RNA polymerase mutants and uses thereof

Abstract

To provide a T7RNA polymerase mutant capable of improving the utilization of a cap analog without requiring the substitution of a wild type promoter, to provide a method for producing the same, and to provide the use of the same in RNA synthesis.SOLUTION: Provided are T7RNA polymerase mutants comprising at least one substitution or deletion at an amino-acid site, wherein the mutants have improved catalytic activities as compared to a wild-type T7RNA polymerase and are capable of increasing capping rates of mRNA products in an in vitro transcription (co-transcriptional capping) process. In addition, a method for producing RNA using this mutant is provided, and more capped mRNA can be obtained by producing an RNA molecule using this method.SELECTED DRAWING: Figure 4

Description

[Technical Field]

[0001] This application relates to the field of biotechnology, and in particular to RNA polymerase mutants, methods for their production, and their use in RNA synthesis. [Background technology]

[0002] The 5' end of intact eukaryotic mRNA contains a 7-methylguanosine (m7G) cap, which is formed in vivo catalyzed by RNA triphosphatase, mRNA guanosine transferase, mRNA methyltransferase, and mRNA nucleoside 2'-O-methyltransferase. The 5' cap structure is involved in processes such as preventing mRNA degradation by exonucleases, reducing mRNA immunogenicity, regulating mRNA half-life, and regulating translation. During the in vitro transcription (IVT) process for producing mRNA, researchers often add cap analogs during production to mimic the 5' cap structure of eukaryotic mRNA.

[0003] The cap structure at the 5' end of mRNA has a significant impact on mRNA stability, translation efficiency, and immunogenicity. As research progresses, cap analogs are becoming increasingly important. Therefore, researchers continue to develop new cap analogs for use in mRNA vaccines and therapeutic RNAs. Currently, cap analogs have evolved to the third generation. First-generation cap analogs have two free 3'-OH groups, which causes reverse incorporation. Therefore, first-generation cap analogs are rarely seen on the market. The most commonly found on the market are the second-generation ARCA cap analog (Formula 1) and the third-generation cap analog (Formula 2). Their main structural formulas are as follows: [ka] [ka]

[0004] ARCA cap analogs are modified cap analogs in which the 3'-OH group near m7G is replaced with -OCH3. This substitution allows RNA polymerase to initiate transcription using only the remaining hydroxyl group, forcing the ARCA cap to be incorporated in the forward direction. Third-generation cap analogs, such as CleanCap AG, can form the Cap1 structure and have significantly improved capping rates compared to second-generation cap analogs. However, this requires replacing the wild-type promoter from the original 5'-TAATACGACTCACTATAGG-3' to 5'-TAATACGACTCACTATAAG-3'. Whether promoter replacement causes safety issues or the generation of new impurities remains to be verified. When using a wild-type promoter, capping rates remain low, and unsuccessfully capped RNA products not only waste raw material but also require subsequent removal by column purification, increasing production costs. Therefore, modifying T7 RNA polymerase can improve cap analog utilization without the need for wild-type promoter replacement, which is of great significance for economical mRNA production and drug safety. Summary of the Invention [Means for solving the problem]

[0005] In a first aspect, the present application provides an RNA polymerase mutant, the amino acid sequence of which comprises at least one amino acid mutation at a position selected from R386, R34, K172, Y178, D388, Q435, N437, or D438 relative to SEQ ID NO:1, and the type of mutation may be selected from a substitution or a deletion.

[0006] In a second aspect, the present application provides a method for manufacturing a semiconductor device comprising: 1) a polynucleotide molecule encoding an RNA polymerase mutant; 2) an expression vector comprising the polynucleotide molecule according to 1); 3) One or more biological materials selected from a host cell containing the polynucleotide molecule according to 1) or a host cell containing the expression vector according to 2) are provided.

[0007] In a third aspect, the present application provides a method for producing the above-mentioned RNA polymerase mutant.

[0008] In a fourth aspect, the present application provides a composition comprising at least one of the RNA polymerase mutants described herein.

[0009] In a fifth aspect, the present application provides a kit comprising at least one of the RNA polymerase mutants described herein.

[0010] In a sixth aspect, the present application further provides the use of the above-mentioned RNA polymerase mutant, composition, or kit in in vitro transcription.

