Method for preparing RNA (Ribonucleic Acid) through in-vitro transcription and application

Abstract

The invention discloses a method for preparing RNA (Ribonucleic Acid) through in-vitro transcription and application. The method comprises the following steps: (1) preparing a linearized DNA template; (2) constructing an in-vitro transcription reaction system, and performing in-vitro transcription under a reaction condition suitable for transcription; and (3) purifying the prepared mRNA. According to the method, RNA polymerase can reduce 3 '-extension and non-specific transcription initiation of an IVT product under the condition that the temperature is increased, and an appropriate amount of L-arginine or salt thereof is added into a reaction system, so that the RNA structure can be effectively prevented from being damaged while the mRNA yield is not reduced, and the generation of dsRNA is further reduced. In addition, the reduction of dsRNA can significantly reduce the immunogenicity of mRNA.

Description

Technical Field

[0001] The invention belongs to the field of molecular biology and provides a method and application of preparing RNA by in vitro transcription (IVT) capable of reducing the formation of dsRNA. Background Art

[0002] In vitro transcription can synthesize RNA in the absence of cells. The principle of in vitro transcription is to use RNA polymerase to transcribe the DNA template into RNA. RNA polymerase is a highly efficient RNA polymerase that can synthesize high-purity and high-yield RNA in vitro. The transcription process usually includes: template recognition, transcription initiation, transcription extension, and transcription termination.

[0003] The in vitro transcription reaction system includes DNA template, RNA polymerase, nucleotide triphosphates (NTPs), buffer and Mg. 2+ ions. The reaction steps of in vitro transcription include: 1) DNA template binds to RNA polymerase to form a DNA-RNA polymerase complex; 2) NTPs enter the complex, and RNA polymerase begins to synthesize RNA; 3) During RNA synthesis, the DNA-RNA polymerase complex slides downward, the DNA template is gradually unwound, and the RNA chain is synthesized; 4) After RNA synthesis is completed, DNase I is added to the reaction system to degrade the DNA template and obtain a pure RNA product. In vitro transcription technology has the characteristics of high efficiency, high yield, and high purity. It can be used in large-scale synthesis of RNA, research on RNA structure and function, preparation of RNA probes, and other fields. At the same time, in vitro transcription technology can also be combined with other technologies, such as RNA modification, RNA labeling, RNA purification, etc., to expand its scope of application.

[0004] The IVT reaction is accompanied by the production of dsRNA. The traditional methods for reducing dsRNA are as follows:

[0005] RP-HPLC method: RP-HPLC provides a fast and reliable alternative to classic RNA, including separation of different RNA types, quantification and purification. RP-HPLC purification can eliminate immune activation and improve the translation of nucleoside-modified protein-coding mRNA. RP-HPLC purified nucleoside-modified mRNA is a powerful carrier used in everything from in vitro stem cell generation to in vivo gene therapy. This method is a method for removing dsRNA after RNA synthesis, which can easily lead to mRNA degradation. Due to the limited column loading capacity, it is difficult to scale up, production capacity is limited, the process is cumbersome, and the cost is high. The high temperature and external force during the purification process affect the integrity of mRNA, resulting in mRNA functional loss. The large amount of organic solvents used in the RP-HPLC process also poses a production safety hazard.

[0006] Hydroxyapatite chromatography: The phosphate groups of nucleic acids can react with the calcium ions of hydroxyapatite and be adsorbed on it. The adsorption force of double-stranded nucleic acids is stronger than that of single-stranded nucleic acids, thereby separating single-stranded mRNA and dsRNA.

[0007] Oligo (dT)-cellulose chromatography: Using oligo dT columns (selectively binding only to RNA with polyA tails), under high salt concentrations, the mutual repulsion between the negatively charged column ligands and RNA can be suppressed, promoting the formation of hydrogen bonds. After the salt ions are removed, the repulsion between the negative charges between the column ligands and RNA is restored, the hydrogen bonds are destroyed, and the RNA is eluted.

[0008] Reducing Mg in IVT reaction system 2+ Concentration: By reducing Mg 2+ concentration, can effectively reduce the production of dsRNA. Experimental results have shown that when Mg 2+ At a concentration of 5 mM, almost no dsRNA was detected. 2+ It is an essential auxiliary factor for RNA polymerase, so low concentrations of Mg 2+ It will seriously affect the production of mRNA. In the mRNA production process, RNA polymerase acts as an engine, has very high enzymatic activity and can also insert modified nucleotides. Generally, T7 RNA polymerase is a magnesium ion-dependent polymerase that requires the assistance of a higher concentration of magnesium ions to bind to the DNA template.

