CN116926149A

CN116926149A - Preparation method of dsRNA and application of dsRNA in preparation of dsRNA standard substance

Info

Publication number: CN116926149A
Application number: CN202310099407.3A
Authority: CN
Inventors: 耿玉杰
Original assignee: Wuhan New Biocalysis Solution Co ltd
Current assignee: Wuhan New Biocalysis Solution Co ltd
Priority date: 2023-02-01
Filing date: 2023-02-01
Publication date: 2023-10-24

Abstract

The application provides a preparation method of dsRNA and application thereof in preparation of dsRNA standard substances, and relates to the technical field of biology. The preparation method of the dsRNA comprises the steps of synthesizing ssRNA through in vitro transcription, and degrading an in vitro transcription template in a reaction product into single nucleotide; annealing to form dsRNA, degrading single-stranded nucleic acid in the annealed product by using S1 nuclease, and purifying the reaction product to obtain the dsRNA. The preparation method alleviates the problem that a method for preparing high-purity dsRNA is lacked in the prior art.

Description

Preparation method of dsRNA and application of dsRNA in preparation of dsRNA standard substance

Technical Field

The application relates to the technical field of biology, in particular to a preparation method of dsRNA and application of the dsRNA in preparation of a dsRNA standard substance.

Background

An mRNA vaccine is a vaccine that utilizes molecular copies of messenger RNA (mRNA) to generate an immune response. Such vaccines deliver antigen-encoding mRNA molecules to immune cells, which use the designed mRNA as a template to construct foreign proteins that are normally produced by a pathogen (e.g., a virus) or cancer cell. These protein molecules stimulate an adaptive immune response, teaching the body to recognize and destroy the corresponding pathogen or cancer cell. mRNA vaccines are composed of lipid nanoparticles and RNA encapsulated therein, protecting the RNA strand and helping it to be absorbed into cells. Because of the new crown epidemic situation, attention is paid to an mRNA vaccine technical route which has remarkable advantages in the aspects of curative effect, development speed period, cost price, expansibility and safety of production and the like.

The preparation and transcription of mRNA stock is a key step in vaccine production, and the quality of mRNA directly determines the clinical manifestation of the vaccine. The whole dsRNA is called double-stranded RNA, and is translated into double-stranded ribonucleic acid. During IVT (in vitro synthesis of mRNA), T7 RNA polymerase may be transcribed using RNA or DNA (template strand and non-template strand) as templates, forming different types of dsRNA byproducts. For mRNA vaccines and mRNA therapeutic drugs, the dsRNA by-products produced during the preparation process not only reduce the efficacy of the vaccines and drugs, but also induce adverse immune reactions, thus ensuring that the product is free of dsRNA impurities, which is an extremely important part in quality control of mRNA biological agents.

dsRNA detection methods include dot blot (immunoblots), ELISA (enzyme-linked immunosorbent assay), HTRF (homogeneous time resolved fluorescence) and HPLC. Except HPLC, dsRNA standard substances are needed in the detection process of other methods, like the process of detecting the content of dsRNA by a dot blot method stated in United states pharmacopoeia, firstly, the dsRNA standard substances and mRNA samples to be detected are dripped on a nylon membrane, after drying and sealing, detection antibody J2 is added, after incubation for 1hr, unbound detection antibodies are washed away, enzyme-labeled secondary antibodies are added, after incubation for 1hr as well, unbound enzyme-labeled secondary antibodies are washed away, substrate reaction color development is added, and the content of dsRNA in the mRNA samples is calculated through comparison of the color development signals of the mRNA samples and the dsRNA standard substances. Standard curves are also drawn with standard in both ELISA and HTRF.

At present, no dsRNA standard is sold in the market, and research and development personnel are required to prepare the dsRNA standard by themselves. dsRNA is usually produced by complementary pairing of two ssrnas (single-stranded RNAs), while ssrnas are synthesized either by chemical synthesis, which is limited in length, typically no more than 160nt, and expensive, or by T7 promoter-mediated in vitro transcription reactions; the T7 in vitro transcription reaction is not limited by the length, and a large amount of ssRNA is prepared, and the cost is obviously lower than that of chemical synthesis, so that the method is more suitable for industrial production. At present, there are literature and product reports on the preparation of dsRNA based on T7 in vitro transcription reaction, and the basic ideas of the schemes are consistent: PCR to obtain transcription template, in vitro transcription, annealing to form double chain, DNase and RNase treatment, alcohol precipitation, re-suspension and quantification.

