In-vitro transcription method based on RNA polymerase

Abstract

The invention provides an in-vitro transcription method based on RNA polymerase and a kit, which are suitable for the fields of mRNA synthesis, gene research and biological pharmacy. According to the method, the problems of low efficiency, poor stability and the like of traditional in-vitro transcription are solved through an optimization technology. The method comprises the following specific steps: providing a linear DNA template containing a T7 promoter; according to the method, site-directed mutagenesis or fusion protein modified high-activity T7RNA polymerase is combined with chemically modified NTPs and a spermine-BSA compound stabilizer to react under the conditions that the temperature is 37-42 DEG C and the pH is 7.5-8.5, and formation of double-stranded RNA is promoted through dynamic temperature control. A reaction system contains Mg < 2 + > / Mn < 2 + > and DTT, and enzyme activity and product integrity are optimized. The kit comprises an improved reagent and a standardized process, the transcription efficiency is improved by 2-5 times, the product yield is greater than or equal to 80%, and the activity is not lost after the kit is stored at 4 DEG C for 1 month. The method has the technical advantages that NTPs degradation resistance is achieved, template preparation is simplified, universality is high, the method is suitable for large-scale production of mRNA vaccines, gene therapy and the like, the production cost is remarkably reduced, and wide application of the in-vitro transcription technology is promoted.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of molecular biology, and particularly relates to an in vitro transcription method based on RNA polymerase, which is suitable for the fields of mRNA synthesis, gene function research and biopharmaceuticals. BACKGROUND

[0002] In vitro transcription technology is an important tool in molecular biology research, and is widely used in the fields of mRNA synthesis, gene function research, vaccine development, etc. Traditional in vitro transcription methods usually use RNA polymerase (such as T7, SP6 or T3 RNA polymerase) to catalyze the synthesis of RNA from a DNA template. However, the existing technology has deficiencies in terms of transcription efficiency, product stability, reaction condition optimization, etc. For example, the activity of conventional RNA polymerase is easily affected by temperature and pH fluctuations, resulting in low transcription efficiency; unmodified ribonucleotides (such as NTPs) may degrade during the reaction, affecting the integrity of the product; non-specific reactions or product degradation in the reaction system also limit its application. In addition, in the context of mRNA vaccines and other scenarios requiring high-purity RNA products, existing methods are difficult to meet the needs of large-scale production. Therefore, it is of great significance to develop an efficient and stable in vitro transcription method with strong controllability and suitable for various application scenarios.

[0003] In view of the defects in the prior art, the present application significantly improves the transcription efficiency and enhances the stability of the product by optimizing the RNA polymerase, improving the nucleotide mixture, introducing a new stabilizer and controlling the reaction conditions. At the same time, by simplifying the template preparation process and standardizing the reaction system, the complexity of the operation is reduced, providing technical support for the wide application of in vitro transcription technology SUMMARY

[0004] The present application proposes an in vitro transcription method based on RNA polymerase, which solves the problems in the prior art.

[0005] The technical solution of the present application is as follows: an in vitro transcription method based on RNA polymerase, comprising the following steps:

[0006] S1. Providing a linear DNA template containing a T7 promoter;

[0007] S2. Adding high-activity RNA polymerase, improved ribonucleotide mixture and stabilizer to the reaction system, and controlling the reaction conditions to be temperature: 37-42℃, pH: 7.5-8.5;

[0008] S3. Performing in vitro transcription reaction and monitoring RNA product generation;

[0009] S4. Achieving efficient transcription by optimizing the reaction system and conditions.

[0010] As a preferred embodiment, the improved ribonucleotide mixture comprises chemically modified NTPs, and the stabilizer is a complex stabilizer comprising spermine and BSA.

[0011] As a preferred embodiment, the high-activity RNA polymerase is a T7 RNA polymerase modified by site-directed mutagenesis or fusion protein, and the activity unit is defined as 1 nmol of ATP incorporated into the polynucleotide moiety within 60 minutes at 37℃.

[0012] As a preferred embodiment, the preparation step of the linear DNA template comprises:

[0013] S11, amplifying the target gene fragment by PCR and adding a T7 promoter sequence at the 5' end of the forward primer;

[0014] S12, linearizing the plasmid using a single enzyme cutting site, and purifying the template DNA by phenol-chloroform extraction and ethanol precipitation.

[0015] As a preferred embodiment, the reaction system further comprises divalent metal ions Mg 2+ or Mn 2+ , at a concentration of 1-5 mM, and a DTT reducing agent at a concentration of 0.5-2 mM.