[0009] Adding urea to the IVT reaction system: Studies have shown that adding 0.8M urea or 1.6M formamide to the IVT reaction can effectively reduce the production of dsRNA. During the IVT process, different concentrations of discrete agents are added to create a mild discrete environment, in which RNA base complementary pairing is destroyed, but the RNA polymerase function is intact. Such a strong denaturing environment can cancel the function of the enzyme, and the intramolecular or intermolecular ribonucleic acid base pairing as a template for dsRNA formation is relatively weak, reducing the formation of dsRNA, but the mRNA yield will also decrease with the increase in the amount of urea added. This study shows that higher concentrations of urea and formamide can effectively reduce dsRNA formation, and have only a slight effect on the mRNA yield generated by IVT. Dispersants within a certain concentration range will not affect the efficiency of T7 RNA polymerase. As the concentration of urea and formamide increases (such as 0.8M urea or 1.6M formamide), urea cannot destroy the double-stranded RNA that has been formed, but can only prevent base pairing during the formation of double-stranded RNA, thereby reducing the formation of dsRNA.

[0010] The use of VSW-3 RNA polymerase can reduce the formation of dsRNA: Currently, in vitro transcription (IVT) is a method for obtaining large-scale mRNA, mainly using T7 RNA polymerase (RNAP). Previous studies have shown that the cell expression level of mRNA (unmodified) produced by VSW-3 RNAP is superior. This is due to the unknown sensitivity of VSW-3 RNA polymerase to the second type of T7 transcription terminator when synthesizing RNA at low temperature, and the RNA product of VSW-3 RNA polymerase has no 3' end extension, which further improves the purity of RNA synthesis, thereby reducing the production of dsRNA. Compared with the conventionally used T7 RNA polymerase, the RNA yield of VSW-3 RNAP is equivalent, but the reaction time is longer (about 12h). Using the explosive growth of environmental microbial genome resources in recent years, Professor Zhu Bin's research group reported the discovery and development of all new phage single-subunit RNA polymerases since the new century, including Syn5 RNA polymerase, KP34 RNA polymerase and VSW-3 RNA polymerase, which has formed an international characteristic research direction.

[0011] RP-HPLC and hydroxyapatite chromatography are difficult to scale up due to limited column loading capacity, limited production capacity, complicated procedures, and high costs. The high temperature and external forces during the purification process affect the integrity of mRNA, resulting in mRNA functional loss and massive mRNA degradation. The large amount of organic solvents used in the RP-HPLC process also poses a safety hazard to production. In addition, reducing Mg in the IVT reaction system 2+ The concentration and addition of urea will seriously affect the yield of mRNA products. The use of VSW-3 RNA polymerase can reduce the formation of dsRNA, but the reaction time is long, which will lead to a lot of time being wasted in the production of mRNA. Summary of the invention

[0012] In view of at least some of the technical problems existing in the prior art, the inventors conducted in-depth research and found that two dsRNA byproducts are produced in the IVT reaction, one from the 3' extension of the IVT product and the other from the production of antisense RNA. When the temperature is increased, RNA polymerase can reduce the 3'-extension and non-specific transcription initiation of the IVT product, and adding an appropriate amount of L-arginine or its salt to the reaction system can effectively prevent the RNA structure from being destroyed without reducing the mRNA yield, further reducing the production of dsRNA. In addition, the reduction of dsRNA can significantly reduce the immunogenicity of mRNA. Specifically, the present invention includes the following contents.

[0013] In a first aspect of the present invention, there is provided a method for preparing RNA by in vitro transcription, comprising the following steps:

[0014] (1) preparing a linearized DNA template;

[0015] (2) constructing an in vitro transcription reaction system, and performing in vitro transcription under reaction conditions suitable for transcription, wherein the in vitro transcription reaction system contains a fixed concentration of arginine or a salt thereof;

[0016] (3) Purify the prepared mRNA.

[0017] In certain embodiments, according to the method for preparing RNA by in vitro transcription of the present invention, the in vitro transcription reaction system comprises: RNA polymerase, a mixture of nucleoside triphosphates and chemical modifications thereof, a buffer, Mg 2+ and arginine or a salt thereof.

