RNA polymerase variants and uses thereof

Abstract

The invention provides an RNA polymerase variant and application thereof, and relates to the technical field of biology. According to the present invention, the wild type T7 RNA polymerase is modified to obtain the T7 RNA polymerase variant, and the capping rate of the RNA product can be substantially improved compared with the wild type T7 RNA polymerase;

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to RNA polymerase variants and their application in the preparation of capped RNA. Background Technology

[0002] A complete mRNA consists of five main parts, from 5' to 3': the 5' cap structure, the 5' untranslated region, the open reading frame encoding the antigen, the 3' untranslated region, and the Poly A tail. In vivo, the 5' cap structure is formed through the catalysis of RNA triphosphatase, mRNA guanylate transferase, mRNA methyltransferase, and mRNA nucleoside 2'-methyltransferase, and is a special structure in eukaryotic post-transcriptional modification that forms mature mRNA. In vitro transcription (IVT) capping can synthesize the cap structure through an enzyme-linked reaction similar to that in vivo, also known as enzymatic capping. It typically uses enzymes such as VCE and FCE as capping catalysts, with the addition of mRNA nucleoside 2'-methyltransferase to initiate the capping reaction. Enzymatic capping is affected by the efficiency of its enzyme-linked catalysis, resulting in a relatively low capping rate. Co-transcriptional capping using cap analogs is currently the mainstream method, achieving a significantly higher capping rate than enzymatic capping.

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

[0004] Formula I Formula II Third-generation capping analogs, such as CleanCap AG, significantly improve the capping rate of their products compared to second-generation capping analogs (capping rate exceeding 90%). However, when using wild-type T7 RNA polymerase as a catalyst, due to steric hindrance and other factors, it is difficult to achieve a 100% capping rate. The generation of uncapped RNA products not only wastes raw materials but also requires subsequent column purification, increasing production costs. Therefore, further improving the mRNA capping rate during co-transcriptional capping is beneficial for the economical production of mRNA. Summary of the Invention

[0005] In a first aspect, this disclosure provides a class of RNA polymerase variants having an amino acid sequence that is at least 95% identical to SEQ ID NO: 1, and containing, relative to SEQ ID NO: 1, at least one mutation selected from the following amino acid sites: K642, S628, T630, G777.

[0006] Secondly, this application provides a class of biological materials selected from one or more of the following: 1) The polynucleotide molecule encoding the above variants; 2) Expression vectors containing polynucleotide molecules as described in 1); 3) Host cells containing the polynucleotide molecules described in 1), or host cells containing the expression vectors described in 2).

[0007] Thirdly, this application provides a method for preparing the aforementioned RNA polymerase variant.

[0008] Fourthly, this application provides a composition comprising at least one RNA polymerase variant as described in this application.

[0009] Fifthly, this application provides a kit comprising at least one RNA polymerase variant as described in this application.

[0010] Sixthly, this application provides the application of the above-mentioned RNA polymerase variant in the in vitro transcription preparation of RNA.

[0011] Seventhly, this application also provides a method for preparing RNA. Invention Details RNA polymerase variants The RNA polymerase variant provided in this application has an amino acid sequence that is at least 95% identical to the amino acid sequence of wild-type T7 RNA polymerase SEQ ID NO: 1, and contains, relative to SEQ ID NO: 1, a substitution or group of substitutions selected from any of the following: K642R+K387S, S628P, T630A or G777S.

[0013] In some embodiments, the variant has at least 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:1.

[0014] In some embodiments, the amino acid sequence of the RNA polymerase variant provided in this application has at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity compared to any of the sequences described in SEQ ID NO: 2, 3, 5, 6. In some embodiments, the amino acid sequence of the variant is as shown in any of SEQ ID NO: 2, 3, 5, 6.

[0015] biomaterials This application provides a polynucleotide encoding the RNA polymerase variant described in this application. Due to codon degeneracy or codon bias of the host cell expressing the polypeptide, the polynucleotide sequence can be any polynucleotide sequence encoding the variant without altering the amino acid sequence. In some embodiments, the polynucleotide sequence encoding the RNA polymerase variant of this application may be selected from SEQ ID NO: 8, 9, 11, 12.

[0016] The expression vector provided in this application comprises a multinucleotide molecule encoding the RNA polymerase variant described in this application. In some embodiments, the expression vector further comprises 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 operatively linked to the multinucleotide molecule encoding the variant.

