RNA polymerase variants and their applications

Abstract

This disclosure provides a class of RNA polymerase variants and their uses. These variants exhibit improved catalytic activity compared to wild-type T7 RNA polymerase, increasing the capping rate of mRNA products during in vitro transcription (co-transcriptional capping). Furthermore, this disclosure provides methods for preparing RNA using these variants. Using this method to prepare RNA molecules, a higher amount of capped mRNA can be obtained.

Description

Technical Field

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

[0002] The 5' end of intact eukaryotic mRNA contains a 7-methylguanosine (m7G) cap, which is formed in vivo by RNA triphosphatases, mRNA guanylyltransferases, mRNA methyltransferases, and mRNA nucleoside 2'-oxymethyltransferases. This 5' cap structure is involved in preventing mRNA degradation by exonucleases, reducing mRNA immunogenicity, regulating mRNA half-life, and controlling translation. During in vitro transcription (IVT) mRNA preparation, researchers often add cap analogs 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 continues to deepen, the importance of cap analogs has become increasingly prominent. To this end, people are constantly developing new cap analogs for use in mRNA vaccines and therapeutic RNA. Today, cap analogs have developed to the third generation. The first generation of cap analogs has two free 3'-OH groups, which causes the cap analogs to be reversely incorporated. Therefore, first-generation cap analogs are rarely seen on the market. The most common ones on the market are the second-generation ARCA cap analogs (Formula 1) and the third-generation cap analogs (Formula 2). Their main structural formulas are shown as examples:

[0004] Formula 1

[0005]

[0006] Formula 2

[0007]

[0008] ARCA cap analog is a modified cap analog with a close m 7The 3'-OH group of G is replaced with -OCH3. This substitution forces RNA polymerase to utilize only the remaining hydroxyl group to initiate transcription, forcing the ARCA cap to be incorporated in the forward orientation. Due to the low capping efficiency of second-generation cap analogs, third-generation cap analogs have been developed, such as CleanCap AG, which can form a Cap1 structure. Compared to second-generation cap analogs, this significantly improves the capping efficiency to >90%. However, this requires replacing the wild-type promoter from 5'-TAATACGACTCACTATAGG-3' to 5'-TAATACGACTCACTATAAG-3'. Whether this promoter replacement will introduce safety issues and the generation of new impurities remains to be demonstrated. If the wild-type promoter is used, the capping efficiency remains low, and uncapped RNA products not only waste raw materials but also require column purification for removal, increasing production costs. Therefore, if T7 RNA polymerase modification can improve the capping efficiency of second-generation cap analogs without replacing the wild-type promoter, it would be of great significance for the economical production of mRNA and drug safety. Summary of the Invention

[0009] In a first aspect, the present disclosure provides a class of RNA polymerase variants, whose amino acid sequence comprises a mutation of at least one amino acid selected from positions G436 and D438 relative to SEQ ID NO: 1, and the mutation type can be selected from substitution or deletion.

[0010] In a second aspect, the present disclosure provides one or more biomaterials selected from the following:

[0011] 1) a polynucleotide molecule encoding an RNA polymerase variant;

[0012] 2) an expression vector comprising the polynucleotide molecule described in 1);

[0013] 3) A host cell comprising the polynucleotide molecule described in 1), or a host cell comprising the expression vector described in 2).

[0014] In a third aspect, the present disclosure provides a method for preparing the aforementioned RNA polymerase variant.

[0015] In a fourth aspect, the present disclosure provides a composition comprising at least one RNA polymerase variant as described in the present disclosure.

[0016] In a fifth aspect, the present disclosure provides a kit comprising at least one RNA polymerase variant as described in the present disclosure.

[0017] In a sixth aspect, the present disclosure further provides use of the above-mentioned RNA polymerase variants, compositions or kits in in vitro transcription.

[0018] In a seventh aspect, the present disclosure also provides a method for preparing RNA or capped RNA. Detailed Description of the Invention

[0020] RNA polymerase variants

[0021] The RNA polymerase variant provided by the present disclosure is a bacteriophage T7 RNA polymerase (T7 RNAP) variant, whose amino acid sequence comprises at least one mutation selected from the following amino acid positions: G436, D438 relative to SEQ ID NO: 1, and the mutation type is selected from substitution or deletion.

[0022] In some embodiments, the variant has a substitution at position G436 selected from the group consisting of: W, H, I, L, N, V, E, A, D, T.

[0023] In some embodiments, the variant has a substitution at position D438 selected from the group consisting of: E, M, W.

[0024] In some embodiments, the amino acid sequence of the variant comprises any one mutation selected from the following positions relative to SEQ ID NO: 1: G436W, G436H, G436I, G436L, G436N, G436V, G436E, G436A, G436D, G436T, D438E, D438M, D438W, R394L+G436H.

[0025] In some embodiments, the disclosure provides RNA polymerase variants having an amino acid sequence that is at least 97%, at least 98%, at least 99% or higher sequence identity to the sequence shown in any one of SEQ ID NOs: 3-16. In some embodiments, the amino acid sequence of the variant is as shown in any one of SEQ ID NOs: 3-16.

[0026] polynucleotide molecules

[0027] The present disclosure provides polynucleotide molecules encoding any of the RNA polymerase variants described herein. In some embodiments, the polynucleotide molecules are as set forth in any of SEQ ID NOs: 19-32. Various modifications may be present in the coding regions of the polynucleotide molecules described herein, as long as the amino acid sequence of the variants disclosed herein does not change with codon degeneracy or with codon preference in the organism in which the variant is expressed.