Commercial kits for dsRNA preparation, such as Promega T7riboMAX, are currently commercially available ^TM Express RNAi kit and Thermo Scientific MEGAscript RNAi kit, both of which in their specifications, show agarose gel electrophoresis results for the production of finished dsRNA (FIGS. 5 and 6), according to which neither of the two commercial kits produced enough single bands of dsRNA product: promega T7riboMAX ^TM All three sizes of dsRNA prepared by the Express RNAi kit have obvious bands, and the product obtained by the final purification step of the Thermo Scientific MEGAscript RNAi kit positive control reaction still has about 1kb band. And neither product had higher resolution analytical tools (e.g., capillary electrophoresis, CE) for further analysis and identification of dsRNA product purity. Therefore, how to improve the purity of dsRNA standards is a currently pending problem.

In view of this, the present application has been made.

Disclosure of Invention

The application aims to provide a preparation method of dsRNA, which is used for alleviating the problem of lack of a method for preparing high-purity dsRNA in the prior art.

The application also aims to provide an application of the preparation method of the dsRNA in preparing a dsRNA standard substance, preparing an mRNA vaccine and preparing an mRNA vaccine quality control product.

In order to solve the technical problems, the application adopts the following technical scheme:

according to one aspect of the present application, there is provided a method of making a dsRNA comprising synthesizing ssRNA by in vitro transcription, and degrading an in vitro transcription template in the reaction product to a single nucleotide; annealing to form dsRNA, degrading single-stranded nucleic acid in the annealed product by using S1 nuclease, and purifying the reaction product to obtain the dsRNA.

Preferably, the in vitro transcription product is obtained by a T7 RNA polymerase catalyzed reaction;

preferably, the transcription template of the in vitro transcription product contains a T7 RNA polymerase promoter sequence at both ends.

Preferably, the transcription template is degraded into individual nucleic acids using at least one nuclease;

preferably, the at least one nuclease comprises Baseline-ZERO ^TM DNase；

Preferably, the at least one nuclease comprises DNase I and a second complementary DNase comprising a single-stranded specific 3 'to 5' exodeoxyribonuclease;

preferably, the second complementary DNase comprises exonuclease I.

Preferably, baseline-ZERO ^TM The reaction condition of DNase degradation transcription template is 36-38 ℃ for 15-60 min;

preferably, baseline-ZERO ^TM The reaction condition of DNase degradation transcription template is that the reaction is carried out for 30min at 37 ℃.

Preferably, the annealing reaction conditions are: maintaining the temperature at 70-80 ℃ for 5-10 min, and then cooling the temperature for 3-4 h to below 40 ℃;

preferably, the temperature is maintained at 75 ℃ for 5min, and then the temperature is reduced for 3-4 h to below 40 ℃.

Preferably, the reaction conditions for degrading the single stranded nucleic acid in the annealed product using S1 nuclease are: reacting for 15-30 min at 36-38 ℃;

preferably, the reaction conditions for degrading ssRNA in the annealed product using S1 nuclease are: the reaction was carried out at 37℃for 20min.

Preferably, the purification reaction product comprises enriching dsRNA in the reaction product;

preferably, the reaction product is ethanol precipitated and the dsRNA in the reaction product is enriched;

preferably, the purification of the reaction product further comprises removal of protein from the reaction product using phenol-chloroform extraction;

preferably, purifying the reaction product further comprises removing free nucleotides using gel filtration chromatography.

Preferably, the dsRNA is at least 80bp in length;

preferably, the dsRNA has a length of 80-1000 bp;

preferably, the dsRNA has a length of 300bp.

According to another aspect of the application, the application also provides an application of the preparation method in preparing a dsRNA standard.

According to another aspect of the application, the application also provides an application of the preparation method in preparing an mRNA vaccine or preparing an mRNA vaccine quality control product.

