RNA polymerase variants that improve RNA capping rates during in vitro transcription

Abstract

The present disclosure provides an RNA polymerase variant capable of improving the RNA capping rate during in vitro transcription, relating to the field of biotechnology. The present disclosure obtains a T7 RNA polymerase variant by modifying a wild-type T7 RNA polymerase, which can significantly improve the capping rate of RNA products compared to the wild-type T7 RNA polymerase.

Description

Technical Field

[0001] The present disclosure relates to the field of biotechnology, and in particular to RNA polymerase variants and applications thereof in preparing capped RNA. Background Art

[0002] A complete mRNA consists of five main parts, from 5' to 3', including: the 5' cap structure, the 5' untranslated region (UTR), the open reading frame encoding the antigen, the 3' untranslated region (UTR), and the PolyA tail. In vivo, the 5' cap structure is formed by RNA triphosphatases, mRNA guanylyltransferases, mRNA methyltransferases, and mRNA nucleoside 2'-methyltransferases. It is a specialized structure that forms during post-transcriptional modification of mature mRNA in eukaryotic organisms. In vitro transcription (IVT) capping, also known as enzymatic capping, can synthesize the cap structure through an enzyme-linked reaction similar to that used in vivo. This process typically uses enzymes such as VCE and FCE as capping catalysts, followed by the addition of mRNA nucleoside 2'-methyltransferases. Enzymatic capping is limited by the efficiency of the enzyme-linked catalysis, resulting in relatively low capping rates. Co-transcriptional capping using cap analogs is also a mainstream method, achieving capping rates far exceeding those of enzymatic capping.

[0003] Today, cap analogs have developed to the third generation. The first generation of cap analogs has two free 3'-OH groups, which causes the cap analogs to be reversely incorporated, so first generation cap analogs are rarely seen on the market. Currently, the most common cap analogs on the market are the second generation ARCA cap analogs (Formula I) and the third generation cap analogs (such as Formula II, CleanCapAG).

[0004]

[0005] Third-generation cap analogs, such as CleanCapAG, have significantly improved capping rates (over 90%) compared to second-generation cap analogs. However, when prepared using wild-type T7 RNA polymerase as a catalyst, steric hindrance and other factors make it difficult to achieve a 100% capping rate. The generation of uncapped RNA products not only wastes raw materials but also requires column purification for removal, increasing production costs. Therefore, further improving the mRNA capping rate during co-transcriptional capping is beneficial to the economical production of mRNA. SUMMARY OF THE INVENTION

[0006] In a first aspect, the present disclosure provides a class of RNA polymerase variants, whose amino acid sequence has at least 95% sequence identity with SEQ ID NO: 1, and comprises a substitution or substitution group selected from any one of the following relative to SEQ ID NO: 1: D388 or D130 / K387.

[0007] In a second aspect, the present disclosure provides one or more biomaterials selected from the following:

[0008] 1) a polynucleotide molecule encoding an RNA polymerase variant;

[0009] 2) an expression vector comprising the polynucleotide molecule described in 1);

[0010] 3) A host cell comprising the polynucleotide molecule described in 1), or a host cell comprising the expression vector described in 2).

[0011] In a third aspect, the present disclosure provides a method for preparing the aforementioned RNA polymerase variant.

[0012] In a fourth aspect, the present disclosure provides a composition comprising at least one RNA polymerase variant as described in the present disclosure.

[0013] In a fifth aspect, the present disclosure provides a kit comprising at least one RNA polymerase variant as described in the present disclosure.

[0014] In a sixth aspect, the present disclosure further provides use of the above-mentioned RNA polymerase variants, compositions or kits in in vitro transcription.