[0016] As a preferred embodiment, during the in vitro transcription reaction, the formation of double-stranded RNA is promoted by a stepwise cooling procedure, which denatures at 95℃ for 5 minutes, and then cools to room temperature at a rate of 0.1℃ / second.

[0017] An in vitro transcription method for a gene sequence encoding a protein, which synthesizes mRNA using an RNA polymerase-based in vitro transcription method, and the mRNA is modified for cell transfection or as a vaccine component.

[0018] An RNA polymerase-based in vitro transcription kit, comprising:

[0019] A high-activity RNA polymerase;

[0020] An improved ribonucleotide mixture;

[0021] A buffer containing Mg 2+ and a stabilizer;

[0022] A template DNA purification reagent;

[0023] A reaction condition control instruction.

[0024] The beneficial effects of the present application are as follows: the transcription efficiency is improved by 2-5 times, the product yield is greater than or equal to 80%, and the stability is enhanced (no loss of activity after 1 month of storage at 4°C) by optimizing enzymes, nucleotides and reaction conditions.

[0025] The chemical modification of NTPs resists degradation, the double-stranded RNA formation technology improves product integrity, the standardized process simplifies operation, is suitable for industrial amplification, significantly reduces production cost, and shows high efficiency, stability and universality in mRNA vaccine, gene therapy and basic research, thereby promoting the wide application of in vitro transcription technology. DETAILED DESCRIPTION

[0026] The embodiments described below are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the present application.

[0027] An in vitro transcription method based on RNA polymerase, comprising the following steps:

[0028] Step S1: linear DNA template preparation:

[0029] The gene of the target coded protein is selected, a T7 promoter sequence is introduced by PCR amplification, and a T7 promoter sequence is added at the 5' end of the forward primer to ensure the accuracy of the RNA polymerase recognition site.

[0030] A single enzyme cutting site is used to linearize the amplified plasmid to avoid the formation of a circular structure of the template affecting the transcription efficiency.

[0031] The phenol-chloroform extraction method is used to remove protein impurities, and ethanol precipitation is used to recover DNA to ensure that the A260 / A280 ratio of the template purity is greater than or equal to 1.8.

[0032] Step S2: reaction system construction:

[0033] The T7 RNA polymerase modified by site-directed mutagenesis or fusion protein technology is selected, and the activity unit is defined as 1 nmol of ATP combined into the polynucleotide part within 60 minutes at 37°C; for example, the mutation site R199H or the GST tag can be introduced to enhance the stability;

[0034] The NTPs containing chemical modification reduce the risk of product degradation and prolong the half-life;

[0035] The spermine (final concentration 0.5 mM) and BSA (final concentration 1 mg / mL) complex stabilizer is used, the spermine protects the enzyme structure by electrostatic interaction, and the BSA prevents non-specific adsorption.

[0036] Mg2+ Concentration 5mM, optimize enzyme active center conformation; add DTT reducing agent, maintain enzyme cysteine residue reduction state;

[0037] Add Tris-HCl buffer at pH 8 to provide the best ionic environment.

[0038] Step S3: In vitro transcription reaction and monitoring:

[0039] Select reaction conditions 40℃ constant temperature incubation, reaction time 3 hours;

[0040] Real-time gel electrophoresis quantitative analysis of RNA generation rate.

[0041] Step S4: Reaction system and condition optimization:

[0042] Use segmented temperature control strategy, initial 40℃ transcription, later reduce temperature to 37℃ to extend enzyme catalytic time;

[0043] Add HEPES buffer 20mM during the reaction, real-time monitoring and maintain pH stability;

[0044] Determine the optimal substrate concentration by NTPs concentration gradient 2mM;

[0045] Step S5: Double-stranded RNA formation promotion

[0046] Perform a step-by-step cooling program: 95℃ denaturation for 5 minutes, slowly cool to room temperature at a rate of 0.1℃ / sec, promote RNA chain correct folding to form double-stranded structure, enhance stability.

[0047] Further, by PCR site-directed mutagenesis introduce specific mutation K235E in T7 RNA polymerase coding sequence, enhance enzyme and promoter binding affinity and catalytic rate.

[0048] Further, T7 enzyme is fused with stable protein expression, improve the enzyme in vitro environment of anti-degradation ability.

[0049] Further, select 2'-O-methyl uridine triphosphate, locked nucleic acid modified CTP, reduce the degradation of nucleases to the product, improve mRNA translation efficiency.