[0011] In a seventh aspect, the present application further provides a method of producing RNA or capped RNA.

[0012] Detailed Description of the Invention RNA polymerase mutants The RNA polymerase mutant provided herein is a bacteriophage T7 RNA polymerase (T7 RNAP) mutant, and the amino acid sequence of the mutant contains at least one mutation at an amino acid position selected from R386, R34, K172, Y178, D388, Q435, N437, and D438 relative to SEQ ID NO: 1, and the type of mutation is selected from substitutions and deletions.

[0013] In some embodiments, the substitution at position R34 of the mutant is A.

[0014] In some embodiments, the type of mutation at the K172 site of the mutant is a deletion.

[0015] In some embodiments, the substitution at the Y178 position of the mutant may be selected from H or D.

[0016] In some embodiments, the substitution at the R386 position of the mutant may be selected from C, W, M, A, I, or F.

[0017] In some embodiments, the substitution at position D388 of the mutant is K.

[0018] In some embodiments, the substitution at position Q435 of the mutant may be selected from A, H, or T.

[0019] In some embodiments, the substitution at the N437 position of the mutant may be selected from F or T.

[0020] In some embodiments, the substitution at the D438 site of the mutant may be selected from T, L, I, P, or V.

[0021] In some embodiments, the amino acid sequence of the variant comprises any of the following substitutions or groups of substitutions compared to SEQ ID NO:1: R386W, R386C, D388K, Q435H, Q435T, Q435A, N437F, N437T, D438T, D438L, D438I, D438P, D438V, R386A+R34A, R386M+R34A, R386M+Y178H, R386W+N437T, R386I+del-K172, R386F+Y178D, R386C+D438P, or D388K+D438P.

[0022] In some embodiments, the amino acid sequence of the RNA polymerase mutants provided herein has at least 97%, at least 98%, or at least 99% sequence identity to the sequence set forth in any one of SEQ ID NOs:2-22. In some embodiments, the amino acid sequence of the mutant is as set forth in any one of SEQ ID NOs:2-22.

[0023] Polynucleotide molecules The polynucleotide molecules provided herein encode any of the RNA polymerase mutants described herein. In some embodiments, the polynucleotide molecules are as set forth in any of SEQ ID NOs: 23-44.

[0024] The polynucleotide molecules described herein may have various modifications in the coding regions so long as the variant amino acid sequence of the present application does not change due to codon degeneracy or preferred codons in the organism in which the variant is expressed.

[0025] Expression vector The expression vectors provided herein are linear or circular DNA molecules that typically contain elements such as a multiple cloning site, resistance genes, and an origin of replication. In some embodiments, the expression vector described herein is pQE-80L.

[0026] In some embodiments, the vectors described herein comprise a polynucleotide molecule encoding a mutant RNA polymerase described herein, and in some embodiments, the vectors further comprise one or more regulatory sequences (such as an enhancer sequence, a promoter sequence, and a terminator sequence) operably linked to the polynucleotide molecule encoding the mutant.

[0027] host cell The host cells provided herein may be any cell useful for expression of the variants of the present application, i.e., any cell that is susceptible following transformation, transfection, or transduction with an expression vector of the present application, including any cellular progeny that differ from the parent cell by mutations that occur during replication.

[0028] In some embodiments, the host cells described herein comprise the polynucleotide molecules or expression vectors described above.

[0029] In some embodiments, the host cell is a prokaryotic cell and may be selected 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, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, 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, Thuringia, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

[0030] Method for producing mutants The present application provides a method for producing the above-mentioned RNA polymerase mutant, comprising the steps of (1) culturing the host cell described herein under conditions suitable for expression of the mutant, and (2) recovering the mutant.

[0031] In some embodiments, methods for recovering the variants may be methods well known in the art, such as centrifugation, filtration, treatment with crystallized protein precipitants (salting out), extraction, sonication, ultrafiltration, dialysis, various chromatographic methods such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC, and combinations of the above methods.

[0032] In some embodiments, the production method further comprises a step of purifying the variant, which may be a method well known in the art, such as chromatography (e.g., ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography), ammonium sulfate precipitation, or the like.

