048] In one embodiment, the kit may be in the form of a column, wherein spermidine may be present in the mobile phase.

[0049] In one aspect, the present invention relates to a method for purifying a synthetic nucleic acid, comprising the steps of: 1) mixing porous silica, spermidine, and a synthetic nucleic acid; 2) centrifuging; 3) adding and mixing an elution solution containing EDTA; and 4) centrifuging to obtain an elution solution.

[0050] In one embodiment, the synthetic nucleic acid may be a nucleic acid synthesized in vitro using a polymerase, may be single-stranded DNA or RNA, may be in vitro transcribed (IVT) RNA synthesized in vitro using an RNA polymerase, and may be single-stranded RNA.

[0051] In one embodiment, step 1) may include a) filling a column with porous silica; b) mixing spermidine and the synthesized nucleic acid; and c) placing the mixture of step b) into the column of step a) and mixing.

[0052] In one embodiment, the porous silica, spermidine, and synthesized nucleic acid can be mixed by stirring at room temperature for 5 to 15 minutes.

[0053] In one embodiment, spermidine and the synthesized nucleic acid can be mixed so that the N / P ratio of the number of amine groups N of spermidine and the number of phosphate groups P of the synthesized nucleic acid is 10 to 30, and the number of amine groups (N) of spermidine can be obtained by multiplying the number of moles of spermidine by 3, and the number of phosphate groups of RNA can be obtained by multiplying the number of moles of nucleic acid by the number of nucleic acid bases (nts).

[0054] In one embodiment, centrifugation can be performed at 2000 to 4000×g for 2 to 5 seconds.

[0055] In one embodiment, if the synthesized nucleic acid is 10 μg or less, an elution solution of 500 nmol of EDTA may be added, and if it is more than 10 μg, an elution solution of 1000 nmol of EDTA may be added.

[0056] In one embodiment, the elution solution may be added and mixed by stirring at room temperature for 5 to 15 minutes.

[0057] In one aspect, the present invention relates to a method for producing a nucleic acid, comprising the steps of: synthesizing a nucleic acid in vitro; mixing porous silica, spermidine, and the synthesized nucleic acid; centrifuging; adding and mixing an elution solution containing EDTA; and centrifuging to obtain an elution solution.

[0058] In one embodiment, the synthetic nucleic acid may be a nucleic acid synthesized in vitro using a polymerase, may be single-stranded DNA or RNA, may be in vitro transcribed (IVT) RNA synthesized in vitro using an RNA polymerase, and may be single-stranded RNA.

[0059] In one embodiment, in the step of mixing the porous silica, spermidine, and synthesized nucleic acid, the porous silica may be filled into a column, centrifuged to remove the buffer, the spermidine and synthesized nucleic acid may be mixed, and then placed into the column and mixed again.

[0060] In one embodiment, spermidine and the synthesized nucleic acid may be mixed so that the N / P ratio of the number of amine groups N of spermidine and the number of phosphate groups P of the synthesized nucleic acid is 10 to 30.

[0061] In one embodiment, if the synthesized nucleic acid is 10 μg or less, an elution solution of 500 nmol of EDTA may be added, and if it is more than 10 μg, an elution solution of 1000 nmol of EDTA may be added.

[0062] In one embodiment, the step of synthesizing the nucleic acid in vitro may be in vitro transcription (IVT) or reverse transcription, wherein IVT may be performed using an RNA polymerase, as long as the synthesis of mRNA from a DNA template encoding RNA is specifically and sufficiently initiated from each cognate RNA polymerase promoter, and full-length mRNA is obtained. The RNA polymerase may be selected from T7 RNA polymerase, SP6 RNA polymerase, and T3 RNA polymerase.

[0063] In the present invention, when less than 0.5% of the total purified nucleic acid is composed of nucleic acid contaminant molecules (double-stranded nucleic acid, double-stranded RNA, etc.) after purifying a synthetic nucleic acid by the method of the present invention, the purified nucleic acid used herein is "substantially free" of contaminant molecules. When the nucleic acid is RNA, the amount and relative amounts of non-contaminant mRNA molecules and RNA contaminant molecules can be determined by HPLC or other methods used in the art to isolate and quantify RNA molecules.

