In-vitro transcription method suitable for wide mRNA length interval, kit and application

Abstract

The invention relates to the technical field of molecular biology, in particular to an in-vitro transcription method suitable for a wide mRNA length interval, a kit and application. In a transcription reaction system adopted by the in-vitro transcription method, the final reaction concentration of a DNA template is 30-60 [mu] g / mL, the reaction concentration of T7 RNA polymerase is 8-12 U / [mu] L, the reaction concentration of Mg < 2 + > is 40-60 mM, and the reaction concentration of a cap analogue is 3.5-4.5 mM. The method can be applied to a wide mRNA length interval, the application range of mRNA in-vitro transcription synthesis is expanded, the synthesis requirements of mRNA with different lengths are met, the flexibility and universality of mRNA in-vitro transcription synthesis are improved, and a more flexible and universal mRNA synthesis method is provided for the fields of gene therapy, vaccine research and development and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of molecular biology, in particular to an in vitro transcription method suitable for a wide range of mRNA length intervals, a kit and application thereof. BACKGROUND

[0002] In the field of mRNA in vitro transcription synthesis, traditional in vitro transcription process is usually only applicable to mRNA of a specific length range. For mRNA of longer or shorter length, the transcription efficiency is low, which affects the flexibility and universality of mRNA in vitro transcription synthesis.

[0003] In the prior art, Song Geshen et al. disclosed a method for producing mRNA stock solution and its application (patent publication number CN117987494A), which selected luciferase gene sequence (length less than 2000bp) as template and optimized the mRNA synthesis method. In this patent, when the Mg 2+ concentration is in the range of 30-50mM, especially when it reaches 40mM, the yield and quality of mRNA can reach the highest level, which is specifically manifested as 257μg RNA per microgram of template, and the integrity of RNA can reach 92.3%.

[0004] In addition, Guo Xiaokai et al. disclosed a preparation method for improving the integrity of full-length S protein mRNA of COVID-19 and its use (patent publication number CN116606867A), which involves S protein mRNA with a length of about 4000nt. In this method, when the Mg 2+ concentration is in the range of 15-40mM, especially when it reaches 24mM, the yield and quality of mRNA can achieve the highest improvement, with a maximum of 234.9μg mRNA per microgram of template, and the highest integrity of 87.1%.

[0005] In addition, Dong Kai et al. disclosed an industrialized production and separation and purification method of self-replicating mRNA stock solution and its application (patent publication number CN118048418A), which optimized the magnesium ion salt type, concentration and the addition of a second salt (sodium acetate) in the buffer through systematic experimental design, so that the yield and integrity of self-replicating mRNA were significantly improved, reaching 210μg and 70.2% respectively.

[0006] Although these methods improve the yield and quality of RNA to some extent, their effectiveness is limited to specific fragment lengths. Each method cannot simultaneously achieve effective improvement in yield and quality under different fragment lengths, which makes it necessary to use different synthesis methods when carrying out different RNA research, thereby increasing the cost and difficulty of use.

[0007] Therefore, there is an urgent need for an in vitro transcription system and method suitable for a wide range of mRNA length intervals to improve the yield and quality of products in the preparation of RNA of different lengths. SUMMARY

[0008] To solve the above technical problems, the present application first provides an in vitro transcription method suitable for a wide range of mRNA length intervals. In the transcription reaction system, the final concentration of the DNA template is 30-60 μg / mL, the reaction concentration of T7 RNA polymerase is 8-12 U / μL, the reaction concentration of Mg 2+ The reaction concentration of Mg is 40-60 mM, and the reaction concentration of cap analog is 3.5-4.5 mM.

[0009] In some embodiments, the final concentration of the DNA template is 30-35 μg / mL, the reaction concentration of T7 RNA polymerase is 9-11 U / μL, the reaction concentration of Mg 2+ The reaction concentration of Mg is 45-55 mM, and the reaction concentration of cap analog is 3.8-4.2 mM. Preferably, the final concentration of the DNA template is 30 μg / mL, the reaction concentration of T7 RNA polymerase is 10 U / μL, the reaction concentration of Mg 2+ The reaction concentration of Mg is 50 mM, and the reaction concentration of cap analog is 4 mM.