[0050] An in vitro transcription method of a gene sequence encoding a protein, comprising:

[0051] The in vitro transcribed and synthesized mRNA is modified by 5' capping and 3' polyadenylation, then introduced into cells through liposomes to realize exogenous protein expression;

[0052] For viral antigen encoding genes, in vitro transcription generates high-purity mRNA, which is encapsulated by liposomes as a nucleic acid vaccine to stimulate immune response.

[0053] An in vitro transcription kit based on RNA polymerase, comprising:

[0054] Highly active T7 RNA polymerase (lyophilized powder, 10U / tube);

[0055] Modified ribonucleotide mixture (containing modified NTPs, 1 mL);

[0056] Stabilizer buffer (containing Mg) 2+ Spermine, BSA, pH 8.0, 5 mL;

[0057] Template DNA purification reagents (phenol-chloroform, ethanol precipitation reagents);

[0058] The steps for using the reaction conditions instruction manual are as follows:

[0059] (1) Template DNA preparation: Perform PCR amplification and linearization according to the instructions;

[0060] (2) Reaction system preparation: Add 10U enzyme, 20μM modified NTPs, and 2mM Mg to a 50μL system. 2+ 1 mg / mL BSA, etc.;

[0061] (3) Start the reaction: Incubate at 40℃ for 2 hours;

[0062] (4) Product recovery: RNA was obtained by phenol-chloroform extraction and isopropanol precipitation.

[0063] Example verification: A linear luciferase gene template containing the T7 promoter was prepared according to step S1. The reaction was carried out using this kit and incubated at 40°C for 2 hours. The product was capped with Cap1 and modified with polyA and then transfected into HeLa cells. Fluorescence detection showed that the expression level was increased by 2.8 times compared with the traditional method.

[0064] Example verification: mRNA was synthesized using this method targeting the SARS-CoV-2 spike protein gene, encapsulated in liposomes, and then used to immunize mice. ELISA detection showed a significant increase in antibody titer.

[0065] This manual describes a highly efficient and stable in vitro transcription system achieved through systematic technical optimization and standardized procedures. Its core innovations lie in enzyme engineering, the application of chemically modified NTPs, dynamic temperature control programs, and the synergistic effect of composite stabilizers. This technology can be widely applied in gene function research, protein production, and nucleic acid drug development, demonstrating significant practical value and industrialization prospects.

[0066] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An in vitro transcription method based on RNA polymerase, characterized in that, Includes the following steps: S1. Provide a linear DNA template containing the T7 promoter; S2. Add highly active RNA polymerase, a modified ribonucleotide mixture, and a stabilizer to the reaction system, and control the reaction conditions as follows: temperature: 37℃-42℃, pH: 7.5-8.5; S3. Perform in vitro transcription and monitor RNA product generation; S4. Achieve efficient transcription by optimizing the reaction system and conditions.

2. The in vitro transcription method based on RNA polymerase according to claim 1, characterized in that, The modified ribonucleotide mixture contains chemically modified NTPs, and the stabilizer is a composite stabilizer containing spermine and BSA.

3. The in vitro transcription method based on RNA polymerase according to claim 1, characterized in that, The highly active RNA polymerase is a T7 RNA polymerase modified by site-directed mutagenesis or fusion protein, and its activity unit is defined as the ability to bind 1 nmol of ATP to a polynucleotide moiety within 60 minutes at 37°C.

4. The in vitro transcription method based on RNA polymerase according to claim 1, characterized in that, The preparation steps of the linear DNA template include: S11. Amplify the target gene fragment by PCR and add the T7 promoter sequence to the 5' end of the forward primer; S12. Linearize the plasmid using a single restriction enzyme site, and purify the template DNA by phenol-chloroform extraction and ethanol precipitation.

5. The in vitro transcription method based on RNA polymerase according to claim 1, characterized in that, The reaction system also includes divalent metal ions Mg. 2+ or Mn 2+ The concentration is 1-5 mM, and the concentration of DTT reducing agent is 0.5-2 mM.

6. The in vitro transcription method based on RNA polymerase according to claim 1, characterized in that, During the in vitro transcription reaction, the formation of double-stranded RNA was promoted by a stepwise cooling program, which involved denaturation at 95°C for 5 minutes, followed by cooling to room temperature at a rate of 0.1°C / second.

7. A method for in vitro transcription of a gene sequence encoding a protein, characterized in that, mRNA is synthesized using the method described in any one of claims 1-5, and the mRNA is modified for use in cell transfection or as a vaccine component.

8. An in vitro transcription kit based on RNA polymerase, comprising: highly active RNA polymerase; Modified ribonucleotide mixture; Contains Mg 2+ Buffer solution for stabilizers; Template DNA purification reagents; Reaction condition control instructions.