[0017] In some embodiments, the expression vector may be a linear or circular DNA molecule, typically containing elements such as a multiple cloning site, an antibiotic resistance gene, and a replication initiation site. In some embodiments, the expression vector described in this application is preferably pQE-80L.

[0018] The host cell provided in this application refers to any cell that is favorable for the expression of the RNA polymerase variants described in this application, that is, any cell that is susceptible after being transformed, transfected or transduced with the expression vectors described in this application, and includes any daughter cells that are different from the parent cells due to mutations that occur during replication.

[0019] In some embodiments, the host cell is a prokaryotic cell, selectable from Gram-positive or Gram-negative bacteria. In some embodiments, the host cell is a Gram-positive bacterium, including but not limited to: *Bacillus*, *Clostridium*, *Enterococcus*, *Bacillus aeruginosa*, *Lactobacillus*, *Lactococcus*, *Bacillus cereus*, *Staphylococcus*, *Streptococcus*, and *Streptomyces*. In some embodiments, the host cell is a Gram-negative bacterium, including but not limited to: *Campylobacter*, *Escherichia coli*, *Flavobacterium*, *Fusobacterium*, *Helicobacter*, *Selenobacter*, *Neisseria*, *Pseudomonas*, *Salmonella*, and *Ureaplasma*. In some embodiments, the host cell is *Escherichia coli*.

[0020] Preparation methods of RNA polymerase variants This application provides a method for preparing the RNA polymerase variant described in this application, comprising: 1) culturing the host cell described in this application under suitable variant expression conditions; and 2) recovering the variant.

[0021] In some embodiments, the method for recovering variants can be a method known in the art, such as centrifugation, filtration, treatment with a crystalline protein precipitant (salting out), extraction, ultrasonic disruption, ultrafiltration, dialysis, various chromatographic methods such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC, and combinations thereof.

[0022] In some embodiments, the preparation method further includes a purification step of the variant, which can be a method known in the art, such as chromatography (e.g., ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, chromatographic focusing, and size exclusion chromatography), isoelectric point focusing electrophoresis, ammonium sulfate precipitation, SDS-PAGE, etc.

[0023] Composition The composition provided in this application comprises at least one RNA polymerase variant described in this application.

[0024] The compositions described in this application may be storage compositions. In some embodiments, in addition to the RNA polymerase variants described above, the compositions described in this application may optionally contain: buffer components (such as Tris base, Tris-HCl, HEPES, MOPS), salts (such as NaCl), enzyme inhibitors (such as EDTA), reducing agents (such as DTT), surfactants (such as Triton X-100), stabilizers (such as glycerol), and other components. In some embodiments, the compositions for storing RNA polymerase variants described in this application contain: RNA polymerase variant, Tris-HCl, NaCl, EDTA, DTT, Triton X-100, and glycerol.

[0025] The compositions of this application may also be in vitro transcription reaction compositions. In some embodiments, the compositions, in addition to the RNA polymerase variants described above, 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 compositions further comprise a DNA template. In some embodiments, the compositions further comprise a cap analogue.

[0026] In some embodiments, the in vitro transcription reaction composition of this application comprises: an RNA polymerase variant, a buffer component, a modified or unmodified nucleoside triphosphate, an RNase inhibitor, a pyrophosphatase, magnesium ions, water, and a cap analog. In some embodiments, the in vitro transcription reaction composition of this application comprises: an RNA polymerase variant, a buffer component, a modified or unmodified nucleoside triphosphate, an RNase inhibitor, a pyrophosphatase, magnesium ions, water, a cap analog, and a DNA template.

[0027] Reagent test kit The kit provided in this application contains at least one RNA polymerase variant described in this application.

[0028] In some embodiments, the kit may also contain 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 also contains a cap analogue. In some embodiments, each component in the kit (if applicable) may be provided in liquid form (e.g., in solution) or in solid form (e.g., dry powder).

[0029] The kit described in this application may include one or more containers containing one or more components described in this application and optional instructions for use.

[0030] Applications or uses This disclosure provides for the use or application of the above-described RNA polymerase variants, compositions, or kits in in vitro transcription.

[0031] This disclosure also provides the use of the above-mentioned RNA polymerase variants, compositions, or kits in a variety of methods, including but not limited to RNA preparation, RNA probe preparation, RNA vaccine preparation, and protein preparation.