[0010] In some embodiments, the reaction time in the in vitro transcription method is 2-4 h, preferably 3 h.

[0011] In some embodiments, the mRNA length interval is 500-100000 nucleotides; preferably 1500-100000 nucleotides; more preferably 1500-9000 nucleotides; and more preferably 4000-8600 nucleotides.

[0012] In particular, the mRNA length interval is 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900, 6000, 6100, 6200, 6300, 6400, 6500, 6600, 6700, 6800, 6900, 7000, 7100, 7200, 7300, 7400, 7500, 7600, 7700, 7800, 7900, 8000, 8100, 8200, 8300, 8400, 8500, 8600, 8700, 8800, 8900, 9000, 9100, 9200, 9300, 9400, 9500, 9600, 9700, 9800, 9900, 10000, 15000, 20000, 25000, 30000, 35000, 40000, 45000, 50000, 55000, 60000, 65000, 70000, 75000, 80000, 85000, 90000, 95000, 100000 nucleotides.

[0013] In some embodiments, the transcription reaction system further comprises a buffer substance, and / or dithiothreitol, and / or spermidine, and / or an RNAase inhibitor, an NTP mixture / a NTP mixture containing base-modified nucleoside triphosphates, and / or a pyrophosphatase; Preferably, the buffer substance is Tris-HCl; preferably, the concentration of the buffer substance is 10-100 mM (more preferably 30-50 mM); Preferably, the concentration of dithiothreitol is 5-20 mM (more preferably 8-12 mM); Preferably, the concentration of spermidine is 1-10 mM (more preferably 1-5 mM); Preferably, the concentration of the RNAase inhibitor is 0.5-5 U / μL (more preferably 0.5-1.5 U / μL); Preferably, the concentration of each nucleoside triphosphate in the NTP mixture / NTP mixture containing base-modified nucleoside triphosphates is 5-25 mM (more preferably 8-12 mM); Preferably, the concentration of pyrophosphatase is 0.001-0.005 U / μL.

[0014] In some embodiments, the transcription reaction system is prepared with sterile enzyme-free water.

[0015] Further, the present application provides an in vitro transcription kit suitable for a wide range of mRNA length intervals, comprising: T7 RNA polymerase, magnesium salt, cap analog, NTP mixture / NTP mixture containing base-modified nucleoside triphosphates, reaction buffer, RNase inhibitor, and water; wherein the concentration of T7 RNA polymerase is 8-12 U / μL, the concentration of Mg 2+ is 40-60 mM, and the concentration of cap analog is 3.5-4.5 mM. Preferably, the concentration of T7 RNA polymerase is 9-11 U / μL, the concentration of Mg 2+ is 45-55 mM, and the concentration of cap analog is 3.8-4.2 mM. More preferably, the concentration of T7 RNA polymerase is 10 U / μL, the concentration of Mg 2+ is 50 mM, and the concentration of cap analog is 4 mM.

[0016] In some embodiments, the T7 RNA polymerase is wild-type or mutant T7 RNA polymerase; and / or, the magnesium salt is magnesium chloride or magnesium acetate; and / or, the cap analog is m7G(5')ppp(5')G, m7G(3oMe)(5')ppp(5')G, m7G(5')ppp(5')ApG, m7G(3oMe)(5')ppp(5')(A)pG, m7G(5')ppp(5')(2oMeA)pG, m7G(3oMe)(5')ppp(5')(2oMeA)pG, m7G(3oMe)(5')ppp(5')(m6A)pG, or a modified cap analog thereof.

[0017] Preferably, the cap analog is m7G(3oMe)(5')ppp(5')(2oMeA)pG (CAP1 modified cap analog), which has the following structure: In some embodiments, the kit further comprises dithiothreitol, and / or spermidine, and / or pyrophosphatase; Preferably, the buffer substance in the reaction buffer is Tris-HCl; preferably, the concentration of the buffer substance is 10-100 mM (more preferably 30-50 mM); Preferably, the concentration of dithiothreitol is 5-20 mM (more preferably 8-12 mM); Preferably, the concentration of spermidine is 1-10 mM (more preferably 1-5 mM); Preferably, the concentration of the RNAase inhibitor is 0.5-5 U / μL (more preferably 0.5-1.5 U / μL); Preferably, the concentration of each nucleoside triphosphate in the NTP mixture / NTP mixture containing base-modified nucleoside triphosphates is 5-25 mM (more preferably 8-12 mM); Preferably, the concentration of the pyrophosphatase is 0.001-0.005 U / μL.