[0018] In certain embodiments, according to the method for preparing RNA by in vitro transcription of the present invention, the chemical modifier comprises N6-methyladenosine, inosine, 5-methylcytosine nucleoside, pseudouridine nucleoside, 5-hydroxymethylcytosine, N1-methyladenosine, and an mRNA cap structure.

[0019] In certain embodiments, according to the method for preparing RNA by in vitro transcription of the present invention, wherein the reaction conditions suitable for transcription have a pH range of 7.5-8.5.

[0020] In certain embodiments, according to the method for preparing RNA by in vitro transcription of the present invention, wherein the reaction conditions suitable for transcription include a reaction temperature range of 35-45°C.

[0021] In certain embodiments, according to the method for preparing RNA by in vitro transcription of the present invention, the concentration of arginine or a salt thereof is 0.1-1000 mM.

[0022] In certain embodiments, the method for preparing RNA by in vitro transcription according to the present invention further comprises a dsRNA detection step and / or an mRNA integrity detection step.

[0023] The second aspect of the present invention provides an in vitro transcription system, wherein the in vitro transcription reaction system comprises: RNA polymerase, a mixture of nucleoside triphosphates and chemical modifications thereof, a buffer, and arginine or a salt thereof.

[0024] The third aspect of the present invention provides an mRNA or a vaccine comprising the same, which is obtained according to the method described in the first aspect.

[0025] Preferably, the mRNA comprises at least one of a 5'-cap structure, a 3'-poly(A) sequence, a 5'-UTR and a 3'-UTR.

[0026] Preferably, the uracil, cytosine or adenine nucleoside of the mRNA contains a modification group.

[0027] Preferably, the chemically modified uridine nucleoside is selected from one or more of 2-fluoro-2-deoxyuridine, pseudouridine, N1-methyl-pseudouridine, 5-methoxyuridine, 2-fluoro-2-deoxy-pseudouridine, 2-fluoro-2-deoxy-N1-methyl-pseudouridine and 2-fluoro-2-deoxy-5-methoxyuridine.

[0028] Preferably, the chemically modified cytidine nucleoside is selected from one or more of N4-acetylcytidine, 2-fluoro-2-deoxycytidine, 5-methylcytidine, 2-fluoro-2-deoxy-5-methylcytidine and 2-fluoro-2-deoxy-N4-acetylcytidine.

[0029] Preferably, the chemically modified adenine nucleoside is selected from one or more of N6-methyladenosine, 2-fluoro-2-deoxyadenosine and 2-fluoro-2-deoxy-N6-methyladenosine.

[0030] Preferably, the chemically modified guanosine nucleoside is selected from one or more of 2-fluoro-2-deoxyguanosine, N7-methyl-guanosine and 2-fluoro-2-deoxy-N7-methyl-guanosine.

[0031] In a fourth aspect of the present invention, a method for reducing the amount of dsRNA during an in vitro transcription reaction is provided, comprising the steps of using a transcription reaction system and conducting a reaction under reaction conditions suitable for transcription, wherein the transcription reaction system comprises arginine or a salt thereof at a concentration of 0.1-1000 mM.

[0032] The fifth aspect of the present invention provides a method for improving the structural stability of mRNA, which comprises the steps of using a transcription reaction system and conducting a reaction under reaction conditions suitable for transcription, wherein the transcription reaction system contains arginine or a salt thereof at a concentration of 0.1-1000 mM.

[0033] The sixth aspect of the present invention provides use of the mRNA or a vaccine comprising the same according to the present invention in the preparation of a product for treating or ameliorating a disease.

[0034] The present invention not only reduces the formation of dsRNA and reduces immunogenicity by adjusting the temperature of the IVT reaction and adding L-arginine, but also improves the structural stability of mRNA and improves the physicochemical functions. If the method of reducing dsRNA residues by simply increasing the temperature is used, the formation of dsRNA dependent on the antisense RNA chain in the IVT product cannot be reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is the result of measuring the dsRNA content in mRNA.

[0036] Figure 2 This is the result of mRNA integrity detection.

[0037] Figure 3 This is the result of measuring the dsRNA content in mRNA (monomethyl pseudouridine modification).