[0033] composition The compositions provided herein comprise at least one of the RNA polymerase mutants described herein.

[0034] The compositions described herein may be compositions for preserving RNA polymerase mutants. In some embodiments, the compositions described herein may optionally contain, in addition to the RNA polymerase mutant, components such as buffer components (e.g., Tris base, Tris-HCl, HEPES, MOPS), salts (e.g., NaCl), enzyme inhibitors (e.g., EDTA), reducing agents (e.g., DTT), detergents (e.g., Triton X-100), and stabilizers (e.g., glycerol). In some embodiments, the compositions described herein for preserving RNA polymerase mutants contain the RNA polymerase mutant, Tris-HCl, NaCl, EDTA, DTT, Triton X-100, and glycerol.

[0035] The composition of the present application may also be an in vitro transcription reaction composition. In some embodiments, the composition further comprises 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 addition to the RNA polymerase mutant described above. In some embodiments, the composition further comprises a DNA template. In some embodiments, the composition further comprises a cap analog.

[0036] In some embodiments, the in vitro transcription reaction compositions described herein comprise an RNA polymerase mutant, buffer components, modified or unmodified nucleoside triphosphates, an RNase inhibitor, pyrophosphatase, magnesium ions, water, and a cap analog. In some embodiments, the in vitro transcription reaction compositions described herein comprise an RNA polymerase mutant, buffer components, modified or unmodified nucleoside triphosphates, an RNase inhibitor, pyrophosphatase, magnesium ions, water, a cap analog, and a DNA template.

[0037] kit The kits provided herein include at least one of the RNA polymerase mutants described herein.

[0038] 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 in the kit (if applicable) can be provided in liquid form (e.g., in solution) or solid form (e.g., as a dry powder).

[0039] Kits described herein can include one or more containers containing one or more components described herein, and optional instructions for use.

[0040] Use or application The present application provides the use of the above-mentioned RNA polymerase mutant, composition, or kit in in vitro transcription.

[0041] The present application further provides uses of the above-described RNA polymerase mutants, compositions, or kits in various methods, including, but not limited to, producing RNA, producing RNA probes, producing RNA vaccines, and producing proteins.

[0042] Methods for Producing RNA The present application provides methods for producing RNA. In some embodiments, the methods include contacting a DNA template, modified or unmodified nucleoside triphosphates with at least one RNA polymerase mutant described herein, and 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.

[0043] The present application further provides methods for producing capped mRNA, in some embodiments, the methods comprising contacting a DNA template, modified or unmodified nucleoside triphosphates, and a cap analog with at least one RNA polymerase mutant described herein and incubating in an in vitro transcription reaction system to obtain a target product.

[0044] In vitro transcription reaction systems and incubation conditions suitable for producing RNA products or capped 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, or whether or not to add exogenous cofactors, taking into account the optimal activity of the RNA polymerase. In some embodiments, the in vitro transcription reaction system described herein includes in vitro transcription reaction reagents, i.e., one or more buffer components, modified or unmodified nucleoside triphosphates, RNase inhibitors, pyrophosphatase, magnesium ions, water, etc. In some embodiments, the incubation temperature in the incubation step described herein is 30°C to 50°C, preferably 37°C. In some embodiments, the incubation time in the incubation step described herein is 20 to 240 minutes, preferably 60 minutes.

[0045] In some embodiments, the RNA products or capped mRNA products produced using the methods described herein have higher yields and / or higher integrity and / or lower dsRNA impurity content and / or more capped mRNA products than those produced using wild-type RNA polymerases.

[0046] In some embodiments, capped mRNA products produced using the methods described herein can have improved cap analog utilization compared to those produced using wild-type RNA polymerases, and the capping rate of the resulting mRNA products can be increased by at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100%.

[0047] Cap analogues The cap analogs used in the methods for producing capped mRNA products or in the in vitro transcription reaction compositions described herein are molecules that are complementary to the nucleotides on a DNA template at the transcription start site.