[0018] Preferably, the kit further comprises sterile enzyme-free water.

[0019] Further, the present application provides the use of the in vitro transcription method or the kit in in vitro transcription of a wide range of mRNA lengths; wherein the final concentration of the DNA template is 30-60 μg / mL, preferably 30-35 μg / mL, more preferably 30 μg / mL.

[0020] Preferably, the DNA template is any one of the DNA sequence of RSV F protein (SEQ ID NO. 1), the DNA sequence of the S protein of the novel coronavirus (SEQ ID NO. 2), and the DNA sequence of the RBD self-replicating RNA (saRNA) of the S protein of the novel coronavirus (SEQ ID NO. 3).

[0021] In some embodiments, the NTP mixture / NTP mixture containing base-modified nucleoside triphosphates comprises at least one of unmodified or modified adenosine triphosphate, unmodified or modified cytosine triphosphate, unmodified or modified guanine triphosphate, and unmodified or modified uracil triphosphate.

[0022] Preferably, the NTP mixture containing base-modified nucleoside triphosphates contains at least one of N1-methyl pseudouridine triphosphate (N1-Me-pUTP), 5-methoxyuridine triphosphate (5-OMe-UTP), pseudouridine triphosphate (pUTP), 5-methylcytidine triphosphate (5-Me-CTP), and 2-thio-uridine triphosphate (2-thio-UTP).

[0023] Compared with the prior art, the present application has the following beneficial effects: 1. It can be applied to a wide range of mRNA length intervals, including long (e.g. 8500 nt) and short (e.g. 1500 nt) mRNAs, improving the flexibility and universality of mRNA in vitro transcription synthesis, meeting the synthesis needs of mRNAs of different lengths.

[0024] 2. By optimizing the components and conditions of the reaction system, the transcription efficiency of long or short mRNAs is improved, thereby improving the yield and quality of mRNA in vitro transcription synthesis.

[0025] 3. There is no need to optimize the reaction system for different lengths of mRNA, and the operation is simple, which improves the efficiency of mRNA in vitro transcription synthesis.

[0026] 4. It expands the application range of mRNA in vitro transcription synthesis, and provides a more flexible and universal mRNA synthesis method for gene therapy, vaccine research and other fields. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 is a linearized template electropherogram.

[0028] Figure 2 is a comparison chart of RNA yield and integrity under different transcription systems.

[0029] Figure 3 is a key parameter analysis chart of the transcription system.

[0030] Figure 4 is the verification result of short-chain mRNA synthesis using the optimal transcription system conditions.

[0031] Figure 5 is the experimental result of medium-chain mRNA synthesis using the optimal transcription system conditions.

[0032] Figure 6 is the experimental result of long-chain mRNA synthesis using the optimal transcription system conditions. DETAILED DESCRIPTION

[0033] For the purposes, technical solutions and advantages of the present application to be clearer, the technical solutions in the present application will be clearly and completely described below. Obviously, the described embodiments are 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 of ordinary skill in the art without creative labor fall within the scope of protection of the present application. In the examples provided in the present specification, if the specific technology or condition is not specified, it is carried out according to the technology or condition described in the literature (for example, patent literatures CN116606867A or CN116286796A) in the art, or according to the product manual. If the reagent or instrument used is not specified by the manufacturer, it is a conventional product that can be purchased through a regular channel.

[0034] The present application relates to molecular biology experiments. If not specifically mentioned, refer to the book "Molecular Cloning" (J. Sambrook, E.F. Fritsch, T. Maniatis, Science Press, 1994). This book and its subsequent published versions are the most commonly used guide reference books for those skilled in the art when performing experimental operations related to molecular biology. In addition, according to different experimental purposes, those skilled in the art complete the corresponding experiments under the guidance of the operation manual attached to various commercial kits or entrust professional companies, such as: gene sequencing, plasmid construction and determination of molecular weight, etc.