[0038] Figure 4 This is the result of mRNA (monomethyl pseudouridine modification) integrity detection.

[0039] Figure 5 These are the results of the determination of dsRNA content in mRNA (modified with monomethyl pseudouridine) prepared by the co-transcriptional capping method.

[0040] Figure 6 This is the result of integrity test of mRNA (modified with monomethyl pseudouridine) prepared by the co-transcriptional capping method.

[0041] Figure 7 These are the results of the determination of dsRNA content in mRNA prepared by the co-transcriptional capping method.

[0042] Figure 8 This is the result of mRNA integrity test prepared by co-transcriptional capping method. DETAILED DESCRIPTION

[0043] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0044] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that the upper and lower limits of the scope and each intermediate value therebetween are specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0045] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0046] Preparation method

[0047] The present invention provides a method for preparing RNA by in vitro transcription, comprising:

[0048] (1) preparing a linearized DNA template;

[0049] (2) constructing an in vitro transcription reaction system, and performing in vitro transcription under reaction conditions suitable for transcription, wherein the in vitro transcription reaction system contains a fixed concentration of arginine or a salt thereof;

[0050] (3) Purify the prepared mRNA.

[0051] In step (1), firstly, a linearized DNA template is prepared, and the plasmid can be linearized using an endonuclease known in the art, which is not particularly limited. The DNA can be linear DNA or circular DNA.

[0052] Step (2) of the present invention is an in vitro transcription (sometimes referred to herein as "IVT") step, which uses a linearized plasmid as a template and performs in vitro transcription to prepare mRNA under reaction conditions suitable for transcription through a constructed transcription system. The transcription system comprises raw materials such as polymerase, triphosphate nucleoside mixture and chemical modifications thereof conventionally used in the art. The polymerase may be, for example, RNA polymerase II, SP6 RNA polymerase, T7 RNA polymerase, etc. The triphosphate nucleoside mixture and chemical modifications thereof may use NTPs, and NTPs include ATP, CTP, GTP and UTP. When it is necessary to synthesize chemically modified mRNA, for example, UTP in the reaction system may be replaced with a corresponding chemically modified uridine nucleoside, for example, UTP may be replaced with one or more of 2-fluoro-2-deoxyuridine, pseudouridine, N1-methyl-pseudouridine, 5-methoxyuridine, 2-fluoro-2-deoxy-pseudouridine, 2-fluoro-2-deoxy-N1-methyl-pseudouridine and 2-fluoro-2-deoxy-5-methoxyuridine.

[0053] The inventors have found through research that a certain concentration of arginine or its salt in the transcription system can effectively prevent the RNA structure from being destroyed while not reducing the mRNA yield, further reduce the production of dsRNA, and the reduction of dsRNA can also significantly reduce the immunogenicity of mRNA. In a preferred embodiment, the transcription system of the present invention contains arginine. In another preferred embodiment, the transcription system of the present invention contains a salt of arginine. The salt of arginine can be in the form of an alkali salt or an acid salt of arginine, which is not particularly limited. When the transcription system uses the corresponding acid salt or alkali salt, it can be adjusted to a suitable pH value as needed.

[0054] In the present invention, the concentration of arginine or its salt is 0.1-1000mM, preferably 10-800mM, and further preferably 10-500mM, for example 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500mM, etc.

[0055] The transcription system of the present invention includes a buffer capable of maintaining the pH value and ionic strength of the reaction system. The buffer may be a buffer system known in the art, examples of which include but are not limited to Tris-HCl buffer and HEPES buffer.

[0056] The transcription system of the present invention may optionally include an enzyme (e.g., DNase) and Mg for purification. 2+ To obtain higher purity and higher yield of mRNA transcripts.

[0057] The present invention further optimizes the transcription reaction conditions and finds that by optimizing the transcription reaction conditions (especially the reaction temperature) in combination with the transcription reaction system of the present invention, the dsRNA residue can be synergistically reduced while ensuring the mRNA yield.

[0058] The reaction temperature of the present invention should not be too high or too low, as too high or too low temperature is not conducive to the transcription reaction. In a preferred embodiment, the temperature range for the transcription reaction is 35-45°C, preferably 37-42°C, such as 37, 38, 39, 40, 41, 42°C, etc.

[0059] In a preferred embodiment, the reaction time is 0.5-12 h, preferably 0.5-8 h, and further preferably 0.5-3 h.