Compared with the prior art, the application has the following beneficial effects:

the preparation method of the dsRNA provided by the application firstly synthesizes ssRNA through in vitro transcription, and then forms the dsRNA after annealing. The inventors have found in previous fumbling experiments that the major impurities generated during this process include ssRNA, oligonucleotides, free nucleotides, etc. In order to remove impurities generated during the preparation of dsRNA, the transcription template is degraded into single nucleotide before annealing, so that the formation of heterozygous double chains with RNA in the following denaturation annealing step due to incompletely degraded residual oligonucleotides can be avoided, and the single nucleotide is easier to remove in the subsequent steps. In order to remove the single-stranded nucleic acid remained in the annealed reaction product, the application adopts S1 nuclease to remove the single-stranded nucleic acid in the annealed product after annealing, and the S1 nuclease is used for removing the single-stranded nucleic acid with stronger specificity and better single-stranded effect. Experiments prove that the dsRNA prepared by the preparation method provided by the application has high purity, and capillary electrophoresis results show that the product obtained by the preparation method provided by the application has single peak, less impurity peaks and high purity. Therefore, the preparation method of the dsRNA is suitable for the application field with higher requirement on the purity of the dsRNA, and can be applied to the preparation of dsRNA standard products and the preparation of mRNA vaccines or mRNA vaccine quality control products.

Drawings

In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present application, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 is a flow chart of the method for preparing dsRNA provided in example 1;

FIG. 2 shows the result of transcription template PCR in example 1;

FIG. 3 shows the agarose gel electrophoresis results of dsRNA prepared in examples 1 to 4;

FIG. 4 shows the results of dsRNA capillary electrophoresis prepared in examples 1 to 4;

FIG. 5 is a kit Promega T7riboMAX ^TM The result of dsRNA agarose gel electrophoresis prepared by Express RNAi kit;

FIG. 6 shows agarose gel electrophoresis results of the products of each step of kit Thermo Scientific MEGAscript RNAi kit;

FIG. 7 shows the results of the capillary electrophoresis purity test of the products of comparative examples 3 to 6.

Detailed Description

The technical solutions of the present application will be clearly and completely described in connection with the embodiments, and it is apparent that the described embodiments are some embodiments of the present application, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.

Herein, dsRNA means double-stranded RNA, ssRNA means single-stranded RNA, dsDNA means double-stranded DNA, ssDNA means single-stranded DNA; DNA and RNA may represent single strand or double strand unless otherwise specified; as used herein, single-stranded nucleic acid may refer to both single-stranded DNA and single-stranded RNA.

According to one aspect of the application, the application provides a method of making a dsRNA. The preparation method provided by the application is to synthesize ssRNA through in vitro transcription, and then form dsRNA after annealing. The inventors found in previous fumbling experiments that the major impurities generated during the preparation of dsRNA include ssRNA, oligonucleotides and free nucleotides, etc. In order to remove impurities generated during the preparation of dsRNA, the application degrades the in vitro transcription template in the reaction product into single nucleotides before the annealing step. In order to remove single-stranded nucleic acid remained in the annealed reaction product, S1 nuclease is adopted to remove single-stranded nucleic acid in the annealed product after annealing, and then the reaction product is purified to obtain dsRNA.

In some alternative embodiments, in vitro transcription is catalyzed by T7 RNA polymerase. In order to achieve a T7 RNA polymerase catalyzed reaction in vitro, the transcription template of the in vitro transcription product preferably contains T7 RNA polymerase promoter sequences at both ends. The transcription template may be obtained, for example, but not limited to, by a PCR reaction, or by directly synthesizing ssDNA and then annealing to obtain transcription template dsDNA, or by directly cleaving the transcription template on the vector.

In some alternative embodiments, the transcription template is obtained by a PCR reaction, and if the T7 RNA polymerase promoter sequence is not present in the template, the T7 RNA polymerase promoter sequence may be added to the transcription template during PCR, e.g., such that the primer contains the T7 RNA polymerase promoter sequence, and then the promoter sequence in the primer is introduced into the PCR product by a PCR reaction; in some alternative embodiments, templates with T7 RNA polymerase promoter sequences may also be selected to serve directly as reaction templates for PCR synthesis of transcription templates. The PCR reaction can be carried out by adopting commercial reagents or kits, constructing a PCR reaction system according to the records of teaching materials, reference documents, commodity instructions or standard documents and the like, and setting proper reaction parameters.