[0032] Preparation method This application provides a method for preparing RNA, comprising contacting a DNA template, a modified or unmodified nucleoside triphosphate, with at least one RNA polymerase variant described in this application, incubating in an in vitro transcription reaction system to obtain a target RNA product. In some embodiments, the RNA product may be dsRNA, ssRNA, mRNA, siRNA, miRNA, piRNA, shRNA, or gRNA.

[0033] This application also provides a method for preparing capped mRNA, the method comprising contacting a DNA template, a modified or unmodified nucleoside triphosphate, a capping analogue, and at least one RNA polymerase variant described in this application, incubating in an in vitro transcription reaction to obtain the target mRNA product.

[0034] Suitable in vitro transcription reaction systems and incubation conditions for generating RNA or mRNA products are well known in the art. Those skilled in the art can determine suitable reaction system pH, reaction temperature, reaction time, salt concentration, or whether to add exogenous cofactors, taking into account the optimal activity of RNA polymerase. In some embodiments, the in vitro transcription reaction system described in this application includes 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, the incubation time in the incubation step described in this application is 20-240 min, preferably 60 min. In some embodiments, the incubation temperature in the incubation step described in this application is 30-50°C, preferably 37°C.

[0035] In some embodiments, the RNA product prepared using the method described in this application has higher yield, and / or higher integrity, and / or less dsRNA impurity content, and / or more capped mRNA product compared to that prepared using wild-type RNA polymerase (SEQ ID NO: 1).

[0036] In some embodiments, the preparation of capped mRNA products using the methods described in this application can improve the utilization rate of capped analogs. Compared with the use of wild-type T7 RNA polymerase, the capping rate 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%.

[0037] Hat-like items The cap analogues used in the methods for preparing capped mRNA products or in in vitro transcription reaction compositions described in this application refer to molecules that are complementary to nucleotide molecules on the DNA template at the transcription start site.

[0038] In some embodiments, the cap analogue may be selected from a dinucleotide cap, a trinucleotide cap, or a tetranucleotide cap. In some embodiments, the cap analogue is a trinucleotide cap, 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, m7GppppUpC, m7GppppUpG, and m7GppppUpU. 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 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 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.

[0039] In some embodiments, the hat analogue described in this application is preferably m7GpppA2′OMepG.

[0040] In vitro transcription reaction reagents The in vitro transcription reaction reagent described in this application includes buffer components, nucleoside triphosphates, RNase inhibitors, inorganic pyrophosphatase, magnesium ions, and water (such as DEPC-water, RNase-free water, DNase-free water, sterile purified water, deionized water, distilled water, etc.).

[0041] In some embodiments, the buffering component may be selected from one or more of the following: 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.

[0042] In some embodiments, the nucleoside triphosphate may be selected from modified or unmodified nucleoside triphosphates (including analogues thereof). In some embodiments, the nucleoside triphosphate may be selected from unmodified ATP, GTP, CTP, or UTP. In some embodiments, the nucleoside triphosphate may 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), and labeled nucleoside triphosphates (the label may be biotin, fluorescent substances, digoxigenin, radioactive elements, etc.).

[0043] In some embodiments, the in vitro transcription reaction reagent described in this application may be selected from any commercially available RNA in vitro transcription reagent.

[0044] Other implementation plans 1. An RNA polymerase variant, wherein the amino acid sequence of the variant includes any mutation selected from the following sites relative to SEQ ID NO: 1: K642R+K387S, S628P, T630A, G777S.

[0045] 2. The variant described in item 1, having an amino acid sequence as shown in any of SEQ ID NO: 2, 3, 5, or 6.

[0046] 3. Biological materials, selected from one or more of the following: 1) A polynucleotide molecule encoding an RNA polymerase variant as described in any of items 1-2; 2) Expression vectors containing polynucleotide molecules as described in 1); 3) A host cell containing a polynucleotide molecule as described in 1), or a host cell containing an expression vector as described in 2).

[0047] 4. A composition comprising a variant as described in either item 1 or item 2.

[0048] 5. A kit containing a variant as described in either item 1 or item 2.

[0049] 6. The variants described in any of items 1 or 2, the compositions described in item 4, or the kits described in item 5.

[0050] 7. A method for preparing RNA, characterized in that the method comprises contacting a DNA template, a modified or unmodified nucleoside triphosphate, and any RNA polymerase variant described in item 1 or 2, incubating in an in vitro transcription reaction system to obtain a target RNA product.