[0035] Term definition: In the present application, the length of mRNA can be divided into short chain, medium chain and long chain, wherein the length of short chain mRNA is about 500-2000 nucleotides (nt); the length of medium chain mRNA is about 2000-5000 nucleotides (nt); and the length of long chain mRNA is about 5000-100000 nucleotides (nt).

[0036] Sterile enzyme-free water: a kind of water treated without microorganisms and nucleases and other impurities, used to ensure that no contaminating substances that may affect the experimental results are introduced during the solution preparation and reagent dilution process in biological experiments, to ensure the purity of the experimental system, especially suitable for biological molecules related operations sensitive to enzymes.

[0037] Tris-HCl: chemical name is tris-hydroxymethyl aminomethane hydrochloride, which is a commonly used biological buffer that can stabilize the pH value of the solution within a certain concentration range, provide a suitable and stable acid-base environment for biochemical reactions, ensure enzyme activity and nucleic acid stability, and is widely used in various biological experiments, especially experiments involving nucleic acid operations.

[0038] Magnesium salt: mainly used as magnesium ion (Mg 2+Magnesium ions play a crucial role as cofactors in biochemical reactions, such as magnesium chloride, which is essential for maintaining the activity of T7 RNA polymerase in transcription reaction systems.

[0039] ATP, CTP, GTP, N1-Me-UT: All belong to nucleotides. ATP (adenosine triphosphate), CTP (cytidine triphosphate), GTP (guanosine triphosphate) are the conventional nucleotide components of RNA, which are used as substrates for RNA polymerase to synthesize mRNA chains during transcription, and are sequentially connected to form RNA molecules according to the DNA template sequence; N1-Me-UTP (N1-methyl uridine triphosphate) is a chemically modified nucleotide, which may have special effects on RNA structure, stability, function, etc., and is involved in the transcription process and affects the properties of the final mRNA.

[0040] DTT (dithiothreitol): It is a strong reducing agent, its main function is to protect the sulfhydryl group (-SH) in the protein molecule from oxidation by reduction, so as to maintain the activity and correct three-dimensional structure integrity of the protein. In biological experimental systems containing proteins, such as reactions involving enzymes such as RNA polymerase, DTT can effectively prevent proteins from being inactivated due to oxidation, ensuring that biological processes such as transcription reactions can proceed smoothly.

[0041] Spermidine (sp0.1): It is a polyamine compound that exists widely in living organisms and has various biological functions. In experimental systems, spermidine may play a role as a regulatory factor. It may interact with nucleic acid molecules and regulate the transcription process by affecting the structural stability of nucleic acids. Specifically, spermidine may bind to the DNA template, changing the conformation of the DNA, making it easier for RNA polymerase to recognize and bind, thereby promoting the formation of the transcription initiation complex, and playing a certain enhancing or regulating role in the transcription process. In addition, spermidine may also interact with other proteins or small molecules, participate in maintaining the ion balance and membrane stability in cells, and indirectly affect the activity of transcription-related enzymes and the synthesis process of mRNA, and synergize with other reagents in the whole mRNA in vitro transcription and related experimental systems.

[0042] YIPP (Pyrophosphatase): A kind of enzyme that can catalyze the hydrolysis of pyrophosphate. In biochemical reactions, especially in processes related to nucleic acid synthesis, such as transcription reactions, pyrophosphatase can catalyze the hydrolysis of pyrophosphate (a byproduct of nucleotide polymerization reaction) into inorganic phosphate, which is beneficial to promote the transcription reaction to generate mRNA, by eliminating the accumulation of pyrophosphate, making the transcription reaction more favorable in thermodynamics, thereby promoting the continuous synthesis of RNA chain, which may play an important role in improving transcription efficiency and yield. At the same time, in the whole experimental system, it cooperates with other reagents such as T7 RNA polymerase, nucleotides, etc., to complete the in vitro transcription process of mRNA together.

[0043] RI (RNAase inhibitor): A substance that can specifically inhibit the activity of RNAase. RNAase exists widely in biological environment and has strong activity, and trace amount of RNAase contamination can cause rapid degradation of RNA molecules. RI inhibits the activity of RNAase by binding to it or changing its active site, thereby protecting the RNA molecules in the experiment from being degraded.