In some alternative embodiments, ssDNA is synthesized directly and then annealed to provide a transcription template, and T7 RNA polymerase promoter sequences are added at both ends of the synthesized ssDNA, and then annealed to provide dsDNA containing T7 RNA polymerase promoter sequences at both ends.

In some alternative embodiments, the transcription template is obtained by direct cleavage of a vector containing the transcription template, such as a plasmid vector.

The degradation of the in vitro transcription templates in the reaction products to individual nucleotides in the present application may be selected from any prior art known in the art capable of accomplishing this function, and the present application is not limited in this regard. While the nuclease used in the traditional method can digest template DNA into oligonucleotides, the application degrades the transcribed template into single nucleotide, which can avoid the formation of heterozygous double strand with RNA in the denaturation annealing step due to incompletely degraded residual oligonucleotides; and during subsequent purification of the reaction product, the single nucleotide produced in this step may be removed along with the single nucleotide remaining from the reaction during in vitro transcription.

In some alternative embodiments, the transcription template is degraded into individual nucleic acids using at least one nuclease, i.e., alternatively, a single nuclease having the ability to degrade dsDNA into individual nucleotides is used to degrade the transcription template into individual nucleic acids; or alternatively degrading the transcription template into individual nucleic acids using a combination of nucleases that have the function of degrading dsDNA into individual nucleotides after combination.

In some alternative embodiments, the enzyme that degrades the DNA into individual nucleotides uses Baseline-ZERO ^TM DNase. Using Baseline-ZERO ^TM The reaction condition for degrading the transcription template by DNase is preferably 36-38 ℃ for 15-60 min, more preferably 37 ℃ for 30min.

In other alternative embodiments, the enzyme that degrades DNA to a single nucleotide comprises DNase I and a second complementary DNase comprising a single-strand specific 3 'to 5' exodeoxyribonuclease, the second complementary DNase being capable of further degrading an oligonucleotide that does not completely degrade DNase I to a single nucleotide. This embodiment can be referred to the combination of nucleases described in WO2007143206A 2. Wherein the second complementary DNase preferably comprises exonuclease I.

The annealing step is used to form dsRNA from ssRNA transcribed in vitro, the annealing reaction conditions are preferably: maintaining the temperature at 70-80 ℃ for 5-10 min, and then cooling the temperature for 3-4 h to below 40 ℃. The temperature is reduced for 3-4 h to below 40 ℃ which means that the reaction temperature is reduced from 70-80 ℃ to below 40 ℃ and needs to be reduced for 3-4 h. The annealing reaction conditions have the following functions: the RNA in the system is heated to thoroughly form a single strand, and then is annealed slowly to form a double-stranded RNA structure. The annealing reaction condition is preferably that the temperature is maintained at 75 ℃ for 5min, and then the temperature is reduced for 3-4 h to below 40 ℃. It will be appreciated that the temperature is reduced to below 40 ℃, typically not below room temperature, for example not below 20 ℃.

The application adopts S1 nuclease to remove ssRNA in annealing products, and the S1 nuclease is a specific single-stranded endonuclease which can degrade ssDNA and ssRNA. At present, RNaseA is adopted to remove ssRNA in the existing dsRNA preparation process, and can specifically degrade the ssRNA in a high-salt buffer solution, and the ssRNA and the dsRNA can be degraded in a low-salt buffer solution or in a higher enzyme concentration. Experiments show that RNaseA cannot effectively remove ssRNA, and the increase of the concentration of RNaseA can completely degrade ssRNA and dsRNA in a reaction product. S1 nuclease specifically degrades single-stranded nucleic acid, because its activity on DNA is five times that on RNA, S1 nuclease is mainly used to remove single-stranded overhangs of DNA fragments, S1 transcript localization, hairpin loop cleavage and in cooperation with Exoneclease III, create unidirectional deletions in DNA fragments. The application creatively uses S1 nuclease to replace RNaseA, and uses the RNaseA to remove single-stranded nucleic acid impurities which are mainly ssRNA in reaction products, and capillary electrophoresis results show that the ssRNA can be thoroughly removed. Preferred reaction conditions for degrading ssRNA in the annealed product using S1 nuclease are: the reaction is carried out for 15 to 30 minutes at 36 to 38 ℃, more preferably for 20 minutes at 37 ℃.

