RNA polymerase variants to increase RNA capping rate in in vitro transcription

Abstract

The invention provides an RNA polymerase variant capable of improving the RNA capping rate in in-vitro transcription, and relates to the technical field of biology. According to the present invention, the wild type T7RNA polymerase is modified to obtain the T7RNA polymerase variant, and the capping rate of the RNA product can be substantially improved compared with the wild type T7RNA 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] The complete mRNA mainly consists of five parts, from 5' to 3', including: 5' cap structure, 5' untranslated region, open reading frame encoding antigen, 3' untranslated region and PloyA tail. In vivo, the 5' cap structure is formed by RNA triphosphatase, mRNA guanosine transferase, mRNA methyltransferase and mRNA nucleoside 2'-methyltransferase. It is a special structure of mature mRNA formed by post-transcriptional modification of eukaryotic organisms. 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. Usually, enzymes such as VCE and FCE are used as capping catalysts, and mRNA nucleoside 2'-methyltransferase is additionally added for capping reaction. Enzymatic capping is affected by its enzyme-linked catalytic efficiency, and the capping rate is relatively low. Co-transcriptional capping by cap analogs is also a mainstream method at present, and the capping rate of this method far exceeds that of enzymatic capping.

[0003] Nowadays, cap analogs have developed to the third generation. The first generation of cap analogs have two free 3'-OH groups, which leads to reverse incorporation of the cap analogs. Therefore, the first generation of 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] Compared with the second-generation cap analogs, the third-generation cap analogs, such as CleanCapAG, have significantly improved their capping rate (capping rate of more than 90%). However, when prepared with wild-type T7 RNA polymerase as a catalyst, due to its steric hindrance and other reasons, it is difficult to achieve a capping rate of 100%. The generation of uncapped RNA products not only causes a waste of raw materials, but also needs to be removed by column purification in the later stage, increasing the production cost. Therefore, further improving the mRNA capping rate in the co-transcriptional capping process is conducive to the economical production of mRNA. SUMMARY OF THE INVENTION

[0007] In a first aspect, the present disclosure provides a class of RNA polymerase variants, whose amino acid sequence has at least 95% sequence identity compared to 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.

[0008] In a second aspect, the present disclosure provides one or more biological materials selected from the following:

[0009] 1) a polynucleotide molecule encoding an RNA polymerase variant;

[0010] 2) an expression vector comprising the polynucleotide molecule as described in 1);

[0011] 3) A host cell comprising the polynucleotide molecule as described in 1), or a host cell comprising the expression vector as described in 2).

[0012] In a third aspect, the present disclosure provides a method for preparing the above-mentioned RNA polymerase variant.

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

[0014] In a fifth aspect, the present disclosure provides a kit comprising at least one RNA polymerase variant as described in the present disclosure.

[0015] In a sixth aspect, the present disclosure also provides use of the above-mentioned RNA polymerase variants, compositions or kits in in vitro transcription.

[0016] In a seventh aspect, the present disclosure also provides a method for preparing RNA or capped RNA. DETAILED DESCRIPTION OF THE INVENTION

[0018] RNA polymerase variants

[0019] 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 of the following relative to SEQ ID NO: 1: D388 or D130 / K387.

[0020] 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.

[0021] In some embodiments, the amino acid sequence of the RNA polymerase variant provided by the present disclosure 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.

[0022] Biomaterials

[0023] The present disclosure provides a polynucleotide molecule encoding the RNA polymerase variant of the present disclosure; wherein the polynucleotide molecule may have various modifications in the coding region, as long as the amino acid sequence of the variant of the present disclosure does not change with the degeneracy of the codons or does not change with the preferred codons in the organism expressing the variant. In some embodiments, the polynucleotide molecule is as shown in any one of SEQ ID NOs: 5-6.

[0024] The disclosure provides an expression vector comprising a polynucleotide molecule encoding an RNA polymerase variant of the disclosure. In some embodiments, the expression vector further comprises one or more regulatory sequences, such regulatory sequences including but not limited to enhancers, promoters, leader peptide sequences, signal peptide sequences, and terminator sequences; wherein the regulatory sequence is operably linked to the polynucleotide molecule encoding the variant. In some embodiments, the expression vector can be a linear or circular DNA molecule, typically comprising elements such as a multiple cloning site, a resistance gene, and a replication initiation site.