[0044] DNA template: A piece of DNA molecule carrying genetic information, which serves as a template for mRNA synthesis in the transcription process. It determines the sequence of the transcription product mRNA, and T7 RNA polymerase synthesizes the mRNA chain complementary to it according to the base sequence information on the DNA template, using ATP, CTP, GTP, N1-Me-UTP, etc. as raw materials, which is the basis of the transcription reaction and determines the specificity and coding information of the final mRNA.

[0045] Cap analog: A substance similar in structure or function to the 5' cap structure of eukaryotic mRNA. The 5' cap structure of eukaryotic mRNA has important influence on the stability, translation initiation efficiency and localization of mRNA in the cell, etc. Cap analogs may mimic the function of natural cap structure in the transcription process, participate in the maturation process of mRNA, help to improve the stability and translation efficiency of mRNA, and thus have a positive impact on the effect of mRNA vaccine and other related products. For example, CAP1 modified cap analog.

[0046] T7 RNA polymerase: A DNA-dependent RNA polymerase that can specifically recognize the T7 promoter sequence on the DNA template, using ATP, CTP, GTP, N1-Me-UTP, etc. as nucleotide substrates, and catalyzing the synthesis of mRNA chain according to the principle of base complementary pairing. It is the key enzyme in the in vitro transcription process, directly determines the synthesis process of mRNA, and plays an accurate regulatory role in the initiation, elongation and termination of transcription. The T7 RNA polymerase in the following examples is wild-type T7 RNA polymerase.

[0047] In the following examples, the preparation steps of the DNA template are as follows: 1. Preparation of plasmid The plasmid was synthesized by GenScript and transformed into stabl 4 bacteria. The bacteria were inoculated into LB culture medium at a ratio of 1:1000 and cultured overnight at 30-35°C on a shaking bed, and the plasmid was extracted using an endotoxin-free large-scale plasmid extraction kit.

[0048] 2. Preparation of linearized template According to the enzyme digestion system in Table 1, the enzyme digestion system was added to a sterile enzyme-free container, mixed well, and then incubated overnight at 50°C in a water bath shaker at 70 rpm.

[0049] Table 1 Enzyme digestion reaction system

[0050] 3. Purification of linearized template using chromatography system A low concentration of sodium chloride solution (0.5-0.8M sodium chloride, pH 7.2-7.5) was used as the equilibrium buffer to fully equilibrate the DEAE anion medium. The linearized template was loaded onto the anion medium, and the medium loading capacity was 0.5-2 mg / ml (DNA amount / volume of medium). Elution was performed using a high concentration of sodium chloride solution (1-2M sodium chloride, pH 7.2-7.5), and the elution peak was collected, which was the purified linearized template. The electrophoresis pattern of the linearized template is shown in Figure 1 . This template was used for subsequent studies.

[0051] Example 1 Optimization of short-chain mRNA co-transcription system The JMP software was used to design a response surface experiment for the key conditions of the short-chain mRNA synthesis system. The DNA template contained the DNA sequence of RSV F protein, 5'UTR sequence, 3'UTR sequence, and PolyA sequence, as shown in SEQ ID NO. 1, and the synthesis system test range is shown in Table 2.

[0052] Table 2 Test range of short-chain mRNA synthesis system

[0053] Further, mRNA yield and integrity were used as response values, and the final concentration of DNA template (μg / mL), T7 RNA polymerase reaction concentration (U / μL), Mg 2+ reaction concentration (mM), cap analog reaction concentration (mM), and synthesis reaction time (h) were used as factors, and the design range values of the factors were limited, and then a central composite design experiment was designed, as shown in Table 3.

[0054] Table 3 Experimental design scheme of key conditions of short-chain mRNA synthesis system

[0055] The synthesis experiments were conducted 28 times according to the experimental conditions in the table above. In the above 28 experiments, the concentrations of the other components in the synthesis system were consistent except for the five variables mentioned above.