[0051] 8. A method for preparing capped mRNA, comprising contacting a DNA template, a modified or unmodified nucleoside triphosphate, a cap analogue, with any of the RNA polymerase variants described in item 1 or 2, incubating in an in vitro transcription reaction system to obtain the target product.

[0052] 9. As described in item 7, the in vitro transcription system also includes a cap analogue.

[0053] 10. The use of RNA polymerase variants as described in item 1 or 2 in the synthesis of RNA drugs.

[0054] Beneficial effects The RNA polymerase variant provided in this application can significantly improve the capping rate of mRNA products in the co-transcriptional capping reaction system compared with wild-type T7 RNA polymerase. This can avoid the waste of raw materials and subsequent purification operations, and save the research and development and production costs of RNA drugs. It is of great significance for the economical production of RNA. Attached Figure Description

[0055] Figure 1 This is a schematic diagram illustrating the construction of recombinant plasmids; Figure 2 The effect of RNA polymerase variants on the integrity of saRNA products. Detailed Implementation

[0056] The technical solution of this application will be further described below with reference to specific embodiments. However, the following embodiments are merely examples of this application and do not represent or limit the scope of protection of this application. The scope of protection of this application is determined by the claims. In the following embodiments, unless otherwise specified, the reagents and consumables used are purchased from ordinary suppliers in the art, and the experimental methods and techniques used are conventional methods and techniques in the art.

[0057] In this application embodiment, 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 at 37°C.

[0058] Example 1: Preparation of RNA polymerase variants A His-tagged protein was added to the front end of the amino acids (SEQ ID NO: 2-6) of wild-type T7 RNA polymerase (SEQ ID NO: 1) and mutant T7 RNA polymerase. After DNA sequence synthesis, PCR amplification was performed (DNA sequence SEQ ID NO: 7-12). The DNA was then introduced into the BseRI and HindIII restriction sites of the expression vector pQE-80L to obtain the recombinant expression vector, which was then transformed using the heat shock method. Escherichia coli BL21(DE3) competent cells were screened for antibiotic resistance using plate coating to obtain recombinant cells. Escherichia coli BL21(DE3) strain; after successful recombination, the obtained strain was activated overnight at 37℃ in LB medium and then added to fermentation broth (LB medium). The strain was cultured until the OD600 value reached 0.6–0.8, then IPTG was added to a final concentration of 0.5 mol / L and cultured for another 7–12 h. The strain was collected by centrifugation at 12000 rpm at 5℃, washed with 0.2 M PBS buffer (pH 7.0), and the strain was obtained. After sonication, affinity chromatography was performed to purify the RNA polymerase stock solution. The correspondence between RNA polymerase and its variants and amino acid sequences is shown in Tables 1-1 and 1-2. Table 1-1: Correspondence between RNA polymerase and amino acid sequence

[0059] Table 1-2: Correspondence between RNA polymerase and amino acid sequence

[0060]

[0061] Example 2: Validation of in vitro transcription reaction The enzyme stock solution was diluted with storage buffer (50 mM Tris-HCl (25℃, pH 7.9), 100 mM NaCl, 0.1 mM EDTA, 2 mM DTT, 0.1% Triton X-100, 50% Glycerol) to an enzyme activity of 400 U / μL. A MIX solution was prepared according to the reaction system (20 μL) in Table 1 and transferred to an EP tube. The MIX solution was then transferred to an octet, mixed, and centrifuged. The octet was placed on a PCR instrument and reacted at 37℃ for 1 h. 36 μL of magnetic beads (Vazyme, catalog number: N412) were added, mixed, and incubated at room temperature for 2–5 min. The mixture was then placed on a magnetic rack to purify the mRNA. After purification, the mRNA was transferred to an RNase-free centrifuge tube to obtain the purified mRNA.

[0062] Prepare the MIX solution (excluding T7 RNA polymerase) in EP tubes according to the reaction system (20 μL) in Table 2; aliquot the MIX solution into octuplets, and then add 1 μL of diluted T7 RNA polymerase to each octuplet containing the MIX solution, mix well, and centrifuge; place the octuplets on a PCR instrument and react at 37℃ for 1.5 h, then add 36 μL of magnetic beads and mix well, incubate at room temperature for 2-5 min; place the mixture on a magnetic rack to purify the mRNA (Vazyme, catalog number: N412), and transfer the purified mRNA to an RNase-free centrifuge tube.