[0015] In a seventh aspect, the present disclosure also provides a method for preparing RNA or capped RNA. Detailed Description of the Invention

[0016] RNA polymerase variants

[0017] The RNA polymerase variant provided by the present disclosure is a bacteriophage T7 RNA polymerase (T7 RNAP) variant, whose amino acid sequence has at least 95% sequence identity with the amino acid sequence of wild-type T7 RNAP SEQ ID NO: 1, and comprises a substitution or substitution group selected from any one of the following relative to SEQ ID NO: 1: D388 or D130 / K387.

[0018] In some embodiments, the amino acid sequence of the variant comprises, relative to SEQ ID NO: 1, a substitution or group of substitutions selected from any one of the following: D388R or D130E / K387S.

[0019] In some embodiments, the amino acid sequence of the RNA polymerase variant provided herein has at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity compared to the sequence described in any one of SEQ ID NOs: 2-3. In some embodiments, the amino acid sequence of the variant is as shown in any one of SEQ ID NOs: 2-3.

[0020] Biomaterials

[0021] The present disclosure provides polynucleotide molecules encoding the RNA polymerase variants described herein; wherein the polynucleotide molecules may contain various modifications in the coding region, as long as the amino acid sequence of the variants disclosed herein does not change with codon degeneracy or with codon preference in the organism in which the variant is expressed. In some embodiments, the polynucleotide molecules are as set forth in any one of SEQ ID NOs: 5-6.

[0022] The present disclosure provides expression vectors comprising polynucleotide molecules encoding the disclosed RNA polymerase variants. In some embodiments, the expression vectors further comprise one or more regulatory sequences, including but not limited to enhancers, promoters, leader peptide sequences, signal peptide sequences, and terminator sequences; wherein the regulatory sequences are operably linked to the polynucleotide molecules encoding the variants. In some embodiments, the expression vectors can be linear or circular DNA molecules, typically comprising elements such as multiple cloning sites, resistance genes, and replication origins.

[0023] In some embodiments, the expression vector described in the present disclosure is preferably pQE-80L.

[0024] The host cells provided by the present disclosure refer to any cells that are favorable for the expression of the variants of the present disclosure, that is, any cells that are susceptible to transformation, transfection or transduction with the expression vectors described in the present disclosure, including any progeny cells that differ from the parent cell due to mutations that occur during replication.

[0025] In some embodiments, the host cell is a prokaryotic cell, which can be selected from gram-positive bacteria or gram-negative bacteria. In some embodiments, the host cell is a gram-positive bacteria, including but not limited to: Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Ocean Bacillus, Staphylococcus, Streptococcus and Streptomyces. In some embodiments, the host cell is a gram-negative bacteria, including but not limited to: Campylobacter, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacter, Silene Bacillus, Neisseria, Pseudomonas, Salmonella and Ureaplasma. In some embodiments, the host cell is Escherichia coli.

[0026] Method for preparing variants

[0027] The present disclosure provides a method for preparing the above-mentioned RNA polymerase variant, comprising: (1) culturing the host cell described in the present disclosure under conditions suitable for expression of the variant; and (2) recovering the variant.

[0028] In some embodiments, the method for recovering the variant can be a method well known in the art, such as centrifugation, filtration, treatment with a crystallization protein precipitant (salting out), extraction, ultrasonication, ultrafiltration, dialysis, various chromatography methods such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC, and combinations thereof.

[0029] In some embodiments, the preparation method further includes a step of purifying the variant, and the purification step can be a method well known in the art, such as chromatography (such as ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, chromatofocusing, and size exclusion chromatography), isoelectric focusing electrophoresis, ammonium sulfate precipitation, SDS-PAGE, etc.

[0030] Composition

[0031] The present disclosure provides compositions comprising at least one RNA polymerase variant described herein.