[0025] In some embodiments, the expression vector described in the present disclosure is preferably pQE-80L.

[0026] 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 are different from the parent cells due to mutations that occur during replication.

[0027] 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, Oceanic 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, Silvae, Neisseria, Pseudomonas, Salmonella and Ureaplasma. In some embodiments, the host cell is Escherichia coli.

[0028] Method for preparing variants

[0029] 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.

[0030] 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 of the above methods.

[0031] In some embodiments, the preparation method also 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.

[0032] Composition

[0033] The present disclosure provides a composition comprising at least one RNA polymerase variant described in the present disclosure.

[0034] 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: 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 composition for storing RNA polymerase variants described in the present disclosure includes: RNA polymerase variants, Tris-HCl, NaCl, EDTA, DTT, Triton X-100 and glycerol.

[0035] 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, also includes one or more in vitro transcription reaction reagents (such as: buffer components, modified or unmodified nucleoside triphosphates, RNA enzyme inhibitors, pyrophosphatase, magnesium ions, water, etc.). In some embodiments, the composition also includes a DNA template. In some embodiments, the composition also includes a cap analog.

[0036] In some embodiments, the in vitro transcription reaction composition described in the present disclosure comprises: RNA polymerase variants, buffer components, modified or unmodified nucleoside triphosphates, RNase inhibitors, pyrophosphatase, magnesium ions, water, and cap analogs. In some embodiments, the in vitro transcription reaction composition described in the present disclosure comprises: RNA polymerase variants, buffer components, modified or unmodified nucleoside triphosphates, RNase inhibitors, pyrophosphatase, magnesium ions, water, cap analogs, and DNA templates.

[0037] Reagent test kit

[0038] The present disclosure provides a kit comprising at least one RNA polymerase variant described in the present disclosure.

[0039] In some embodiments, the kit may also include 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 includes a cap analog. 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).

[0040] The kits described herein may include one or more containers holding one or more components described herein and optionally instructions for use.

[0041] Application or use

[0042] The present disclosure provides applications or uses of the above-mentioned RNA polymerase variants, compositions, or kits in in vitro transcription.

[0043] 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, the preparation of RNA probes, the preparation of RNA vaccines, the preparation of proteins, etc.

[0044] Methods for preparing RNA

[0045] The present disclosure provides a method for preparing 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 in the present disclosure, incubating in an in vitro transcription reaction system, and obtaining a target RNA product. In some embodiments, the RNA product can be dsRNA, ssRNA, mRNA, siRNA, miRNA, piRNA, shRNA or gRNA.

[0046] 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, a cap analog, and at least one RNA polymerase variant described in the present disclosure, incubating in an in vitro transcription reaction, and obtaining a target mRNA product.

[0047] In vitro transcription reaction systems and incubation conditions suitable for generating RNA products or mRNA products are well known in the art. A person of ordinary skill in the art can determine the appropriate pH value, reaction temperature, reaction time, salt concentration, or whether to add exogenous auxiliary factors, etc. of the reaction system, 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.

[0048] In some embodiments, the RNA products prepared using the methods described in the present disclosure have higher yields, and / or higher integrity, and / or less dsRNA impurity content, and / or more capped mRNA products, etc., compared to using wild-type RNA polymerase (SEQ ID NO: 1).

[0049] In some embodiments, the use of the methods described in the present disclosure to prepare capped mRNA products can increase the utilization rate of the cap analogs, and 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% compared to using wild-type T7 RNA polymerase.

[0050] Hat analogs

[0051] The cap analog used in the method for preparing a capped mRNA product or the cap analog in an in vitro transcription reaction composition described in the present disclosure refers to a molecule that can be complementary to a nucleotide molecule on a DNA template at the transcription start site.

[0052] 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 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.

[0053] In some embodiments, the cap analog described in the present disclosure is preferably m7GpppA2′OMepG.

[0054] In vitro transcription reaction reagents

[0055] 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.

[0056] 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, malic acid buffer, MES buffer, histidine buffer, PIPES buffer, bis-tris buffer or ethanolamine buffer.

[0057] 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 markers (the markers can be biotin, fluorescent substances, digoxin, radioactive elements, etc.).

[0058] 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.