[0048] In some embodiments, the cap analog may be selected from a dinucleotide cap, a trinucleotide cap, or a tetranucleotide cap. In some embodiments, the cap analog is a trinucleotide cap and may 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 may be selected from m7GpppApA, m7GpppApC, m7GpppApG, m7GpppApU, m7GpppCpA, m7GpppCpC, m7GppppCpG, m7GppppCpU, m7GppppGpA, m7GppppGpC, m7GppppGpG, m7GppppGpU, m7GpppUpA, m7GppppUpC, m7GpppUpG, and m7GpppUpU. In some embodiments, the trinucleotide cap may be 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 m7G3'OMepppA2'OMepA, m7G3'OMepppA2'OMepC, m7G3'OMepppA2'OMepG, m7G3'OMepppA2'OMepU, m7G3'OMepppC2'OMepA, m7G3'OMepppC2'OMepC, m7G3'OMepppC2'OMepG, m7G3'OMepppC 2'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 may be 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.

[0049] In some embodiments, the cap analog described herein is preferably m7GpppA2'OMepG.

[0050] In some embodiments, when a trinucleotide cap GAG (e.g., m7GpppA2'OMepG) is used to produce a capped mRNA product, or in an in vitro transcription reaction composition that includes such a cap analog, the first nucleotide at the +1 position of a DNA template molecule (sense strand) is G and the second nucleotide at the +2 position is G. In some embodiments, the nucleotide residue in the m7GpppA2'OMepG cap analog can complementarily pair with the +1 position of the antisense strand of the DNA template molecule (see Figure 2).

[0051] In vitro transcription reaction reagents The in vitro transcription reaction reagents described herein include buffer components, nucleoside triphosphates, RNase inhibitors, inorganic pyrophosphatase, magnesium ions, water (e.g., DEPC-water, RNase-free water, DNase-free water, sterile purified water, deionized water, distilled water, etc.), and the like.

[0052] In some embodiments, the buffer component may be selected from one or more of a phosphate buffer, a Tris buffer, a MOPS buffer, a HEPES buffer, a citrate buffer, an acetate buffer, a malate buffer, a MES buffer, a histidine buffer, a PIPES buffer, a bis-tris buffer, and an ethanolamine buffer.

[0053] In some embodiments, the nucleoside triphosphate may be selected from modified or unmodified nucleoside triphosphates (including analogs thereof). In some embodiments, the nucleoside triphosphate may be selected from unmodified ATP, GTP, CTP, and UTP. In some embodiments, the nucleoside triphosphate may be selected from modified nucleoside triphosphates, where the type of modification on the nucleoside includes, but is not limited to, m1A (N1-methyladenosine), m6A (N6-methyladenosine), m5C (5-methylcytidine), 5moU (5-methoxyuridine), ψ (pseudouridine), m1ψ (N1-methylpseudouridine), and nucleoside triphosphates having a marker (the marker may be biotin, a fluorescent substance, digoxin, a radioactive element, etc.).

[0054] In some embodiments, the in vitro transcription reaction reagents described herein may be selected from any commercially available RNA in vitro transcription reagent.