[0032] The composition described in the present disclosure can be a storage composition. In some embodiments, the composition described in the present disclosure, in addition to the above-mentioned RNA polymerase variants, may optionally include: a buffer component (such as Tris base, Tris-HCl, HEPES, MOPS), a salt (such as NaCl), an enzyme inhibitor (such as EDTA), a reducing agent (such as DTT), a surfactant (such as Triton X-100), a stabilizer (such as glycerol), and the like. In some embodiments, the composition for storing RNA polymerase variants described in the present disclosure comprises: RNA polymerase variant, Tris-HCl, NaCl, EDTA, DTT, Triton X-100, and glycerol.

[0033] The composition of the present disclosure can also be an in vitro transcription reaction composition. In some embodiments, the composition, in addition to the above-mentioned RNA polymerase variants, further comprises one or more in vitro transcription reaction reagents (such as: buffer components, modified or unmodified nucleoside triphosphates, RNAse inhibitors, pyrophosphatase, magnesium ions, water, etc.). In some embodiments, the composition further comprises a DNA template. In some embodiments, the composition further comprises a cap analog.

[0034] In some embodiments, the in vitro transcription reaction composition described in the present disclosure comprises: an RNA polymerase variant, a buffer component, modified or unmodified nucleoside triphosphates, an RNase inhibitor, pyrophosphatase, magnesium ions, water, and a cap analog. In some embodiments, the in vitro transcription reaction composition described in the present disclosure comprises: an RNA polymerase variant, a buffer component, modified or unmodified nucleoside triphosphates, an RNase inhibitor, pyrophosphatase, magnesium ions, water, a cap analog, and a DNA template.

[0035] Reagent test kit

[0036] The present disclosure provides a kit comprising at least one RNA polymerase variant described in the present disclosure.

[0037] In some embodiments, the kit may further comprise one or more in vitro transcription reaction reagents (e.g., buffer components, modified or unmodified nucleoside triphosphates, RNase inhibitors, pyrophosphatase, magnesium ions, water, etc.). In some embodiments, the kit further comprises a cap analog. In some embodiments, each component of the kit (if applicable) may be provided in liquid form (e.g., in solution) or in solid form (e.g., dry powder).

[0038] The kits described herein may include one or more containers holding one or more components described herein and, optionally, instructions for use.

[0039] Application or use

[0040] The present disclosure provides applications or uses of the above-mentioned RNA polymerase variants, compositions, or kits in in vitro transcription.

[0041] The present disclosure also provides uses of the above-mentioned RNA polymerase variants, compositions, or kits in various methods, including but not limited to the preparation of RNA, preparation of RNA probes, preparation of RNA vaccines, preparation of proteins, etc.

[0042] Methods for preparing RNA

[0043] The present disclosure provides a method for producing RNA. In some embodiments, the method comprises contacting a DNA template, a modified or unmodified nucleoside triphosphate, with at least one RNA polymerase variant described herein, and incubating the mixture in an in vitro transcription reaction system to obtain a target RNA product. In some embodiments, the RNA product can be dsRNA, ssRNA, mRNA, siRNA, miRNA, piRNA, shRNA, or gRNA.

[0044] The present disclosure also provides a method for preparing capped mRNA. In some embodiments, the method comprises contacting a DNA template, modified or unmodified nucleoside triphosphates, and a cap analog with at least one RNA polymerase variant described herein, and incubating the mixture in an in vitro transcription reaction to obtain a target mRNA product.

[0045] In vitro transcription reaction systems and incubation conditions suitable for generating RNA products or mRNA products are well known in the art. Those skilled in the art can determine the appropriate pH value, reaction temperature, reaction time, salt concentration of the reaction system, or whether to add exogenous auxiliary factors, etc., taking into account the optimal activity of RNA polymerase. In some embodiments, the in vitro transcription reaction system described in the present disclosure comprises in vitro transcription reaction reagents: one or more buffer components, modified or unmodified nucleotide phosphates, RNAse inhibitors, pyrophosphatase, magnesium ions, water, etc. In some embodiments, in the incubation step described in the present disclosure, the incubation time is 20-240 min, preferably 60 min. In some embodiments, in the incubation step described in the present disclosure, the incubation temperature is 30-50°C, preferably 37°C.