[0059] Other implementations:

[0060] 1. An RNA polymerase variant, whose amino acid sequence, relative to SEQ ID NO: 1, comprises a substitution or substitution group selected from any one of the following: D388R or D130E / K387S.

[0061] 2. The variant as described in item 1, whose amino acid sequence is shown in any one of SEQ ID NOs: 2-3.

[0062] 3. A polynucleotide molecule encoding the RNA polymerase variant as described in any one of item 1 or 2.

[0063] 4. An expression vector comprising the polynucleotide molecule as described in item 3.

[0064] 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.

[0065] 6. A composition comprising a variant as described in any one of item 1 or 2.

[0066] 7. A kit comprising the variant as described in any one of item 1 or 2.

[0067] 8. Use of the variant as described in any one of item 1 or 2, the composition as described in item 6 or the kit as described in item 7 in in vitro transcription.

[0068] 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, incubating in an in vitro transcription reaction system, and obtaining a target RNA product.

[0069] 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 them in an in vitro transcription reaction system to obtain a target product.

[0070] The RNA polymerase variant provided by the present disclosure can significantly improve the capping rate of mRNA products in the co-transcriptional capping reaction system compared with the wild-type T7 RNA polymerase, avoid the waste of raw materials and subsequent purification operations, save the research and development and production costs of RNA drugs, and is of great significance for the economical production of RNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 Schematic diagram of the construction of recombinant plasmid. Example

[0073] The technical solution of the present disclosure is further described below in conjunction with specific embodiments. However, the following embodiments 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 embodiments, unless otherwise specified, the reagents and consumables used are purchased from ordinary suppliers in the field, and the experimental methods and technical means used are conventional methods and means in the field.

[0074] In the disclosed embodiments, the 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 the reaction condition of 37°C.

[0075] Example 1 Preparation of RNA polymerase variants

[0076] A His tag protein was added to the front end of the amino acids of the wild-type T7 RNA polymerase (SEQ ID NO: 1) and the mutant T7 RNA polymerase (SEQ ID NO: 2-3), and PCR amplification was performed after DNA sequence synthesis (DNA sequence SEQ ID NO: 4-6), and then the BseRI and HindIII restriction sites of the expression vector pQE-80L were introduced to obtain a recombinant expression vector, which was transferred into the competent state of Escherichia coli BL21 (DE3) by heat shock method, and after antibiotic resistance plate coating screening, a recombinant Escherichia coli BL21 (DE3) strain was obtained; after the successful recombination was verified, the obtained strain was activated overnight at 37°C in LB medium and then added to the fermentation broth (LB medium) and cultured to OD The 600 value is 0.6-0.8, and IPTG with a final concentration of 0.5 mol / L is added to continue culturing for 4-6 hours, and then the strain is collected by centrifugation at 12000 rpm and 5°C, and the strain is 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 is performed to obtain RNA polymerase stock solution.

[0077] The correspondence between RNA polymerase and its variants and amino acid sequences is shown in Table 1:

[0078] Table 1

[0079]

[0080]

[0081] Example 2 Preparation of mRNA by in vitro transcription reaction

[0082] The enzyme stock solution was diluted with storage buffer (50mM Tris-HCl (25℃, pH 7.9), 100mM NaCl, 0.1mM EDTA, 2mM DTT, 0.1% Triton X-100, 50% Glycerol) to an enzyme activity of 300U / μL. MIX solution (except T7 RNA polymerase) was prepared in EP tubes according to the reaction system (20μL) in Table 2 or Table 3, and the MIX solution was dispensed into eight strips, and then 1μL of the diluted T7 RNA polymerase was pipetted into the eight strips containing the MIX solution, mixed, and centrifuged; the eight strips were placed on a PCR instrument for reaction at 37℃ for 1h, and then 36μL of magnetic beads were added, mixed, and incubated at room temperature for 2-5min; the mixed solution was placed on a magnetic rack to purify mRNA (Vazyme, catalog number: N412), and after purification, it was transferred to an RNase-free centrifuge tube to obtain purified mRNA.