In some alternative embodiments, purifying the reaction product comprises enriching the reaction product for dsRNA, optionally using an alcohol precipitation reaction product, optionally using ethanol or isopropanol, preferably using 70% v/v (final concentration of the reaction system) ethanol to enrich the reaction product for dsRNA.

In some alternative embodiments, purifying the reaction product further comprises removing the protein from the reaction product, which may be removed by phenol-chloroform extraction.

In some alternative embodiments, purifying the reaction product further comprises removing free nucleotides from the reaction product. In the construction of in vitro transcription systems, the amount of one of the substrates, NTP, is often excessive in order to increase the efficiency of the T7 in vitro transcription enzyme, and the step described above does not remove free NTP. At the same time, free nucleotides are also produced during the steps of dsDNA degradation and ssRNA degradation, and the presence of free nucleotides can interfere with accurate quantification of the final dsRNA product. Thus, the dsRNA production method preferably further comprises removing free nucleotides. The free nucleotides are preferably removed using gel filtration chromatography, more preferably using a centrifuge column packed with Sephadex G-25.

The preparation method of the dsRNA is preferably used for preparing dsRNA with the length of more than 80bp, and more preferably used for preparing dsRNA with the length of 80-1000 bp. The preparation method provided by the application can prepare dsRNA with or without modification, and the application is not limited to the dsRNA. The modification includes, but is not limited to, at least one of modification of bases, modification of linkages between bases, and modification of dsRNA end points. The modification may be added in a manner generally and well known in the art, optionally during the step of in vitro transcription, for example by incorporating modified nucleotides into the dsRNA during in vitro transcription.

In some alternative embodiments, the method of making the dsRNA is performed according to the following steps:

1. in vitro transcription template amplification:

(a) And (2) PCR: the main purpose of this step is to add the T7 RNA polymerase promoter to both ends of the transcribed sequence by a PCR reaction. If there is no T7 promoter sequence at both ends of the sequence to be amplified on the template, it is necessary to introduce a T7 promoter sequence on the primer. The T7 RNA polymerase promoter sequence is 5'-TAATACGACTCACTATAGGN-3' (seq_1). The 3 rd base G at the 3 'end is the first base to be incorporated into the RNA strand, followed by the second base G (the 2 nd base at the 3' end) to be incorporated into the RNA strand.

(b) And (3) PCR product recovery: agarose gel electrophoresis of appropriate concentration was selected according to the size of the PCR product to determine if the fragment size was correct. If the size is correct, the residual sample is subjected to glue recovery, and after glue recovery, the concentration is measured by nanodrop.

2. In vitro transcription and product purification:

(a) In vitro transcription is performed using an in vitro transcription kit: in vitro transcription reaction system and time reference kit instruction, after IVT reaction, baseline-ZERO is used ^TM DNase or other nucleases that degrade DNA into individual nucleotides remove the transcribed template, and complete degradation of the DNA transcribed template into individual nucleotides prevents the formation of a hybrid duplex with RNA during the subsequent denaturation annealing step due to incomplete degradation of the remaining oligonucleotides.

(b) Denaturation annealing: heating the transcribed product at 75 ℃ for 5min, then turning off the heating device, slowly cooling for 3-4 hr, and then cooling to below 40 ℃ to perform the next reaction.

(c) The nuclease removes ssRNA, and the S1 nuclease specifically degrades single-stranded nucleic acid, so that the yield of dsRNA of a target product is not affected while the single-stranded RNA is effectively removed.

(d) And (3) dsRNA purification: the purification is mainly to remove protein impurities such as enzyme in a reaction system, remove protein through phenol-chloroform extraction, and enrich dsRNA products through ethanol low-temperature precipitation.

(e) Removal of free nucleotides: free nucleotides were removed using a centrifuge column packed with Sephadex G-25.

(f) Nanodrop concentration measurement. Packaging, and storing at-80deg.C

According to another aspect of the application, the application also provides the application of the preparation method of the dsRNA in preparing a dsRNA standard substance. The dsRNA obtained by the preparation method provided by the application has low impurity content of ssRNA, oligonucleotide, free nucleotide and the like, and is suitable for the application field with higher requirement on the purity of the dsRNA, so that the preparation method can be applied to the preparation of dsRNA standard products.