[0046] In some embodiments, RNA products prepared using the methods described herein have higher yields, higher integrity, lower dsRNA impurity levels, and / or more capped mRNA products, compared to those prepared using wild-type RNA polymerase (SEQ ID NO: 1).

[0047] In some embodiments, the use of the methods described herein to prepare capped mRNA products can increase the utilization rate of the cap analog, and the capping efficiency of the obtained mRNA products can be increased by at least 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, or 20% compared to using wild-type T7 RNA polymerase.

[0048] Hat analogues

[0049] The cap analog used in the method for preparing a capped mRNA product or the cap analog in an in vitro transcription reaction composition disclosed in the present invention refers to a molecule that can be complementary to a nucleotide molecule on a DNA template at the transcription start site.

[0050] In some embodiments, the cap analog can be selected from a dinucleotide cap, a trinucleotide cap, or a tetranucleotide cap. In some embodiments, the cap analog is a trinucleotide cap, which can be selected from GAA, GAC, GAG, GAU, GCA, GCC, GCG, GCU, GGA, GGC, GGG, GGU, GUA, GUC, GUG, and GUU. In some embodiments, the trinucleotide cap is selected from m7GpppApA, m7GpppApC, m7GpppApG, m7GpppApU, m7GpppCpA, m7GpppCpC, m7GpppCpG, m7GpppCpU, m7GpppGpA, m7GpppGpC, m7GpppGpG, m7GpppGpU, m7GpppUpA, m7GpppUpC, m7GpppUpG, and m7GpppUpU. In some embodiments, the trinucleotide cap is selected from m7G3′OMepppApA, m7G3′OMepppApC, m7G3′OMepppApG, m7G3′OMepppApU, m7G3′OMepppCpA, m7G3′OMepppCpC, m7G3′OMepppCpG, m7G3′OMepppCpU, m7G3′OMepppGpA, m7G3′OMepppGpC, m7G3′OMepppGpG, m7G3′OMepppGpU, m7G3′OMepppUpA, m7G3′OMepppUpC, m7G3′OMepppUpG, and m7G3′OMepppUpU. In some embodiments, the trinucleotide cap is selected from the group consisting of m7G3′OMepppA2′OMepA, m7G3′OMepppA2′OMepC, m7G3′OMepppA2′OMepG, m7G3′OMepppA2′OMepU, m7G3′OMepppC2′OMepA, m7G3′OMepppC2′OMepC, m7G3′OMepppC2′OMepG, m7G3′OMeppp C2′OMepU, m7G3′OMepppG2′OMepA, m7G3′OMepppG2′OMepC, m7G3′OMepppG2′OMepG, m7G3′OMepppG2′OMepU, m7G3′OMepppU2′OMepA, m7G3′OMepppU2′OMepC, m7G3′OMepppU2′OMepG, and m7G3′OMepppU2′OMepU.In some embodiments, the trinucleotide cap is selected from the group consisting of m7GpppA2′OMepA, m7GpppA2′OMepC, m7GpppA2′OMepG, m7GpppA2′OMepU, m7GpppC2′OMepA, m7GpppC2′OMepC, m7GpppC2′OMepG, m7GpppC2′OMepU, m7GpppG2′OMepA, m7GpppG2′OMepC, m7GpppG2′OMepG, m7GpppG2′OMepU, m7GpppU2′OMepA, m7GpppU2′OMepC, m7GpppU2′OMepG, and m7GpppU2′OMepU.

[0051] In some embodiments, the cap analog described herein is preferably m7GpppA2′OMepG.

[0052] In vitro transcription reaction reagents

[0053] The in vitro transcription reaction reagents described in the present disclosure include buffer components, nucleoside triphosphates, RNase inhibitors, inorganic pyrophosphatase, magnesium ions, water (such as DEPC-water, RNase-free water, DNase-free water, sterile purified water, deionized water, distilled water, etc.), etc.