[0064] The present invention is described in more detail through the following examples. However, the following examples are intended only to concretize the content of the present invention and are not intended to limit the present invention.

[0065] Example 1. Binding analysis of mesoporous silica particles and IVT RNA by spermidine

[0066] 1-1. IVT RNA synthesis

[0067] IVT RNA (in vitro transcribed RNA) was produced as the target RNA by synthesizing RNA in vitro using T7 RNA polymerase. Specifically, plasmid DNA encoding EGFP (Addgene, Watertown, MA, USA) with 3'UTR, 5'UTR, and poly(A) tail sequences was linearized by EcoRI restriction enzyme (Takara, Tokyo, Japan), and then approximately 2 μg of linearized plasmid was added to 50 μL of a reaction solution containing 2 mM rants mixture (Promega, Madison, WI, USA), 100 U T7 RNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA), and 40 U recombinant RNase inhibitor (Takara) dissolved in IVT buffer containing 50 mM Tris-HCl (pH 7.5), 15 mM MgCl2, 2 mM spermidine (Sigma-Aldrich, St. Louis, MO, USA), and 5 mM DTT (Sigma-Aldrich). The in vitro transcription reaction mixture was incubated at 37°C for 2 hours, and 5 U DNase I (Takara) was added and incubated for 2 hours to induce DNA degradation. After incubation at 37°C for 30 minutes, 20 mM EDTA was added to the reaction mixture to stop the enzymatic reaction, and the synthesized RNA was isolated by precipitation with 8 M LiCl solution (Sigma-Aldrich), and then dissolved in nuclease-free water.The concentration of the IVT RNA (in vitro transcribed RNA) (996 nts) (Table 1) obtained in this way was measured using a UV-Vis spectrophotometer (Ultrospec 2100 pro spectrophotometer; Biochrom Ltd., Cambridge, UK) at a wavelength of 260 nm.

[0068] Sequence (5'->3')IVTRNAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAAGCGGCCGCGACTCTTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0069] 1-2. Confirmation of binding of IVT RNA and porous silica by spermidine

[0070] A substance that induces binding between the IVT RNA synthesized in Example 1-1 and porous silica was explored. Specifically, a material having a size of approximately 150 μm and a surface area of ​​approximately 1000 m was investigated. 2 / g of porous silica (SBA-15, cat. #913855, Sigma-Aldrich) was mixed in a 10 mM HEPES (pH 7.2) buffer solution at a concentration of 30 mg / mL to prepare a porous silica slurry, and a solution of 20 mM sodium ions (NaCl), magnesium ions (MgCl2), calcium ions (CaCl2) and IVT buffer (50 mM Tris-HCl (pH 7.5), 15 mM MgCl2, 2 mM spermidine and 5 mM DTT) was prepared. The porous silica slurry prepared above (0.6 mg), 20 μL of each of the prepared solutions and the IVT RNA (998 nts, 2 μg) synthesized in Example 1-1 were mixed, and the mixed solution was mixed in a microcentrifuge tube (1.5 mL) for 10 minutes. After centrifugation at 3000×g for 3 seconds, the supernatant was separated and the presence of IVT RNA in the supernatant was confirmed using the UREA PAGE experiment.

[0071] As a result, when sodium ions (NaCl), magnesium ions (MgCl2), or calcium ions (CaCl2) were added, IVT RNA was mostly detected in the supernatant, and it was found that IVT RNA bound to porous silica only when mixed with IVT buffer (Fig. 2A and B). Since the composition of IVT buffer is 50 mM Tris-HCl (pH 7.5), 15 mM MgCl2, 2 mM spermidine, and 5 mM DTT (Dithiothreitol), when analyzed which of these substances caused IVT RNA to bind to porous silica, it was found that IVT RNA bound to porous silica only when spermidine was present (Fig. 2C).