According to another aspect of the application, the application also provides an application of the preparation method of the dsRNA in preparing an mRNA vaccine or preparing an mRNA vaccine quality control product. The prepared dsRNA has high purity, and can be used as a dsRNA standard substance in the preparation process of an mRNA vaccine, for example, the dsRNA standard substance can be used for drawing a standard curve in the step of detecting the dsRNA in the mRNA vaccine by ELISA and HTRF, or can be applied to detecting the content of the dsRNA by a dot blot method.

The technical solution and advantageous effects of the present application are further described below in connection with preferred embodiments.

Example 1

The embodiment provides a preparation method of 300bp dsRNA, and the preparation flow is shown in figure 1.

(1) PCR obtains transcription templates:

(a) PCR primer design:

the main purpose of this step is to add a T7 RNA polymerase promoter sequence to both ends of the transcribed sequence by a PCR reaction, the T7 promoter sequence being 5'-TAATACGACTCACTATAGGN-3' (seq_1). Wherein N represents a degenerate base, representing that the position is arbitrary A/T/C/G, and starting with 3', the 3 rd base G is the first base incorporated into the RNA strand, followed by the second base G (the 2 nd base at the 3' end) incorporated into the RNA strand. Taking this experiment as an example, two primers were used, dsRNA-F and 300bp dsRNA-R, the sequences of which are shown in Table 1.

TABLE 1

The PCR template is plasmid pcDNA3-300bp dsRNA plasmid. The primer dsRNA-F is a sequence from a plasmid template, in which the downstream sequence is T7 promoter TAATACGACTCACTATAGG, so that a 300bp dsRNA transcription template with T7 promoters at two ends can be obtained by PCR.

(b) PCR reaction

The kit used for PCR is toyobo, cat#: KOD-201, PCR system and PCR procedure are shown in Table 2 and Table 3, respectively.

TABLE 2 PCR System

KOD plus	1μL
		dNTPs(2mM)	5μL
MgSO ₄ (25mM)	2μL
		dsRNA-F(10μM)	1.5μL
300bp dsRNA-R(10μM)	1.5μL
		3-300bp plasmid of pcDNA	50ng
10×KOD-plus buffer	5μL
		Non-ribozyme water	Add to 50μL

TABLE 3 PCR procedure

(c) PCR product gel recovery

1% agarose (Bio-A620014) gel electrophoresis was used to determine whether the fragment size was about 370bp, and the nucleic acid dye (Northenan, GR 501-01) was DL2000 (Takara, cat# 3427A). The results of the gel electrophoresis are shown in FIG. 2. After verifying the correct size, the remaining samples were subjected to gel recovery (Universal DNA purification recovery kit from Tiangen, cat. Number: DP 214-03), and after gel recovery, the concentration was determined by nanodrop. The concentration of the PCR product obtained in this step was 163.118 ng/. Mu.L.

(2) In vitro transcription

In vitro transcription was performed using T7 RNA Polymerase (HC) kit (Promega, P4074). The in vitro transcription system is shown in Table 4.

TABLE 4 unmodified dsRNA in vitro transcription System

(3)Baseline-ZERO ^TM DNase treatment to remove DNA transcription templates

After the IVT reaction was completed, 2. Mu.L (2 MBU) of baseline-zero DNaseI, 4. Mu.L of 10 Xbuffer, 4. Mu.L of DEPC-H were added ₂ O，37℃30min。

(4) Denaturation annealing: heating the transcribed product at 75 deg.c for 5min and then returning to room temperature for 3-4 hr.

(5) Removal of single stranded RNA: the reaction system is shown in Table 5.

TABLE 5 reaction system

(6) dsRNA purification

200. Mu.L DEPC-H was added ₂ O was added to a total volume of 300. Mu.L. Adding equal volume of phenol-chloroform, shaking for 20s, and mixing. The centrifugation is carried out at maximum speed for 15min at 4 ℃. The aqueous phase was transferred to another clean 1.5mL EP tube. 1/10 volume of 3M sodium acetate (pH 5.2) and 2.5 volumes of absolute ethanol were added. And (3) after being mixed evenly, standing for more than 30 minutes at the temperature of minus 20 ℃. The centrifugation is carried out at the highest rotational speed for 30min and at 4 ℃.