[0054] In some embodiments, the buffer component can be selected from one or more of: phosphate buffer, Tris buffer, MOPS buffer, HEPES buffer, citrate buffer, acetate buffer, malate buffer, MES buffer, histidine buffer, PIPES buffer, bis-tris buffer or ethanolamine buffer.

[0055] In some embodiments, the nucleoside triphosphates can be selected from modified or unmodified nucleoside triphosphates (including analogs thereof). In some embodiments, the nucleoside triphosphates can be selected from unmodified ATP, GTP, CTP, UTP. In some embodiments, the nucleoside triphosphates can be selected from modified nucleoside triphosphates, and the modification types on the nucleoside include but are not limited to m1A (N1-methyladenosine), m6A (N6-methyladenosine), m5C (5-methylcytidine), 5moU (5-methoxyuridine), ψ (pseudouridine), m1ψ (N1-methyl-pseudouridine), nucleoside triphosphates with labels (labels can be biotin, fluorescent substances, digoxin, radioactive elements, etc.).

[0056] In some embodiments, the in vitro transcription reaction reagents described in the present disclosure can be selected from any commercially available RNA in vitro transcription reagents.

[0057] Other implementation options:

[0058] 1. An RNA polymerase variant, wherein the amino acid sequence thereof comprises, relative to SEQ ID NO: 1, a substitution or substitution group selected from any one of the following: D388R or D130E / K387S.

[0059] 2. The variant as described in item 1, whose amino acid sequence is shown in any one of SEQ ID NOs: 2-3.

[0060] 3. A polynucleotide molecule encoding the RNA polymerase variant according to any one of item 1 or 2.

[0061] 4. An expression vector comprising the polynucleotide molecule as described in item 3.

[0062] 5. A host cell comprising the polynucleotide molecule as described in item 3 or a host cell comprising the expression vector as described in item 4.

[0063] 6. A composition comprising the variant according to any one of item 1 or 2.

[0064] 7. A kit comprising the variant according to any one of item 1 or 2.

[0065] 8. Use of the variant according to any one of item 1 or 2, the composition according to item 6 or the kit according to item 7 in in vitro transcription.

[0066] 9. A method for preparing RNA, comprising contacting a DNA template, modified or unmodified nucleoside triphosphates with the RNA polymerase variant described in any one of item 1 or 2, and incubating the mixture in an in vitro transcription reaction system to obtain a target RNA product.

[0067] 10. A method for preparing capped mRNA, comprising contacting a DNA template, modified or unmodified nucleoside triphosphates, a cap analog, and the RNA polymerase variant described in any one of item 1 or 2, and incubating the mixture in an in vitro transcription reaction system to obtain a target product.

[0068] The RNA polymerase variants provided in the present disclosure can significantly improve the capping rate of mRNA products in a co-transcriptional capping reaction system compared to wild-type T7 RNA polymerase, thereby avoiding the waste of raw materials and subsequent purification operations, saving the R&D and production costs of RNA drugs, and having important significance for the economical production of RNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 Schematic diagram of the construction of recombinant plasmid.

[0070] Example

[0071] The technical solutions of the present disclosure are further illustrated below with reference to specific examples. However, the following examples are merely examples of the present disclosure and do not represent or limit the scope of protection of the present disclosure. The scope of protection of the present disclosure shall be subject to the claims. In the following examples, unless otherwise specified, all reagents and consumables used were purchased from common suppliers in the field, and the experimental methods and technical means used were conventional methods and means in the field.

[0072] In the embodiments of the present disclosure, enzyme activity is defined as the amount of enzyme required to generate 1 μmol of product or convert 1 μmol of substrate within 1 minute under reaction conditions of 37°C.