[0060] The method of the present invention further comprises a dsRNA detection step and / or an mRNA integrity detection step. The dsRNA detection step can use a double antibody sandwich enzyme-linked immunosorbent assay (sandwich ELISA) to detect the dsRNA content in the in vitro transcription system or the synthesized mRNA stock solution.

[0061] Those skilled in the art should understand that other steps or operations may be included before or after the above steps (1)-(3), or between any of these steps, for example, to further optimize and / or improve the method described in the present invention.

[0062] In vitro transcription system

[0063] The second aspect of the present invention provides a reaction system for in vitro transcription synthesis of mRNA, which at least comprises arginine or its salt at a concentration of 0.1-1000mM. Based on this, the present invention also provides a kit for in vitro transcription synthesis of mRNA, which comprises RNA polymerase, a mixture of nucleoside triphosphates and chemical modifications thereof, a buffer and arginine or its salt.

[0064] mRNA or vaccines containing the same

[0065] The present invention also provides mRNA or a vaccine containing the same, which is prepared by the method of the present invention. In a specific embodiment, the mRNA includes at least one of a 5'-cap structure, a 3'-polyadenylic acid sequence, a 5'-UTR and a 3'-UTR. In a preferred embodiment, the uracil, cytosine or adenine nucleoside of the mRNA contains a chemically modified group, wherein the chemically modified uridine nucleoside is selected from one or more of 2-fluoro-2-deoxyuridine, pseudouridine, N1-methyl-pseudouridine, 5-methoxyuridine, 2-fluoro-2-deoxy-pseudouridine, 2-fluoro-2-deoxy-N1-methyl-pseudouridine and 2-fluoro-2-deoxy-5-methoxyuridine; the chemically modified cytosine nucleoside is selected from one or more of N4-acetylcytidine, 2-fluoro -2-deoxycytidine, 5-methylcytidine, 2-fluoro-2-deoxy-5-methylcytidine and 2-fluoro-2-deoxy-N4-acetylcytidine; the chemically modified adenine nucleoside is selected from one or more of N6-methyladenosine, 2-fluoro-2-deoxyadenosine and 2-fluoro-2-deoxy-N6-methyladenosine; the chemically modified guanine nucleoside is selected from one or more of 2-fluoro-2-deoxyguanosine, N7-methyl-guanosine and 2-fluoro-2-deoxy-N7-methyl-guanosine.

[0066] use

[0067] The present invention further provides use of mRNA or a vaccine comprising the same in preparing a product for treating or ameliorating a disease, such as a therapeutic agent.

[0068] Example 1

[0069] The following shows the preparation of mRNA without nucleoside modification.

[0070] 1. Preparation of linearized DNA template

[0071] The plasmid was linearized using BspQI endonuclease and the linearized plasmid was used as a template.

[0072] 2.IVT reaction

[0073] Using the linearized plasmid as a template, T7 RNA polymerase, NTP and other raw materials were used for in vitro transcription to prepare mRNA. The final concentration gradient of L-arginine was 0 mM, 20 mM, 40 mM, 60 mM, and 80 mM, and the pH was 8.0. The reaction was carried out at 37°C and 42°C for 3 h, respectively, and then the prepared mRNA was purified by lithium chloride precipitation method.

[0074] 3. Detection of dsRNA residues in IVT reaction products

[0075] Double antibody sandwich enzyme-linked immunosorbent assay (sandwich ELISA) is used to detect the content of dsRNA in the in vitro transcription system or the synthesized mRNA stock solution. The capture antibody is used to coat the microplate to form a solid-phase antibody. The dsRNA calibrator and the sample to be tested are added to the solid-phase antibody microplate, and then the detection antibody is added. Finally, the enzyme-labeled reagent labeled with horseradish peroxidase is added to form a "coated antibody-antigen-enzyme-labeled detection antibody" complex. After washing, the color developing solution is added for color development. The color developing solution is converted into blue under the catalysis of the HRP enzyme and finally converted into yellow under the action of acid. The depth of the color is positively correlated with the amount of dsRNA in the sample. The dsRNA detection results are as follows: Figure 1 The results of mRNA integrity test are shown in Figure 2 shown.

[0076] Example 2

[0077] The following shows the preparation of mRNA modified with monomethylpseudouridine.