The supernatant was discarded, 70% glacial ethanol in DEPC water was added, the mixture was inverted, the precipitate was washed (white), and centrifuged at 12000rpm,5min,4 ℃. Repeating the ethanol cleaning step, removing ethanol, air drying at room temperature to semitransparent state, adding 40 μl TE buffer, and dissolving.

Note that: during the 70% ice-ethanol wash the pellet, care should be taken not to pour the pellet.

(7) Removal of free NTP:

(7.1) shaking the packing in the heavy suspension G-25 centrifuge column, unscrewing the cap, and twisting off the bottom seal. 720g was centrifuged for 30s.

(7.2) the supernatant was carefully aspirated, 250. Mu.L of TE buffer was added, the suspension was shaken to resuspend the filler, and 720g was centrifuged for 30s.

(7.3) carefully pipette off the supernatant and place the column into a clean 1.5mL EP tube. The sample was carefully dropped into the middle of the upper of the padding. The sample was collected by centrifugation at 720g for 1 min.

(8) Nanodrop concentration measurement. The agarose gel electrophoresis and capillary electrophoresis detection purities are shown in figure 3 and figure 4 respectively, and the products are stored at-80 ℃ after sub-packaging.

Example 2

The difference between this example and example 1 is that the dsRNA was pUTP (pseudo uridine) -modified dsRNA, which was modified in the in vitro transcription step, and rUTP in the in vitro transcription system of unmodified dsRNA was replaced with pUTP, and the in vitro transcription system was as shown in Table 6, and the other steps were the same as in example 1. pUTP-modified dsRNA was obtained.

TABLE 6pUTP modified dsRNA in vitro transcription System

Example 3

This example differs from example 1 only in that the dsRNA was N1-Me-pUTP (N1-methylpseuduridines) modified dsRNA was modified in the in vitro transcription step, rUTP in the in vitro transcription system of the unmodified dsRNA was replaced with N1-Me-pUTP, the in vitro transcription system was as shown in Table 7, and the other steps were the same as in example 1. To obtain the N1-Me-pUTP modified dsRNA.

TABLE 7N1-Me-pUTP modified dsRNA in vitro transcription System

Example 4

This example differs from example 1 only in that the dsRNA was 5-OMe-UTP (5-methyluridine) -modified dsRNA, which was modified in the in vitro transcription step, and rUTP in the in vitro transcription system of the unmodified dsRNA was replaced with 5-OMe-UTP, as shown in Table 8, and the other steps were the same as in example 1. Obtaining the 5-OMe-UTP modified dsRNA.

Table 8 5-OMe-UTP modified dsRNA in vitro transcription System

The agarose gel electrophoresis results of the dsRNAs prepared in examples 1 to 4 are shown in FIG. 3, in which lane 1 in FIG. 3 is the unmodified dsRNA prepared in example 1, lane 2 is the pUTP modified dsRNA prepared in example 2, lane 3 is the N1-Me-pUTP modified dsRNA prepared in example 3, and lane 4 is the 5-OMe-UTP modified dsRNA prepared in example 4.

The detection results of dsRNA capillary electrophoresis prepared in examples 1-4 are shown in FIG. 4, and it can be seen from FIG. 4 that the product prepared by the preparation method of the example has fewer impurity peaks and high purity.

Comparative example 1

The comparative example is a kit Promega T7riboMAX ^TM Express RNAi kit. T7riboMAX was used by non-denaturing agarose gel analysis ^TM The Express RNAi system produces dsRNA molecules of different sizes. About 4X 10 on 1.8% agarose/1×TAE gel ¹¹ Various dsRNA molecules were used and the gel was stained with 0.5. Mu.g/ml ethidium bromide and observed. The results are shown in FIG. 5. Lane name: lane 1,1kb DNA Ladder (catalog number: G5711); lane 2, 74ng 180bp dsRNA; lane 3,200ng 500bp dsRNA; lane 4,400ng 1000bp dsRNA; lanes 5,100bp DNA Ladder (catalog number: G2101). Description: dsRNA migrate slower than dsDNA.