[0073] Example 1 Preparation of RNA polymerase variants

[0074] A His tag protein was added to the front end of the amino acids of wild-type T7 RNA polymerase (SEQ ID NO: 1) and mutant T7 RNA polymerase (SEQ ID NO: 2-3). After DNA sequence synthesis, PCR amplification was performed (DNA sequence SEQ ID NO: 4-6). Then, the recombinant expression vector was introduced into the BseRI and HindIII restriction sites of the expression vector pQE-80L to obtain a recombinant expression vector. The recombinant Escherichia coli BL21 (DE3) was transformed into competent Escherichia coli BL21 (DE3) using the heat shock method. After screening by antibiotic resistance plate coating, the recombinant Escherichia coli BL21 (DE3) strain was obtained. After verification of successful recombination, the obtained strain was activated overnight in LB medium at 37°C and then added to fermentation broth (LB medium) and cultured to an OD of 0. The 600 value was 0.6 to 0.8, IPTG was added at a final concentration of 0.5 mol / L and culture was continued for 4 to 6 hours, and the strain was collected by centrifugation at 12000 rpm and 5°C, and washed and collected with 0.2 M PBS buffer with a pH value of 7.0 to obtain the strain; after ultrasonic disruption, affinity chromatography purification was performed to obtain the RNA polymerase stock solution.

[0075] The corresponding relationship between RNA polymerase and its variants and amino acid sequences is shown in Table 1:

[0076] Table 1

[0077]

[0078]

[0079] Example 2 Preparation of mRNA by in vitro transcription reaction

[0080] Dilute the enzyme stock solution with storage buffer (50mM Tris-HCl (25°C, pH 7.9), 100mM NaCl, 0.1mM EDTA, 2mM DTT, 0.1% Triton X-100, 50% Glycerol) to an enzyme activity of 300U / μL. Prepare the MIX solution (excluding T7 RNA polymerase) according to the reaction system (20μL) in Table 2 or Table 3 into EP tubes. Dispense the MIX solution into eight strips. Pipet 1μL of the diluted T7 RNA polymerase into each of the eight strips containing the MIX solution, mix thoroughly, and centrifuge. Place the eight strips in a PCR instrument and incubate at 37°C for 1 hour. Afterwards, add 36μL of magnetic beads, mix thoroughly, and incubate at room temperature for 2-5 minutes. Place the mixture on a magnetic rack to purify mRNA (Vazyme, Cat. No. N412). After purification, transfer the mixture to an RNase-free centrifuge tube to obtain purified mRNA.

[0081] Table 2: Reaction system ratio (D130E / K387S, D388R)

[0082]

[0083]

[0084] Table 3: Reaction ratio (D130E / K387S, D388R)

[0085]

[0086] Example 3 Capping Rate Detection

[0087] (1) The purified mRNA in Example 2 was combined with the probe. The reaction system is shown in Table 4, and the reaction conditions are shown in Table 5: Table 4: Reaction system ratio

[0088]

[0089] Table 5: Reaction conditions

[0090] temperature time 95℃ 2min 70～16℃ -0.1℃ / s, ≈180 cycles 16℃ ∞

[0091] (2) RNase H digestion: Prepare the enzyme digestion reaction system (Thermo Scientific, Catalog No. EN0201) according to Table 6, vortex thoroughly to mix, and place in a PCR instrument for reaction at 25°C for 20 min:

[0092] Table 6: Enzyme digestion reaction system ratio

[0093] Components 25 pmol volume Previous step product 21 μL RNase H Reaction Buffer (10×) 3μL RNase H (5 U / μl) 3μL <![CDATA[RNase-freeH2O]]> 3μL Overall system 30μL

[0094] (3) SA magnetic bead binding

[0095] ① Magnetic bead washing: Take 9 μL of SA magnetic beads into a centrifuge tube and place it on a magnetic rack. After the solution becomes clear, use a pipette to discard the supernatant; remove the centrifuge tube from the magnetic rack, add 200 μL of RNase-free H2O to rinse, place it on a magnetic rack, after the solution becomes clear, use a pipette to discard the supernatant, and add 200 μL of RNase-free H2O to repeat the rinse.