[0078] 1. Preparation of linearized DNA template

[0079] The plasmid was linearized using BspQI endonuclease and the linearized plasmid was used as a template.

[0080] 2.IVT reaction

[0081] Using the linearized plasmid as a template, T7 RNA polymerase, NTP and other raw materials were used for in vitro transcription to prepare mRNA. The final concentration gradient of L-arginine hydrochloride was 0mM, 20mM, 40mM, 60mM, and 80mM. The pH of the system was adjusted to 7.5-8.0. The reaction was carried out at 37°C and 42°C for 3h, respectively, and then the prepared mRNA was purified by ammonium acetate precipitation method.

[0082] 3. Detection of dsRNA residues in IVT reaction products

[0083] Double antibody sandwich enzyme-linked immunosorbent assay (sandwich ELISA) is used to detect the content of dsRNA in the in vitro transcription system or the synthesized mRNA stock solution. The capture antibody is used to coat the microplate to form a solid-phase antibody. The dsRNA calibrator and the sample to be tested are added to the solid-phase antibody microplate, and then the detection antibody is added. Finally, the enzyme-labeled reagent labeled with horseradish peroxidase is added to form a "coated antibody-antigen-enzyme-labeled detection antibody" complex. After washing, the color developing solution is added for color development. The color developing solution is converted into blue under the catalysis of the HRP enzyme and finally converted into yellow under the action of acid. The depth of the color is positively correlated with the amount of dsRNA in the sample. The dsRNA detection results are as follows: Figure 3 The results of mRNA integrity test are shown in Figure 4 shown.

[0084] Example 3

[0085] The following shows the preparation of monomethylpseudouridine-modified mRNA prepared by the co-transcriptional capping method.

[0086] 1. Preparation of linearized DNA template

[0087] The plasmid was linearized using BspQI endonuclease and the linearized plasmid was used as a template.

[0088] 2.IVT reaction

[0089] Using the linearized plasmid as a template, T7 RNA polymerase, NTP and other raw materials were used for in vitro transcription to prepare mRNA. The final concentration gradient of L-arginine was 0 mM, 20 mM, 40 mM, 60 mM, and 80 mM. The reaction was carried out at 37°C and 42°C for 3 h, respectively, and then the prepared mRNA was purified by ammonium acetate precipitation.

[0090] 3. Detection of dsRNA residues in IVT reaction products

[0091] Double antibody sandwich enzyme-linked immunosorbent assay (sandwich ELISA) is used to detect the content of dsRNA in the in vitro transcription system or the synthesized mRNA stock solution. The capture antibody is used to coat the microplate to form a solid-phase antibody. The dsRNA calibrator and the sample to be tested are added to the solid-phase antibody microplate, and then the detection antibody is added. Finally, the enzyme-labeled reagent labeled with horseradish peroxidase is added to form a "coated antibody-antigen-enzyme-labeled detection antibody" complex. After washing, the color developing solution is added for color development. The color developing solution is converted into blue under the catalysis of the HRP enzyme and finally converted into yellow under the action of acid. The depth of the color is positively correlated with the amount of dsRNA in the sample. The dsRNA detection results are as follows: Figure 5 The results of mRNA integrity test are shown in Figure 6 shown.

[0092] Example 4

[0093] The following shows the preparation of unnucleoside-modified mRNA prepared by the co-transcriptional capping method.

[0094] 1. Preparation of linearized DNA template

[0095] The plasmid was linearized using SpeI endonuclease and the linearized plasmid was used as a template.

[0096] 2.IVT reaction

[0097] Using the linearized plasmid as a template, T7 RNA polymerase, NTP and other raw materials were used for in vitro transcription to prepare mRNA. The final concentration gradient of L-arginine was 0 mM, 20 mM, 40 mM, 60 mM, and 80 mM. The reaction was carried out at 37°C and 42°C for 3 h, respectively, and then the prepared mRNA was purified by ammonium acetate precipitation.