[0072] 1-3. Optimization of binding induction of IVT RNA and porous silica

[0073] To derive the amount of spermidine required to induce RNA binding to porous silica, the number of phosphate groups contained in RNA (998 nts) was represented as 'P', and the number of amine groups contained in spermidine was represented as 'N', and the N / P ratio was calculated. At this time, the number of moles of RNA in the sample was calculated by dividing the mass of IVT RNA (996 nts) by the molecular weight of RNA (320 kDa), and the number of phosphate groups (P) present in the RNA sample was obtained by multiplying the number of moles of RNA by the number of RNA bases (996 nts). The number of amine groups (N) of spermidine was obtained by multiplying the number of moles of spermidine by 3 (3 amine groups per 1 spermidine). The N / P ratio was obtained by dividing the number of amine groups (N) by the number of phosphate groups (P) of RNA. The binding of RNA to porous silica was analyzed as in the above example by adding spermidine with the N / P ratio derived in this way adjusted to 0, 1.25, 5, 10, and 20.

[0074] As a result, when the N / P ratio was less than 5, IVT RNA was not significantly bound to porous silica, and when the N / P ratio was 5 or more, IVT RNA was significantly bound to porous silica. In particular, when the N / P ratio was 20, no RNA was present in the supernatant, confirming that all IVT RNA was bound to porous silica when the N / P ratio was 20 (Fig. 2D).

[0075] Example 2. Comparison of IVT RNA binding efficiency when spermidine is present in the stationary or mobile phase.

[0076] Since we confirmed that spermidine can bind IVT RNA to silica particles in batch mode of chromatography, we analyzed the difference in separation efficiency of IVT RNA when applied to chromatography, when the stationary phase is bound to silica and fixed and when the mobile phase is in a buffer solution. To make a stationary phase by binding spermidine to porous silica particles, 50 mg of porous silica particles (SBA-15, Sigma-Aldrich) were amine-functionalized with 80 μL of 3-aminopropyl triethoxysilane (APTS, Sigma-Aldrich) and 50 μL of saturated ammonium hydroxide solution (Sigma-Aldrich) dissolved in 2 mL of ethanol, stirred overnight at 25 °C, and then washed the silica particles once with 2 mL of ethanol and once with 2 mL of methanol. The washed particles were dried under vacuum and dispersed in 3 mL of N,N-dimethyl formamide (DMF, Samchun Chemical, Seoul, Korea) with 1.2 mmol of succinic anhydride (Sigma-Aldrich) and 1.2 mmol of N,N-diisopropylethylamine (DIEA, Sigma-Aldrich). The particles were then stirred at 25 °C for 3 h to effect succinylation, and the silica particles were washed with 2 mL of DMF, methylene chloride (MC, Daejung Chemicals, Gyeonggi-do, Korea), and methanol and dried under vacuum. 0.4 mmol of diisopropylcarbodiimide (DIC, Sigma-Aldrich), 0.4 mmol of hydroxy benzotriazole (HOBT, Sigma-Aldrich), 0.48 mmol of DIEA, and 0.48 mmol of spermidine was added to succinylated porous silica dispersed in 1.5 mL of DMF and reacted overnight at 25°C with stirring. The silica particles were washed with 2 mL of DMF, MC, and methanol and dried under vacuum. The weight increment of the silica particles on the stationary phase where spermidine was bound to silica was measured to confirm that the amount of spermidine loaded on the porous silica was 0.69 mmol / g. In the group using the spermidine-bound porous silica as the stationary phase, 20 μL of the spermidine-bound silica slurry (30 mg / mL) was mixed with 20 μL of IVT RNA (998 nts, 0.2 μg). In the group using spermidine as the mobile phase for comparison, the same amount of spermidine as the stationary phase was added to the mobile phase. As in the above example, the silica slurry prepared with 10 mM HEPES (pH 7.2) was filled into a spin column (Pierce™ spin column, Thermo Fisher Scientific) and centrifuged at 3000×g for 3 s. Then, IVT RNA (998 nts, 2 μg) and spermidine corresponding to an N / P ratio of 20 were mixed in a volume of 20 μL and mixed at room temperature for 10 min. After centrifugation at 3000×g for 3 s, the filtrate (flowthrough) was collected. RNA present in the filtrate was confirmed by agarose gel electrophoresis.