[0096] ② Reaction conditions: Remove the centrifuge tube from the magnetic stand, add the enzyme-digested product to the SA magnetic beads (solid), pipette 20-30 times, mix thoroughly, place on a roller and incubate at room temperature for 30 minutes to allow the magnetic beads and the enzyme-digested product to fully combine.

[0097] (4) Rinse and wash

[0098] ① Place the product from the previous step on a magnetic stand for 2-3 minutes until the solution becomes clear and discard the supernatant with a pipette;

[0099] ② Add 200 μL of rinsing solution to rinse, taking care not to blow away the magnetic beads, let it stand for 0.5 to 1 minute, and discard the supernatant with a pipette;

[0100] ③Repeat step ②;

[0101] ④ Remove the centrifuge tube from the magnetic stand, add 30uL of elution buffer, and pipette 10-20 times to mix evenly to evenly disperse the magnetic beads and fully elute;

[0102] ⑤ Place in a PCR instrument, react at 85℃ for 3 minutes, then immediately place on a magnetic stand. After the solution is clarified (0.5-1 minute), aspirate the supernatant into a new centrifuge tube. The supernatant is the desired product.

[0103] ⑥ After pretreatment, the product of the system in Table 2 was diluted 20 times with 1× Dilution Buffer, and 20-50 μL was sent to the S1 card holder of the Qsep400 ultra-micro nucleic acid protein analyzer for capillary electrophoresis to detect the capping rate. After pretreatment, the product of the system in Table 3 was sent to the Thermo scientific Vanquish Flex-Qrbitrap Exploris120 to detect the capping rate (ion mode: negative ion; scan mode: full scan; scan range: 600-3000). The capping rate calculation formula is:

[0104] mRNA capping rate (%)=(capped mRNA / (capped mRNA+uncapped mRNA))×100%.

[0105] Table 7: RNA polymerase variant capping rate (Table 2 system product) improvement effect comparison 1

[0106] Serial number name Cap rate 1 WT 87.2％ 2 D130E / K387S 90.5％ 3 D388R 100％

[0107] Table 8: Comparison of RNA polymerase variant capping efficiency (products from the system in Table 3) II

[0108] Serial number name Cap rate 1 WT 90.38％ 2 D130E / K387S 97％ 3 D388R 94.72％

[0109] The test results showed that compared with the WT group, under the systems in Table 2 and Table 3, the enzyme variants in the D130E / K387S and D388R groups could significantly increase the capping rate of mRNA products, among which the D388R in the Table 2 system could reach 100% (Table 7).

Claims

1. RNA polymerase variant, characterized in that The amino acid sequence of the variant is shown in SEQ ID NO: 2 or 3.

2. A polynucleotide molecule encoding the RNA polymerase variant according to claim 1.

3. An expression vector comprising the polynucleotide molecule as claimed in claim 2.

4. A host cell comprising the polynucleotide molecule according to claim 2 or a host cell comprising the expression vector according to claim 3.

5. A composition, characterized in that The composition comprises the variant of claim 1.

6. A kit, characterized in that The kit comprises the variant of claim 1.

7. Use of the variant according to claim 1, the composition according to claim 5 or the kit according to claim 6 in in vitro transcription.

8. A method for preparing RNA, comprising contacting a DNA template, modified or unmodified nucleoside triphosphates with the RNA polymerase variant of claim 1, and incubating the mixture in an in vitro transcription reaction system to obtain a target RNA product.

9. A method for preparing capped mRNA, comprising contacting a DNA template, modified or unmodified nucleoside triphosphates, and a cap analog with the RNA polymerase variant of claim 1, and incubating the mixture in an in vitro transcription reaction system to obtain a target product.

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