[0098] 3. Detection of dsRNA residues in IVT reaction products

[0099] Double antibody sandwich enzyme-linked immunosorbent assay (sandwich ELISA) is used to detect the content of dsRNA in the in vitro transcription system or the synthesized mRNA stock solution. The capture antibody is used to coat the microplate to form a solid-phase antibody. The dsRNA calibrator and the sample to be tested are added to the solid-phase antibody microplate, and then the detection antibody is added. Finally, the enzyme-labeled reagent labeled with horseradish peroxidase is added to form a "coated antibody-antigen-enzyme-labeled detection antibody" complex. After washing, the color developing solution is added for color development. The color developing solution is converted into blue under the catalysis of the HRP enzyme and finally converted into yellow under the action of acid. The depth of the color is positively correlated with the amount of dsRNA in the sample. The dsRNA detection results are as follows: Figure 7 The results of mRNA integrity test are shown in Figure 8 shown.

[0100] Although the present invention has been described with reference to exemplary embodiments, it should be understood that the present invention is not limited to the disclosed exemplary embodiments. Various adjustments or changes may be made to the exemplary embodiments of the present specification without departing from the scope or spirit of the present invention. The scope of the claims should be based on the broadest interpretation to cover all modifications and equivalent structures and functions.

Claims

1. A method for preparing RNA by in vitro transcription, characterized in that The following steps are involved: (1) preparing a linearized DNA template; (2) constructing an in vitro transcription reaction system, and performing in vitro transcription under reaction conditions suitable for transcription, wherein the in vitro transcription reaction system contains a fixed concentration of arginine or a salt thereof; (3) Purify the prepared mRNA.

2. The method for preparing RNA by in vitro transcription according to claim 1, characterized in that The in vitro transcription reaction system comprises: a mixture of nucleoside triphosphates and chemical modifications thereof, RNA polymerase, a buffer and arginine or a salt thereof.

3. The method for preparing RNA by in vitro transcription according to claim 2, characterized in that The concentration of the arginine or its salt is 0.1-1000 mM.

4. The method for preparing RNA by in vitro transcription according to claim 1, characterized in that In the reaction conditions suitable for transcription, the pH range of the reaction is 7.5-8.5, and the temperature range of the reaction is 35-45°C.

5. The method for preparing RNA by in vitro transcription according to claim 2, characterized in that Chemical modifiers include N6-methyladenosine, inosine, 5-methylcytosine, pseudouridine, 5-hydroxymethylcytosine, N1-methyladenosine, and mRNA cap structure.

6. The method for preparing RNA by in vitro transcription according to claim 1, characterized in that The method further comprises a dsRNA detection step and / or an mRNA integrity detection step.

7. An in vitro transcription system, characterized in that: The in vitro transcription reaction system comprises: RNA polymerase, a mixture of nucleoside triphosphates and chemical modifications thereof, a buffer and arginine or a salt thereof.

8. mRNA or a vaccine comprising the same, characterized in that Obtained according to the method according to any one of claims 1 to 6; Preferably, the mRNA comprises at least one of a 5'-cap structure, a 3'-poly(A) sequence, a 5'-UTR and a 3'-UTR; Preferably, the uracil, cytosine or adenine nucleoside of the mRNA contains a modification group; Preferably, the chemically modified uridine nucleoside is selected from one or more of 2-fluoro-2-deoxyuridine, pseudouridine, N1-methyl-pseudouridine, 5-methoxyuridine, 2-fluoro-2-deoxy-pseudouridine, 2-fluoro-2-deoxy-N1-methyl-pseudouridine and 2-fluoro-2-deoxy-5-methoxyuridine; Preferably, the chemically modified cytosine nucleoside is selected from one or more of N4-acetylcytidine, 2-fluoro-2-deoxycytidine, 5-methylcytidine, 2-fluoro-2-deoxy-5-methylcytidine and 2-fluoro-2-deoxy-N4-acetylcytidine; Preferably, the chemically modified adenine nucleoside is selected from one or more of N6-methyladenosine, 2-fluoro-2-deoxyadenosine and 2-fluoro-2-deoxy-N6-methyladenosine; Preferably, the chemically modified guanosine nucleoside is selected from one or more of 2-fluoro-2-deoxyguanosine, N7-methyl-guanosine and 2-fluoro-2-deoxy-N7-methyl-guanosine.

9. A method for reducing the amount of dsRNA during an in vitro transcription reaction, characterized in that: The method comprises the steps of using a transcription reaction system and performing an in vitro transcription reaction under reaction conditions suitable for transcription, wherein the transcription reaction system comprises arginine or a salt thereof at a concentration of 0.1-1000 mM.

10. Use of the mRNA according to claim 8 or a vaccine comprising the same in the preparation of a product for treating or ameliorating a disease.

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