[0077] As a result, when a stationary phase in which spermidine was bound to porous silica was used, IVT RNA did not bind to the silica particles, whereas when spermidine was present in the mobile phase, IVT RNA bound to the porous silica when the N / P ratio was 6 or higher (Fig. 3). In other words, it was confirmed that IVT RNA effectively bound to porous silica when spermidine was present in the mobile phase.

[0078] Example 3. Comparison of IVT RNA binding efficiency according to silica type

[0079] To determine the RNA binding effect according to the silica structure, porous silica and spherical silica were used to determine the RNA binding effect according to the silica shape. 20 μL of porous silica slurry (30 mg / mL) or spherical silica (cat. #78991, Sigma-Aldrich), each prepared by mixing 30 mg / mL in 10 mM HEPES (pH 7.2) buffer solution, was filled into a Pierce™ spin column (ThermoFisher Scientific) and centrifuged at 3000×g for 3 seconds to remove the buffer in the slurry. IVT RNA (998 nts, 2 μg) was mixed with spermidine, 10 mM HEPES buffer (pH 7.2), and 10 mM NaCl at an N / P ratio of 20 in a volume of 20 μL, transferred to a silica-filled spin column, and incubated with shaking at 25 °C for 10 min. The spin column was centrifuged at 3000 × g for 3 s to obtain the filtrate (flowthrough), and 20 μL of 300 mM NaCl elution buffer containing 10 mM HEPES (pH 7.2) was treated, stirred for 10 min, and centrifuged at 3000 × g for 3 s to obtain the eluate. RNA present in the collected filtrate and eluate was confirmed by agarose gel electrophoresis.

[0080] As a result, RNA was found to bind to porous silica when the N / P ratio was 20 or higher, whereas RNA did not bind to spherical silica even when the N / P ratio was 100. In addition, the amount of RNA eluted using electrolyte was found to be significantly lower in spherical silica than in porous silica (Fig. 4). Therefore, it was confirmed that porous silica is a more suitable resin for binding and desorption of IVT RNA using spermidine.

[0081] Example 4. Comparison of IVT RNA binding efficiency according to polyamine type

[0082] Three types of linear polyamines (ethylenediamine, spermidine, and spermine) were used to derive the most effective polyamine for the adsorption and desorption of IVT RNA. Specifically, a porous silica slurry (0.6 mg) was packed into a spin column (Pierce™ spin column, Thermo Fisher Scientific), and the buffer solution contained in the slurry was removed by centrifugation. IVT RNA (998 nts, 2 μg) was mixed with ethylenediamine, spermidine, and spermine at concentrations of 1 mM, 2 mM, 4 mM, and 8 mM, respectively, in a volume of 20 μL, and mixed at room temperature for 10 min. After centrifuging at 3000×g for 3 seconds, the filtrate was obtained by centrifuging for a second, and 20 μL of 300 mM NaCl elution buffer containing 10 mM HEPES (pH 7.2) was treated, stirred for 10 minutes, and centrifuged at 3000×g for 3 seconds to collect the eluant. The RNA present in the collected filtrate and eluate was confirmed by agarose gel electrophoresis.

[0083] As a result, the polyamines that induced the binding of IVT RNA were found to be spermidine and spermine, and upon elution, RNA bound to porous silica was desorbed only by spermidine (Fig. 5). Therefore, it was confirmed that spermidine is the most suitable polyamine for effectively binding and desorbing IVT RNA to porous silica.