Comparative example 2

This comparative example is kit Thermo Scientific MEGAscript RNAi kit. dsRNA agarose gel electrophoresis was performed as shown in FIG. 6, with 400 Xdiluted samples, 1% agarose gel separation, and ethidium bromide staining.

Comparative example 3

The difference between this comparative example and example 1 is that the step (3) and the step (5) are combined into one step, DNaseI is used for removing the transcription template DNA, RNaseA is used for removing the single-stranded RNA, and the reaction system and reaction conditions are as follows:

TABLE 9 reaction System and conditions

Comparative example 4

The product of comparative example 3 was treated with high concentration of RNaseA, and the reaction system and reaction conditions were as follows:

TABLE 10 reaction systems and conditions

Comparative example 5

The product of comparative example 3 was RNaseIII treated, and the reaction system and reaction conditions were as follows:

TABLE 11 reaction system and conditions

Comparative example 6

The product of comparative example 3 was treated with S1 nuclease, and the reaction system and reaction conditions were the same as in example 1.

Comparative example 1 and comparative examples 1-2 demonstrate that the dsRNA obtained in the present application is of higher purity. In addition, the application also adopts a technical means of higher resolution-Capillary Electrophoresis (CE) to analyze the purity of the product dsRNA (figure 4), and the result shows that the obtained product is almost single peak and has higher purity.

The results of dsRNA capillary electrophoresis of comparative examples 3 to 6 are shown in FIG. 7, and the green curve is the dsRNA sample prepared using RNaseA (comparative example 3); the yellow curve is a sample (comparative example 4) after high concentration RNaseA treatment of the product of comparative example 3, and all peaks in the sample in the region A and the region B disappear, which indicates that the peaks in the region A and the region B are RNA; the red curve is for the sample after RNaseIII treatment of the product of comparative example 3 (comparative example 5) with the peak of region A disappeared, since RNaseIII specifically degraded dsRNA, indicating that the peak of region A disappeared was dsRNA; the purple curve is for the sample after S1 nuclease treatment of the product of comparative example 3 (comparative example 6), the peak of region B disappeared, which is the residual ssRNA impurity, because S1 nuclease specifically degrades single stranded nucleic acid. As can be seen from FIG. 7, the S1 nuclease has the best effect of removing single-stranded nucleic acid from the sample, the ssRNA cannot be effectively removed by the low-concentration RNaseA treatment, and the dsRNA is degraded by the high-concentration RNaseA, so that the effect of removing single-stranded nucleic acid by using the S1 nuclease is optimal.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present application, and not for limiting the same; although the application has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the application.

Claims

1. A method of making a dsRNA comprising: firstly, synthesizing ssRNA through in vitro transcription, and degrading an in vitro transcription template in a reaction product into single nucleotide; annealing to form dsRNA, degrading single-stranded nucleic acid in the annealed product by using S1 nuclease, and purifying the reaction product to obtain the dsRNA.

2. The method of claim 1, wherein the in vitro transcription product is obtained by a T7 RNA polymerase catalyzed reaction;

3. The method of claim 1, wherein the transcription template is degraded into individual nucleic acids using at least one nuclease;

preferably, the at least one nuclease comprisesBaseline-ZERO ^TM DNase；

preferably, the second complementary DNase comprises exonuclease I.

4. The process according to claim 3, wherein Baseline-ZERO ^TM The reaction condition of DNase degradation transcription template is 36-38 ℃ for 15-60 min;

5. The method of claim 1, wherein the annealing reaction conditions are: maintaining the temperature at 70-80 ℃ for 5-10 min, and then cooling the temperature for 3-4 h to below 40 ℃;

6. The method according to claim 1, wherein the reaction conditions for degrading the single-stranded nucleic acid in the annealed product using S1 nuclease are: reacting for 15-30 min at 36-38 ℃;

7. The method of claim 1, wherein purifying the reaction product comprises enriching dsRNA in the reaction product;

8. The method of any one of claims 1-7, wherein the dsRNA is at least 80bp in length;

preferably, the dsRNA has a length of 80-1000 bp;

preferably, the dsRNA has a length of 300bp.

9. Use of the method of any one of claims 1-8 for the preparation of a dsRNA standard.

10. Use of the method of any one of claims 1-8 for the preparation of an mRNA vaccine, or for the preparation of a quality control product of an mRNA vaccine.