[0055] Other embodiments: 1. An RNA polymerase mutant, the amino acid sequence of which comprises at least one substitution or deletion at an amino acid position selected from R386, R34, K172, Y178, D388, Q435, N437, and D438 relative to SEQ ID NO:1. 2. The mutant according to paragraph 1, (1) the substitution at position R386 is selected from C or W; (2) the substitution at position D388 is K; (3) the substitution at position Q435 is selected from A, H, and T; (4) the substitution at position N437 is selected from F and T; (5) the substitution at position D438 is selected from T, L, I, P, and V; (6) The substitution at position R34 is selected from A; (7) the mutation at position K172 is a deletion; (8) The substitution at position Y178 is selected from H and D. 3. The variant according to paragraph 1, wherein the amino acid sequence comprises a mutation at any site selected from R386W, R386C, D388K, Q435A, Q435H, Q435T, N437F, N437T, D438T, D438L, D438I, D438P, D438V, R386A+R34A, R386M+R34A, R386M+Y178H, R386W+N437T, R386I+del-K172, R386F+Y178D, D388K+D438P, and R386C+D438P relative to SEQ ID NO:1. 4. The mutant according to item 1, wherein the amino acid sequence of the mutant is set forth in any one of SEQ ID NOs: 2 to 22. 5. A polynucleotide molecule encoding a variant according to any one of paragraphs 1 to 4. 6. An expression vector comprising a polynucleotide molecule according to paragraph 5. 7. A host cell comprising a polynucleotide molecule according to paragraph 5 or an expression vector according to paragraph 6. 8. A method for producing the mutant according to any one of items 1 to 4, comprising: (1) culturing the host cell according to item 7; (2) recovering the mutant. 9. A composition comprising a variant according to any one of paragraphs 1 to 4. 10. The composition of paragraph 9, further comprising a cap analog. 11. The composition of paragraph 9 or 10, further comprising a DNA template. 12. A kit comprising the mutant according to any one of items 1 to 4. 13. Use of the variant according to any one of paragraphs 1 to 4, the composition according to any one of paragraphs 9 to 11, or the kit according to paragraph 12 in in vitro transcription. 14. A method for producing RNA, comprising the steps of contacting a DNA template and modified or unmodified nucleoside triphosphates with the RNA polymerase mutant described in any one of items 1 to 4, and incubating the mixture in an in vitro transcription reaction system to obtain a target RNA product. 15. A method for producing capped mRNA, comprising the steps of contacting a DNA template, modified or unmodified nucleoside triphosphates, and a cap analog with an RNA polymerase mutant described in any one of items 1 to 4, and incubating the mixture in an in vitro transcription reaction system to obtain a target product. 16. The method of claim 15, wherein the cap analog is a trinucleotide cap, preferably m7GpppA2'OMepG. 17. The composition according to item 11 or the method according to any one of items 14 to 16, wherein the DNA template has 2'-deoxyguanosine residues at the +1 and +2 positions. 18. Use of a variant described in any one of paragraphs 1 to 4, a composition described in any one of paragraphs 9 to 11, or a kit described in paragraph 12 in transcribing a DNA template having 2'-deoxyguanosine residues at the +1 and +2 positions of the template. [Effects of the Invention]

[0056] The present application provides an RNA polymerase mutant and a method for producing the same. By modifying wild-type T7 RNA polymerase, an RNA polymerase mutant with higher catalytic efficiency can be obtained. This mutant can improve the capping rate of mRNA products during in vitro transcription, resulting in a larger amount of capped mRNA product. The present application also provides a method for producing capped mRNA. This method can increase the yield of capped mRNA products and reduce the amount of cap analogs used, thereby avoiding raw material waste and reducing production costs. This method is extremely important for the economical production of RNA. [Brief explanation of the drawings]

[0057] [Figure 1] FIG. 1 is a schematic diagram of a double-stranded DNA template. [Figure 2]FIG. 2 shows the complementary pairing of a cap analog with a DNA template. [Figure 3] FIG. 3 is a schematic diagram showing the construction of recombinant plasmids. [Figure 4] Figure 4 shows the effect of RNA polymerase and its mutants (R386W, Q435A, Q435H, Q435T, N437F, N437T, D438T, D438L, D438I, D438P, D438V, R386A+R34A, R386M+R34A, R386M+Y178H, R386W+N437T, R386I+Del172, R386F+Y178D) on mRNA capping rate. [Figure 5] Figure 5 shows the effect of RNA polymerase and its mutants (WT, R386C, R386W, D388K, R386C+D438P, R386W+N437T, D388K+D438P, N437T, D438P) on the mRNA capping rate. DETAILED DESCRIPTION OF THE INVENTION

[0058] In the examples of this application, one unit (U) of enzyme activity is the enzyme activity of 1 nmol of [ 3 It is defined as the amount of enzyme required to incorporate [H]ATP into an acid-insoluble precipitate.

[0059] Example 1: Preparation of RNA polymerase mutants The RNA polymerases shown in Table 1 DNA sequences (SEQ ID NOs: 23-44) were synthesized and PCR-amplified. The resulting recombinant expression vectors were then inserted into the BseRI and HindIII restriction enzyme sites of the expression vector pQE-80L. The resulting vectors were then transformed into E. coli BL21(DE3) by chemical transformation, plated onto ampicillin-resistant LB plates, and incubated overnight in a 37°C incubator. Plasmid extraction and sequencing of single colonies were performed to obtain recombinant engineered bacteria containing the target gene. The sequenced E. coli recombinant strains were inoculated into LB medium and cultured overnight. They were then inoculated into fermentation broth (LB medium) at 1-5% V / V and grown until the OD 600 reached 0.6-0.8. IPTG was added to a final concentration of 0.5 mol / L, and the mixture was cultured for 4 to 6 hours. After centrifugation at 12,000 rpm and 4°C, the culture was harvested and washed with 0.2 M PBS buffer (pH 7.0) to obtain the cells. After ultrasonic disruption, the cells were purified by affinity chromatography to obtain the RNA polymerase stock solution.