[0084] Example 5. Comparison of IVT RNA desorption efficiency according to elution solution

[0085] To compare the desorption efficiency of IVT RNA according to the buffer solution for desorbing IVT RNA bound to porous silica, a spin column (Pierce™ spin column, Thermo Fisher Scientific) was filled with a porous silica slurry (0.6 mg), and the buffer solution contained in the slurry was removed through centrifugation. Then, IVT RNA (998 nts, 2 μg) and spermidine corresponding to an N / P ratio of 20 were mixed in a volume of 20 μL and mixed for 10 minutes at room temperature. After centrifugation at 3000×g for 3 seconds, the filtrate was obtained. Afterwards, 20 μL of each of a sodium chloride (NaCl) solution containing 10 mM HEPES (pH 7.2), an ethylenediaminetetraacetic acid (EDTA) solution containing 10 mM HEPES (pH 7.2), and a urea solution containing 10 mM HEPES (pH 7.2) were added to the spin column as an elution solution to detach the IVT RNA bound to the porous silica, and mixed at room temperature for 10 minutes. The mixture was centrifuged at 3000×g for 3 seconds to obtain an eluant, and the RNA present in the eluant was confirmed by agarose gel electrophoresis.

[0086] As a result, it was shown that RNA was present in the eluates extracted using the three elution solutions (Fig. 6), and it was confirmed that IVT RNA bound to porous silica in the presence of spermidine was desorbed by sodium chloride, EDTA, and urea.

[0087] Example 6. Derivation of optimal elution solution for IVT RNA desorption

[0088] 6-1. IVT RNA recovery rate analysis

[0089] The RNA recovery rate was compared when RNA was desorbed with the sodium chloride and EDTA elution solution used for IVT RNA desorption. Specifically, 100 μL of porous silica slurry (30 mg / mL) was charged into a spin column (Pierce™ spin column, Thermo Fisher Scientific), and the buffer solution contained in the slurry was removed through centrifugation. IVT RNA (998 nts, 10 μg) and spermidine corresponding to an N / P ratio of 20 were mixed in a volume of 100 μL and mixed in the spin column at room temperature for 10 minutes. After that, 100 μL of sodium chloride elution solution (1 mM, 5 mM, 7 mM, 10 mM, 20 mM, 30 mM, and 40 mM) or EDTA elution solution (50 mM, 70 mM, 100 mM, 300 mM, 500 mM, 700 mM, and 1000 mM) containing 10 mM HEPES (pH 7.2) was added to the spin column starting from the solution with the lowest concentration and mixed at room temperature for 10 minutes. The eluate was obtained by centrifugation at 3000×g for 3 seconds, and 100 μL of the elution solution with the second lowest concentration was added, mixed at room temperature for 10 minutes, and centrifuged at 3000×g for 3 seconds to obtain the eluate. After obtaining the eluate by gradually increasing the concentration of the elution solution in this way, the RNA present in each eluate was analyzed by agarose gel electrophoresis. The amount of RNA present in the eluate was quantified by fluorescence using the Quant-it™ RiboGreen RNA Test Kit (Invitrogen), and the RNA concentration in the sample was quantitatively analyzed by comparing the fluorescence intensity with the reference curve.

[0090] As a result, it was shown that the amount of RNA eluted from the porous silica increased as the concentration of EDTA or NaCl increased (Fig. 7A), and the amount of RNA recovered using EDTA was 80% of the amount of RNA initially injected, but the amount of RNA recovered using sodium chloride did not reach 20%, showing that EDTA showed a recovery rate about 4 times higher than that of sodium chloride (Fig. 7B).

[0091] 6-2. Analysis of the amount of EDTA required for IVT RNA elution

[0092] The amount of EDTA required to elute a specific amount of IVT RNA adsorbed on porous silica was analyzed. Specifically, 100 μL of porous silica slurry (30 mg / mL) was filled into a spin column (Pierce™ spin column, Thermo Fisher Scientific), and the buffer solution contained in the slurry was removed through centrifugation. 3, 5, 7, 10, or 20 μg of IVT RNA was mixed with spermidine at an N / P ratio of 20, and then mixed with the porous silica at room temperature for 10 minutes. Centrifugation was performed at 3000×g for 3 seconds, and the filtrate was obtained. To detach IVT RNA bound to porous silica, 500 nmol and 1000 nmol EDTA containing 10 mM HEPES (pH 7.2) were sequentially dispensed as elution solutions to obtain elutions at different concentration levels as in Example 6-1, and the RNA was analyzed by agarose gel electrophoresis. The amount of RNA present in the elution confirmed on the agarose gel was calculated by quantifying the band thickness using Image J software, and the recovery rate was calculated by RiboGreen analysis.