[0083] Table 2: Reaction system ratio (D130E / K387S, D388R)

[0084]

[0085]

[0086] Table 3: Reaction ratio (D130E / K387S, D388R)

[0087]

[0088] Example 3 Capping rate detection

[0089] (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

[0090]

[0091] Table 5: Reaction conditions

[0092] temperature time 95℃ 2min 70～16℃ -0.1℃ / s,≈180cycles 16℃ ∞

[0093] (2) RNase H digestion: Prepare the digestion reaction system (Thermo Scientific, Catalog No.: EN0201) according to Table 6, vortex thoroughly to mix well, and place in a PCR instrument for reaction at 25°C for 20 min:

[0094] Table 6: Enzyme digestion reaction system ratio

[0095] Components 25pmol volume Previous step product 21μL RNaseH Reaction Buffer (10×) 3μL RNaseH (5U / μl) 3μL <![CDATA[RNase-freeH2O]]> 3μL Overall system 30μL

[0096] (3) SA magnetic bead binding

[0097] ① Magnetic bead washing: Take 9μL SA magnetic beads into a centrifuge tube and place it on a magnetic rack. When the solution becomes clear, use a pipette to discard the supernatant; remove the centrifuge tube from the magnetic rack, add 200μL RNase-free H2O to rinse, place it on a magnetic rack, when the solution becomes clear, use a pipette to discard the supernatant, and then add 200μL RNase-free H2O to repeat the rinse.

[0098] ② Reaction conditions: Remove the centrifuge tube from the magnetic rack, add the enzyme cleavage product to the SA magnetic beads (solid), pipette 20-30 times, mix thoroughly, place on a tumbling instrument and incubate at room temperature for 30 minutes to allow the magnetic beads and the enzyme cleavage product to fully combine.

[0099] (4) Rinse and wash

[0100] ① Place the product from the previous step on a magnetic rack for 2 to 3 minutes until the solution becomes clear and discard the supernatant with a pipette;

[0101] ② Add 200 μL of rinsing solution to rinse, taking care to avoid blowing away the magnetic beads, let it stand for 0.5 to 1 minute, and discard the supernatant with a pipette;

[0102] ③Repeat step ②;

[0103] ④ Remove the centrifuge tube from the magnetic stand, add 30uL of elution buffer, and mix evenly by pipetting 10-20 times to make the magnetic beads evenly dispersed and fully eluted;

[0104] ⑤ Place in a PCR instrument, react at 85℃ for 3 minutes, and then immediately place on a magnetic rack. After the solution is clarified (0.5-1 minute), pipette the supernatant into a new centrifuge tube. The supernatant is the desired product.

[0105] 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 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 Thermo scientific Vanquish Flex-Qrbitrap Exploris120 to detect the capping rate (ion mode: negative ion; scanning mode: Full scan; scanning range: 600-3000). The capping rate calculation formula is:

[0106] mRNA capping rate (%)=(capped mRNA / (capped mRNA+uncapped mRNA))×100%.

[0108] Table 6: RNA polymerase variant capping rate (Table 2 system product) improvement effect comparison I

[0109] Serial Number name Cap rate 1 WT 87.2％ 2 D130E / K387S 90.5％ 3 D388R 100％

[0110] Table 7: Comparison of the improvement effect of RNA polymerase variant capping rate (product of Table 3 system) II

[0111] Serial Number name Cap rate 1 WT 90.38％ 2 D130E / K387S 97％ 3 D388R 94.72％

[0112] 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 6).

Claims

1. An RNA polymerase variant, characterized in that The amino acid sequence of the variant comprises, relative to SEQ ID NO: 1, a substitution or substitution group selected from any one of the following: D388R or D130E / K387S.

2. The RNA polymerase variant according to claim 1, wherein the amino acid sequence is shown in any one of SEQ ID NOs: 2-3.

3. A polynucleotide molecule encoding the RNA polymerase variant according to any one of claims 1 or 2.

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

5. A host cell comprising the polynucleotide molecule as claimed in claim 3 or a host cell comprising the expression vector as claimed in claim 4.

6. A composition, characterized in that The composition comprises the variant as claimed in any one of claims 1 or 2.

7. A kit, characterized in that The kit comprises the variant according to any one of claims 1 or 2.

8. Use of the variant according to any one of claims 1 or 2, the composition according to claim 6 or the kit according to claim 7 in in vitro transcription.

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

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 according to any one of claims 1 or 2, and incubating them in an in vitro transcription reaction system to obtain a target product.

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