[0056] Example 2: Purification and Yield Detection of Short-Chain mRNA mRNA recovery and purification: Equal volumes of 8M LiCl solution were added to each of the in vitro transcribed mRNA samples. After mixing, the samples were incubated at -80℃ for 30 min, then centrifuged at 21000×g for 20 min using a centrifuge pre-cooled to 4℃. The supernatant was discarded, and the precipitate was collected. The precipitate was then washed with 1 mL of 70% ethanol solution, centrifuged at 21000×g for 10 min, and the supernatant was discarded, collecting the mRNA precipitate. Finally, the mRNA precipitate was dissolved in 1 mL of sterile, enzyme-free water. The concentration of the collected mRNA (μg / mL) was measured using a Nano Drop ultraviolet spectrophotometer. The mRNA yield (μg) was calculated based on the volume (mL) of sterile, enzyme-free water added to dissolve the mRNA precipitate. The results are shown below. Figure 2 And Table 4.

[0057] Example 3: Integrity Detection of Short-Chain mRNA The integrity of collected mRNA was detected using an Agilent 5200 capillary electrophoresis system. Taking the Agilent DNF-471 RNA Kit (15 nt) as an example: RNA samples were diluted to the detection range. The RNA ladder and RNA samples were denatured at 70°C for 2 min and then transferred to ice to cool. 22 μL of RNA Diluent Marker (15 nt) (DM) solution was added to each sample well and ladder, and 24 μL of BF-25 Blank solution was added to each blank well. After mixing the denatured RNA sample, 2 μL was added to each sample well and mixed. 2 μL of the denatured RNA ladder was added to well (H)12 and mixed. The sample plate was centrifuged to remove air bubbles and placed in the sample drawer for detection. The DNF-471-33 program was used to detect mRNA integrity. The results are shown below. Figure 2 And Table 4.

[0058] Table 4. mRNA integrity, yield, and amplification fold under different transcription systems

[0059] Example 4: Analysis of Key Conditions for Short-Chain mRNA Synthesis System Based on the above results, the "trait-standard least squares method" is selected and the data model fitting is performed for "yield and integrity" respectively. The relationship model between each influencing factor and response value is obtained through the specific experimental results, and the mRNA yield and integrity specification limit is set. According to the results of the delineator, 10,000 simulation experiments are performed, and when the "defect rate" of the simulation experiment result is reduced to the lowest, the optimal operating range of the five key parameters in Example 1 can be obtained.

[0060] The results are: the final concentration of DNA template reaction is 30-60 μg / mL, the optimal is 30 μg / mL, the reaction concentration of T7 RNA polymerase is 8-12 U / μL, the optimal is 10 U / μL, the reaction concentration of Mg 2+ The reaction concentration of 40-60 mM, the optimal is 50 mM, the reaction concentration of cap analog is 3.5-4.5 mM, the optimal is 4 mM, the synthesis reaction time is 2-4 h, the optimal is 3 h. The analysis of key parameters is shown in Figure 3

[0061] Example 5 Verification experiment of key conditions of short-chain mRNA synthesis system According to the key parameter range determined in Example 4, the in vitro transcription synthesis experiment of RSV F protein mRNA was carried out under the optimal conditions, and the system is shown in Table 5.

[0062] Table 5 Composition of in vitro transcription system of short-chain mRNA

[0063] After synthesis, the yield and integrity detection was carried out according to the method of Example 2 and Example 3, and the total yield of 50 μL synthesis system was 490.8 μg, and each μg template could produce 327.2 μg mRNA, and the integrity was 93.9%, and the results are shown in Figure 4

[0064] Example 6 Test the applicability of the optimized mRNA synthesis system in the preparation of medium-chain mRNA According to the key parameter range determined in Example 4, the in vitro transcription synthesis experiment of RSV F protein mRNA was carried out under the optimal conditions, and the system is shown in Table 5.

[0065] Table 6 Composition of in vitro transcription system of medium-chain mRNA

[0066] ​​The yield was detected after synthesis according to the method of Example 2, and the total yield of 8 ml of the synthesis system was 49.3 mg, and 205.4 ug of mRNA could be produced per ug of template.