[0093] As a result, less than 10 μg of loaded RNA was effectively eluted by 500 nmol EDTA elution, whereas most of the RNA loaded in an amount of 20 μg was not eluted by 500 nmol EDTA and was eluted by 1000 nmol EDTA (Fig. 7C), confirming that 500 nmol EDTA can desorb IVT RNA from porous silica bound to less than 10 μg of IVT RNA, and 1000 nmol EDTA can desorb IVT RNA from porous silica bound to more than 10 μg of IVT RNA.

[0094] 6-3. Integrity Analysis of Detached IVT RNA

[0095] The integrity of the RNA eluted in Example 6-2 was analyzed using a capillary electrophoresis automatic nucleic acid analyzer (TapeStation, Agilent Technologies). As a result, the RNA eluted with EDTA was found to have the same RNA length and shape as the initially introduced RNA (input RNA) (Fig. 7D), confirming that EDTA does not affect the integrity of the RNA.

[0096] Example 7. Confirmation of the Selective Purification Effect of Single-Stranded RNA Using Porous Silica, Spermidine, and EDTA Elution Solutions

[0097] It was confirmed whether the RNA purification method using the porous silica, spermidine, and EDTA elution solution of the present invention can selectively purify only the target single-stranded RNA from nucleotides, enzyme proteins, ions, and double-stranded RNA, which are residual substances or products of incorrect enzyme activity during mRNA synthesis. Specifically, 100 μL of porous silica slurry (30 mg / mL) was filled into a spin column (Pierce™ spin column, Thermo Fisher Scientific), and the buffer solution contained in the slurry was removed through centrifugation. 10 μg of IVT RNA (998 nts) without any separate purification process was mixed with 100 μL of spermidine corresponding to an N / P ratio of 20, and then placed in the spin column and mixed for 10 minutes at room temperature. 100 μL of EDTA (5 mM, 6 mM, 7 mM, 8 mM, 9 mM, and 10 mM) solutions containing 10 mM HEPES (pH 7.2) were added to the spin column starting from the lowest concentration solution, mixed at room temperature for 10 minutes, and centrifuged at 3000×g for 3 seconds to obtain the eluate. After obtaining the eluate by gradually increasing the concentration of the eluate, the total RNA present in the eluate and the amount of double-stranded RNA contained therein were quantified by RiboGreen analysis, sandwich ELISA analysis using an antibody-based dsRNA ELISA kit (Exalpha Biologicals, Shirley, MA, USA) that utilizes double-strand-specific binding J2 antibody, and dot blot analysis. At this time, cellulose-based chromatography using conventional cellulose fibers was used as a control, and the eluent used at this time was 16% (v / v) ethanol.

[0098] As a result, the amount of double-stranded RNA contained in the RNA solution eluted with 5 mM EDTA was 0.26% of the total RNA mass eluted, which was lower than the 0.6% double-stranded RNA content present in the initially added IVT RNA. RNA eluted by gradually increasing the EDTA concentration had a higher double-stranded RNA content than when eluted with 5 mM EDTA (Fig. 8A). In addition, as a result of analyzing total RNA and dsRNA in each eluted solution, the RNA recovery rate was approximately 40% when RNA was eluted with 5 mM EDTA from porous silica, whereas the recovery rate when eluted with 16% (v / v) ethanol from cellulose resin was less than 30%. It was confirmed that 92% of double-stranded RNA was removed compared to the initially added RNA when 5 mM EDTA solution was used (Fig. 8C).

[0099] Through this, it was confirmed that the method of eluting RNA with EDTA using the spermidine-based porous silica chromatography of the present invention has excellent IVT RNA purification efficiency and can remove dsRNA contaminants more effectively than the conventional cellulose-based chromatography, and is therefore a suitable method for selectively purifying only the target single-stranded RNA.