[0060] The correspondence between RNA polymerase mutants and amino acid sequences is shown in Table 1. [Table 1] JPEG2026010073000005.jpg252170JPEG2026010073000006.jpg254170JPEG2026010073000007.j pg246170JPEG2026010073000008.jpg248170JPEG2026010073000009.jpg250170JPEG20260100730 00010.jpg249170JPEG2026010073000011.jpg251170JPEG2026010073000012.jpg248170JPEG202 6010073000013.jpg249170JPEG2026010073000014.jpg245170JPEG2026010073000015.jpg251166

[0061] Example 2 Production of mRNA by in vitro transcription The enzyme stock solution was diluted with storage buffer (50 mM Tris-HCl (25°C, 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 20 μL mix was prepared according to the reaction system (Table 2) and transferred to an EP tube. The mix was added to an 8-strip tube, mixed uniformly, and centrifuged. The 8-strip tube was placed in a PCR machine and incubated at 37°C for 1 hour. 36 μL of magnetic beads (Vazyme, product number N412) were added, mixed uniformly, and incubated at room temperature for 2–5 minutes. The mixture was then placed in a magnetic rack for mRNA purification. After purification, the mixture was transferred to an RNase-free centrifuge tube to obtain purified mRNA. [Table 2]

[0062] Example 3: Detection of capping rate After pretreatment with an mRNA Capping Rate Detection Kit (Vazyme, product number: DD3510-01), the capping rate of the mRNA product was detected using LC-MS.

[0063] (1) The purified mRNA in Example 2 was bound to a probe. The reaction system is shown in Table 3, and the reaction conditions are shown in Table 4. [Table 3] [Table 4] (2) RNase H enzyme cleavage: The enzyme cleavage reaction system was prepared according to Table 5, thoroughly mixed by vortexing, and placed in a PCR device for reaction at 25°C for 20 minutes. [Table 5] (3) Binding of SA magnetic beads: (1) Washing magnetic beads: 9 μL of SA magnetic beads (product number Cat. No.: SM017005) were placed in a centrifuge tube and placed on a magnetic rack. Once the solution was clear, the supernatant was aspirated and discarded. The centrifuge tube was removed from the magnetic rack and washed with 200 μL of RNase-free HO. The tube was then placed on the magnetic rack. Once the solution was clear, the supernatant was aspirated and discarded with a pipette. 200 μL of RNase-free HO was added for another wash. (2) Reaction conditions: The centrifuge tube was removed from the magnetic rack, and the enzyme cleavage product was added to the SA magnetic beads (solid). The mixture was mixed uniformly by pipetting 20 to 30 times. The mixture was then placed on a roller and incubated for 30 minutes at room temperature while rotating, allowing the magnetic beads and the enzyme cleavage product to completely bind to each other. (4) Rinse and elution: (1) Place the product from the previous step on a magnetic rack, and after 2-3 minutes, when the solution becomes clear, aspirate the supernatant with a pipette and discard it. (2) While being careful not to disperse the beads, add 200 μL of rinse solution to rinse, leave for 0.5-1 minute, and then aspirate the supernatant with a pipette and discard it. (3) Repeat step (2), (4) Remove the centrifuge tube from the magnetic rack, add 30 μL of elution solution, and pipette 10 to 20 times to mix evenly and disperse the beads evenly and completely elute. (5) Place the PCR tube in a PCR machine and incubate at 85°C for 3 minutes. Immediately place the tube on a magnetic rack. Once the solution becomes clear (0.5-1 minute), aspirate the supernatant and transfer it to a new centrifuge tube. This supernatant is the desired product. (6) The above product was transferred to a Thermo Scientific Vanquish Flex-Qrbitrap Exploris 120, and the capping rate (mobile phase: Phase A: 2% hexafluoroisopropanol-1% N'N-diisopropylethylamine aqueous solution, Phase B: 2% hexafluoroisopropanol-1% N'N-diisopropylethylamine methanol solution, chromatography column: Nano ChromCore C18 3 μm, 4.6*100 mm, ion mode: negative ion, scan mode: full scan, scan range: 600-3000) was detected. The formula for calculating the capping rate was as follows: mRNA capping rate (%) = (capped mRNA / (capped mRNA+uncapped mRNA)) × 100%.