[0067] The mRNA was further purified by affinity chromatography, and the specific steps were as follows: 1) The obtained mRNA synthesis solution was loaded on the affinity medium containing oligo dt, and the medium loading was 1-2 mg / ml (RNA amount / medium volume), and the affinity medium was equilibrated with a buffer solution containing a high concentration of sodium chloride (0.5-1 M sodium chloride, pH 7.2-7.5); 2) Washed with a buffer solution containing a lower concentration of sodium chloride (0.1-0.3 M sodium chloride, pH 7.2-7.5) to remove incomplete mRNA; 3) Eluted with sterile water for injection, pH 7.2-7.5, collected the elution peak, and obtained the purified mRNA solution. After affinity chromatography, the integrity of the mRNA was 85.2%, and the results were shown in Figure 5 .

[0068] From the above results, compared with the data disclosed in CN116606867A, the in vitro transcription reaction system can obtain higher integrity, and the amount of mRNA produced per ug of template is also significantly improved, which has obvious advantages, that is, the final concentration of DNA template reaction is 30 ug / mL, the concentration of T7 RNA polymerase reaction is 10 U / ul, the concentration of Mg 2+ The synthesis system with a reaction concentration of 50 mM for Mg

[0069] Example 7 Test the applicability of the optimized mRNA synthesis system in the preparation of long-chain mRNA According to the key parameter range determined in Example 4, the DNA sequence of the RBD self-replicating RNA (saRNA) of the new crown S protein was used as a template for mRNA in vitro transcription synthesis experiment under the optimal conditions. The DNA template contains the DNA sequence of the RBD self-replicating RNA (saRNA) of the new crown S protein, 5'UTR sequence, 3'UTR sequence and PolyA sequence, as shown in SEQ ID NO. 3. The system is shown in Table 7.

[0070] Table 7 Composition of long-chain mRNA in vitro transcription system

[0071] After synthesis, the yield and integrity were detected according to the methods of Example 2 and Example 3, and the yield of saRNA was 915.5 ug, the amount of mRNA produced per ug of template was 305.2 ug, and the integrity was 80.8%, and the results were shown inFigure 6 .

[0072] From the above results, compared with the data disclosed in CN 118048418 A, the in vitro transcription reaction system can obtain higher integrity under the same purification method, and the amount of mRNA produced per μg template is also significantly improved. That is, the final concentration of DNA template reaction is 30 μg / mL, the concentration of T7 RNA polymerase reaction is 10 U / μL, the concentration of Mg 2+ The synthesis system with the reaction concentration of 50 mM and the cap analog reaction concentration of 4 mM is suitable for the preparation of long-chain mRNA.

[0073] In summary, the present application is initially aimed at short-chain mRNA (1800 nt) (RSV preF) transcription system formula optimization, and the best transcription system formula is obtained through prediction and experimental verification. On this basis, medium-chain (4000 nt) and long-chain mRNA (8500 nt) tests are attempted, and the results surprisingly find that the transcription system formula is also suitable for medium-chain and long-chain mRNA, and the effect is significantly better than the prior art.

[0074] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. An in vitro transcription method suitable for a wide range of mRNA length intervals, characterized in that, The transcription reaction system used has a final concentration of DNA template of 30-60 μg / mL, a reaction concentration of T7 RNA polymerase of 8-12 U / μL, a reaction concentration of Mg 2+ The reaction concentration of the cap analog is 3.5-4.5 mM.

2. The in vitro transcription method according to claim 1, characterized in that, The final concentration of the DNA template reaction is 30-35 μg / mL, the concentration of the T7 RNA polymerase reaction is 9-11 U / μL, the concentration of the Mg 2+ The reaction concentration is 45-55 mM, and the reaction concentration of the cap analog is 3.8-4.2 mM; Preferably, the DNA template reaction final concentration is 30 μg / mL, the T7 RNA polymerase reaction concentration is 10 U / μL, the Mg 2+ The reaction concentration is 50 mM, and the cap analog reaction concentration is 4 mM.

3. The in vitro transcription method according to claim 1 or 2, characterized in that, The reaction time in the in vitro transcription method is 2-4h, preferably 3h.