Claims

1. A composition for purifying synthetic nucleic acid comprising porous silica and spermidine.

2. In paragraph 1, A composition for purifying synthetic nucleic acids, wherein the particle size of the porous silica is 100 to 200 μm.

3. In paragraph 1, The surface area of ​​the above porous silica is 800 to 1500 m 2 / g, a composition for purifying synthetic nucleic acids.

4. In paragraph 1, The above synthetic nucleic acid is a composition for purifying a synthetic nucleic acid, which is a nucleic acid synthesized in a test tube using a polymerase.

5. In paragraph 1, A composition for purifying synthetic nucleic acids, wherein the composition further comprises a nucleic acid elution solution containing EDTA (Ethylenediaminetetraacetic acid).

6. In paragraph 1, A composition for purifying a synthetic nucleic acid, wherein the synthetic nucleic acid is a single-stranded nucleic acid.

7. A kit for purifying a synthetic nucleic acid comprising the composition of paragraph 1. 8.1) Step of mixing porous silica, spermidine and synthesized nucleic acid; 2) Centrifugation step; 3) A step of adding and mixing an elution solution containing EDTA; and 4) A method for purifying a synthetic nucleic acid, comprising the step of obtaining an effluent by centrifugation.

9. In paragraph 8, A method for purifying a synthetic nucleic acid, wherein the above-mentioned synthesized nucleic acid is a single-stranded nucleic acid.

10. In paragraph 8, Step 1) above: a) A step of filling a column with porous silica; b) a step of mixing spermidine and synthesized nucleic acid; and c) A method for purifying a synthetic nucleic acid, comprising the step of introducing the mixture of step b) into the column of step a) and mixing.

11. In paragraph 8, A method for purifying a synthetic nucleic acid, comprising mixing the above porous silica, spermidine, and a synthetic nucleic acid by stirring at room temperature for 5 to 15 minutes.

12. In paragraph 8, A method for purifying a synthetic nucleic acid, wherein spermidine and a synthetic nucleic acid are mixed so that the N / P ratio of the number of amine groups of the spermidine and the number of phosphate groups of the synthetic nucleic acid is 10 to 30.

13. In paragraph 8, A method for purifying a synthetic nucleic acid, wherein the centrifugation is performed at 2000 to 4000×g for 2 to 5 seconds.

14. In paragraph 8, A method for purifying a synthetic nucleic acid, wherein if the synthesized nucleic acid is 10 μg or less, an elution solution of 500 nmol of EDTA is added, and if it exceeds 10 μg, an elution solution of 1000 nmol of EDTA is added.

15. In paragraph 8, A method for purifying synthetic nucleic acids, comprising adding the above-mentioned elution solution and stirring at room temperature for 5 to 15 minutes to mix. 16.1) Step of synthesizing nucleic acid in vitro; 2) A step of mixing porous silica, spermidine and synthesized nucleic acid; 3) Centrifugation step; 4) A step of adding and mixing an elution solution containing EDTA; and 5) A method for producing nucleic acid, comprising the step of obtaining an effluent by centrifugation.

17. In paragraph 16, A method for producing a nucleic acid, wherein the nucleic acid is a single-stranded nucleic acid.

18. In paragraph 16, Step 1) above: a) A step of filling a column with porous silica; b) a step of mixing spermidine and synthesized nucleic acid; and c) A method for producing a nucleic acid, comprising the step of introducing the mixture of step b) into the column of step a) and mixing.

19. In paragraph 16, A method for producing a nucleic acid, wherein spermidine and a synthesized nucleic acid are mixed so that the N / P ratio of the number of amine groups N of the spermidine and the number of phosphate groups P of the synthesized nucleic acid is 10 to 30.

20. In paragraph 16, A method for producing a nucleic acid, wherein if the synthesized nucleic acid is 10 μg or less, an elution solution of 500 nmol of EDTA is added, and if it exceeds 10 μg, an elution solution of 1000 nmol of EDTA is added.

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