[0063] Table 2: Reaction System Proportions

[0064] Example 3: Capping Rate Detection (1) The purified mRNA from Example 2 was bound to the probe. The reaction system is shown in Table 3, and the reaction conditions are shown in Table 4. Table 3: Reaction System Proportions

[0065] Table 4: Reaction Conditions

[0066] (2) RNase H digestion: Prepare the digestion reaction system according to Table 5 (Thermo Scientific, catalog number: EN0201), vortex thoroughly to mix well, and place in a PCR instrument. Incubate at 25°C for 20 min. Table 5: Composition of the enzyme digestion reaction system

[0067] (3) SA magnetic bead bonding ① Magnetic bead cleaning: Take 9 μL of SA magnetic beads into a centrifuge tube, place it on a magnetic rack, and after the solution becomes clear, use a pipette to remove 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, and after the solution becomes clear, use a pipette to remove the supernatant, and then add 200 μL of RNase-free H2O to repeat the rinsing once more.

[0068] ②Reaction conditions: Remove the centrifuge tube from the magnetic rack, add the enzyme digestion product to the SA magnetic beads (solid), pipette and mix thoroughly 20-30 times, place on a tumbler and incubate at room temperature for 30 minutes to allow the magnetic beads to fully combine with the enzyme digestion product.

[0069] (4) Rinsing and elution ① Place the product from the previous step on a magnetic rack for 2-3 minutes until the solution becomes clear, then use a pipette to remove the supernatant; ② Add 200 μL of rinsing solution to rinse, being careful not to blow away the magnetic beads, let stand for 0.5~1 min, and then use a pipette to remove the supernatant; ③ Repeat step ②; ④ Remove the centrifuge tube from the magnetic rack, add 30 μL of elution buffer, and mix thoroughly by pipetting 10-20 times to ensure even dispersion of the magnetic beads and complete elution; ⑤ Place it in a PCR instrument and react at 85℃ for 3 min. Immediately after that, place it on a magnetic rack. After the solution becomes clear (0.5~1 min), aspirate the supernatant into a new centrifuge tube. The supernatant is the desired product. ⑥ The product from step ⑤ was sent to a Thermo Scientific Vanquish Flex-QrbitrapExploris 120 microscope for capping rate detection (ion mode: negative ion; scan mode: full scan; scan range: 600-3000). The capping rate was calculated using the following formula: mRNA capping rate (%) = (capped mRNA / (capped mRNA + uncapped mRNA)) ×100%.

[0070] Table 6: Comparison of the effects of RNA polymerase variants on improving capping rate

[0071] The test results showed that, compared with the WT group, all mutants significantly increased the capping rate of mRNA products, with mutant T630A reaching 99.0%.

Claims

1. An RNA polymerase variant, characterized in that, The amino acid sequence of the variant has at least 95% sequence identity with SEQ ID NO: 1, and, relative to SEQ ID NO: 1, contains a substitution or group of substitutions selected from any of the following: K642R+K387S, S628P, T630A or G777S.

2. The variant of claim 1, wherein the amino acid sequence of the variant is shown in any one of SEQ ID NO: 2, 3, 5, 6.

3. A biomaterial, characterized in that, The biomaterial is selected from one or more of the following: 1) A multinucleotide molecule encoding an RNA polymerase variant as described in claim 1 or 2; 2) Expression vectors containing polynucleotide molecules as described in 1); 3) A host cell containing a polynucleotide molecule as described in 1), or a host cell containing an expression vector as described in 2).

4. A composition, characterized in that, Includes the variants as described in claim 1 or 2.

5. A reagent kit, characterized in that, Includes the variants as described in claim 1 or 2.

6. The use of the variants of claim 1 or 2, the composition of claim 4, or the kit of claim 5 in the in vitro transcription preparation of RNA.

7. A method for preparing RNA, characterized in that, The method involves contacting a DNA template, a modified or unmodified nucleoside triphosphate, and the RNA polymerase variant described in claim 1 or 2, incubating them in an in vitro transcription reaction system to obtain the target RNA product.

8. A method for preparing capped mRNA, comprising contacting a DNA template, a modified or unmodified nucleoside triphosphate, a capping analogue with the RNA polymerase variant of claim 1 or 2, incubating in an in vitro transcription reaction system to obtain the target product.

9. The method of claim 7, wherein the in vitro transcription system further comprises a cap analogue.

10. The use of the RNA polymerase variant as described in claim 1 or 2 in RNA drug synthesis.

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