4. The in vitro transcription method according to any one of claims 1 to 3, characterized in that, The length of the mRNA is 500-100000 nucleotides; preferably 1500-100000 nucleotides; more preferably 1500-9000 nucleotides; and more preferably 4000-8600 nucleotides.

5. The in vitro transcription method according to any one of claims 1 to 4, wherein, The transcription reaction system further comprises a buffer substance, and / or dithiothreitol, and / or spermidine, and / or an RNAase inhibitor, an NTP mixture / a NTP mixture containing base-modified nucleoside triphosphates, and / or a pyrophosphatase; Preferably, the buffer substance is Tris-HCl; and preferably, the concentration of the buffer substance is 10-100 mM. Preferably, the concentration of dithiothreitol is 5-20 mM. Preferably, the concentration of spermidine is 1-10 mM. Preferably, the concentration of the RNAase inhibitor is 0.5-5 U / μL. Preferably, the concentration of each nucleoside triphosphate in the NTP mixture / NTP mixture containing base-modified nucleoside triphosphates is 5-25 mM. Preferably, the concentration of the pyrophosphatase is 0.001-0.005 U / μL.

6. An in vitro transcription kit suitable for a broad range of mRNA length intervals, comprising: T7 RNA polymerase, a magnesium salt, a cap analog, an NTP mixture, a reaction buffer, an RNase inhibitor, and water; characterized in that the concentration of the T7 RNA polymerase is 8 to 12 U / μL, the concentration of the magnesium salt is 40 to 60 mM, and the concentration of the cap analog is 3.5 to 4.5 mM. 2+ T7 RNA polymerase, a magnesium salt, a cap analog, an NTP mixture, a reaction buffer, an RNase inhibitor, and water; characterized in that the concentration of the T7 RNA polymerase is 8 to 12 U / μL, the concentration of the magnesium salt is 40 to 60 mM, and the concentration of the cap analog is 3.5 to 4.5 mM. Preferably, the concentration of T7 RNA polymerase is 9-11 U / μL, the concentration of Mg 2+ is 45-55 mM, and the concentration of cap analog is 3.8-4.2 mM. More preferably, the concentration of T7 RNA polymerase is 10 U / pL, the concentration of Mg 2+ is 50 mM, and the concentration of cap analog is 4 mM.

7. The in vitro transcription kit according to claim 6, characterized in that, The T7 RNA polymerase is a wild type or mutant T7 RNA polymerase. And / or, the magnesium salt is magnesium chloride or magnesium acetate; And / or, the cap analog is m7G(5')ppp(5')G, m7G(3oMe)(5')ppp(5')G, m7G(5')ppp(5')ApG, m7G(3oMe)(5')ppp(5')(A)pG, m7G(5')ppp(5')(2oMeA)pG, m7G(3oMe)(5')ppp(5')(2oMeA)pG, m7G(3oMe)(5')ppp(5')(m6A)pG, or a modified cap analog thereof.

8. The in vitro transcription kit according to claim 6 or 7, characterized in that, The kit further comprises dithiothreitol, and / or spermidine, and / or a pyrophosphatase; Preferably, the buffer substance in the reaction buffer is Tris-HCl; and preferably, the concentration of the buffer substance is 10-100 mM. Preferably, the concentration of dithiothreitol is 5-20 mM. Preferably, the concentration of spermidine is 1-10 mM. Preferably, the concentration of the RNAase inhibitor is 0.5-5 U / μL. Preferably, the concentration of each nucleoside triphosphate in the NTP mixture / NTP mixture containing base-modified nucleoside triphosphates is 5-25 mM. Preferably, the concentration of the pyrophosphatase is 0.001-0.005 U / μL.

9. Use of the in vitro transcription method according to any one of claims 1 to 5 or of the kit according to any one of claims 6 to 8 for in vitro transcription over a broad range of mRNA lengths; wherein, The final concentration of the DNA template is 30-60 μg / mL, preferably 30-35 μg / mL, and more preferably 30 μg / mL.

10. Use according to claim 9, characterized in that, The length of the mRNA is 500-100000 nucleotides; preferably 1500-100000 nucleotides; more preferably 1500-9000 nucleotides; and more preferably 4000-8600 nucleotides. And / or, the reaction time of the in vitro transcription is 2-4h, preferably 3h.

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