[0064] The detection results are shown in Figure 4. When the ratio of cap analog to starting NTP was 0.33:1, all of the T7 RNA polymerase mutants in Example 1 were able to effectively improve the capping rate of mRNA products, with R386W+N437T increasing the capping rate to 100%.

[0065] Example 4 Production of mRNA by in vitro transcription reaction mRNA was produced by in vitro transcription with reference to Example 2 above, and the amount of cap analog added was further reduced to 0.24 μL (see Table 6 for details), while the other reaction conditions were the same. The capping rate was further detected with reference to Example 3. [Table 6]

[0066] The results are shown in Figure 5. Compared to wild-type T7 RNAP, all mutations effectively increased the capping rate. Among them, the T7 RNAP duplication mutations R386C+D438P, R386W+N437T, and D388K+D438P all significantly increased the capping rate at a single site. The capping rate of D388K+D438P increased to 95.5%, an improvement of more than 10%, compared to the 84% capping rate of the single site D388K and the 68.5% capping rate of the single site D438P.

Claims

1. An RNA polymerase mutant, wherein the amino acid sequence of the mutant comprises at least one substitution or deletion at an amino acid position selected from R386, R34, K172, Y178, D388, Q435, N437, and D438 relative to SEQ ID NO:

1.

2. (1) the substitution at position R386 is selected from C or W; (2) the substitution at position D388 is K; (3) the substitution at position Q435 is selected from A, H, and T; (4) the substitution at position N437 is selected from F and T; (5) the substitution at position D438 is selected from T, L, I, P, and V; (6) The substitution at position R34 is selected from A; (7) the mutation at position K172 is a deletion; (8) The mutant according to claim 1, characterized in that the substitution at position Y178 is selected from H and D.

3. The mutant according to claim 1, characterized in that the amino acid sequence of the mutant contains a mutation at any site selected from R386W, R386C, D388K, Q435A, Q435H, Q435T, N437F, N437T, D438T, D438L, D438I, D438P, D438V, R386A+R34A, R386M+R34A, R386M+Y178H, R386W+N437T, R386I+del-K172, R386F+Y178D, D388K+D438P, and R386C+D438P relative to SEQ ID NO:

1.

4. The mutant of claim 1, wherein the amino acid sequence of the mutant is set forth in any one of SEQ ID NOs: 2 to 22.

5. A polynucleotide molecule encoding the mutant of claim 1.

6. An expression vector comprising the polynucleotide molecule of claim 5.

7. A host cell comprising the polynucleotide molecule of claim 5.

8. (1) culturing the host cell according to claim 7; (2) recovering the mutant.

9. A composition comprising the mutant of claim 1.

10. A kit comprising the mutant of claim 1.

11. Use of the variant of claim 1, the composition of claim 9, or the kit of claim 10 in in vitro transcription.

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

13. A method for producing capped mRNA, the method comprising the steps of contacting a DNA template, modified or unmodified nucleoside triphosphates, and a cap analog with the RNA polymerase mutant described in claim 1, incubating in an in vitro transcription reaction system, and obtaining a target product.

14. 14. The method of claim 13, wherein the cap analog is a trinucleotide cap.

15. 15. The method of claim 14, wherein the trinucleotide cap is m7GpppA2'OMepG.

16. 16. The method of claim 12, wherein the DNA template has a 2'-deoxyguanosine residue at the +1 and +2 positions.

17. Use of the mutant of claim 1, the composition of claim 9, or the kit of claim 10 in transcribing a DNA template having 2'-deoxyguanosine residues at the +1 and +2 positions of the template.

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