[0028] expression vector

[0029] The expression vector provided in the present disclosure refers to a linear or circular DNA molecule, which generally contains elements such as a multiple cloning site, a resistance gene, a replication origin site, etc. In some embodiments, the expression vector described in the present disclosure is pQE-80L.

[0030] In some embodiments, the vectors described herein comprise a polynucleotide molecule encoding an RNA polymerase variant of the present disclosure. In some embodiments, the vectors further comprise one or more regulatory sequences (such as enhancer, promoter, and terminator sequences) operably linked to the polynucleotide molecule encoding the variant.

[0031] host cells

[0032] The host cells provided by the present disclosure can be 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 vector of the present disclosure, including any cell progeny that differs from the parent cell due to mutations that occur during replication.

[0033] In some embodiments, the host cell described in the present disclosure comprises the aforementioned polynucleotide molecule or the expression vector.

[0034] In some embodiments, the host cell is a prokaryotic cell, which can be selected from gram-positive bacteria or gram-negative bacteria. In some embodiments, the host cell is a gram-positive bacteria, including but not limited to: Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Ocean Bacillus, Staphylococcus, Streptococcus and Streptomyces. In some embodiments, the host cell is a gram-negative bacteria, including but not limited to: Campylobacter, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacter, Silene Bacillus, Neisseria, Pseudomonas, Salmonella and Ureaplasma. In some embodiments, the host cell is Escherichia coli BL21 (DE3).

[0035] Method for preparing variants

[0036] 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.

[0037] 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, etc., HPLC, and combinations of the above methods.

[0038] In some embodiments, the preparation method further comprises a step of purifying the variant, and the purification step can be a method well known in the art, such as chromatography (e.g., ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, and ammonium sulfate precipitation).

[0039] Composition

[0040] The present disclosure provides compositions comprising at least one RNA polymerase variant described herein.

[0041] The compositions described herein can be compositions for storing RNA polymerase variants. In some embodiments, in addition to the aforementioned RNA polymerase variants, the compositions described herein may optionally include: buffer components (e.g., Tris base, Tris-HCl, HEPES, MOPS), salts (e.g., NaCl), enzyme inhibitors (e.g., EDTA), reducing agents (e.g., DTT), surfactants (e.g., Triton X-100), stabilizers (e.g., glycerol), and other components. In some embodiments, the compositions described herein for storing RNA polymerase variants include: RNA polymerase variants, Tris-HCl, NaCl, EDTA, DTT, Triton X-100, and glycerol.

[0042] The compositions disclosed herein may also be in vitro transcription reaction compositions. In some embodiments, in addition to the aforementioned RNA polymerase variants, the compositions further comprise one or more in vitro transcription reaction reagents (e.g., a buffer component, modified or unmodified nucleoside triphosphates, an RNase inhibitor, pyrophosphatase, magnesium ions, water, etc.). In some embodiments, the compositions further comprise a DNA template. In some embodiments, the compositions further comprise a cap analog.

[0043] In some embodiments, the in vitro transcription reaction composition described in the present disclosure comprises: an RNA polymerase variant, a buffer component, modified or unmodified nucleoside triphosphates, an RNase inhibitor, pyrophosphatase, magnesium ions, water, and a cap analog. In some embodiments, the in vitro transcription reaction composition described in the present disclosure comprises: an RNA polymerase variant, a buffer component, modified or unmodified nucleoside triphosphates, an RNase inhibitor, pyrophosphatase, magnesium ions, water, a cap analog, and a DNA template.

[0044] Reagent test kit

[0045] The present disclosure provides a kit comprising at least one RNA polymerase variant described in the present disclosure.

[0046] 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 may further comprise a cap analog. In some embodiments, each component of the kit (if applicable) may be provided in liquid form (e.g., in solution) or in solid form (e.g., dry powder).

[0047] The kits described herein may include one or more containers holding one or more components described herein and, optionally, instructions for use.

[0048] Application or use

[0049] The present disclosure provides use of the aforementioned RNA polymerase variants, compositions, or kits in in vitro transcription.

[0050] The present disclosure also provides uses of the above-mentioned RNA polymerase variants, compositions, or kits in various methods, including but not limited to the preparation of RNA, preparation of RNA probes, preparation of RNA vaccines, preparation of proteins, etc.

[0051] Methods for preparing RNA

[0052] 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 herein, and incubating the mixture in an in vitro transcription reaction system to obtain a target RNA product. In some embodiments, the RNA product can be dsRNA, ssRNA, mRNA, siRNA, miRNA, piRNA, shRNA, or gRNA.

[0053] The present disclosure also provides a method for preparing capped mRNA. In some embodiments, the method comprises contacting a DNA template, modified or unmodified nucleoside triphosphates, and a cap analog with at least one RNA polymerase variant described herein, and incubating the mixture in an in vitro transcription reaction system to obtain a target product.

[0054] In vitro transcription reaction systems and incubation conditions suitable for generating RNA products or capped 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 of the reaction system, or whether to add exogenous auxiliary factors, etc., taking into account the optimal activity of RNA polymerase. In some embodiments, the in vitro transcription reaction system described in the present disclosure comprises in vitro transcription reaction reagents: one or more buffer components, modified or unmodified nucleoside triphosphates, RNase inhibitors, pyrophosphatase, magnesium ions, water, etc. In some embodiments, in the incubation step described in the present disclosure, the incubation temperature is 30~50°C, preferably 37°C. In some embodiments, in the incubation step described in the present disclosure, the incubation time is 20~240 min, preferably 60 min.

[0055] In some embodiments, RNA products or capped mRNA products prepared using the methods described herein have higher yields, and / or higher integrity, and / or lower dsRNA impurity content, and / or more capped mRNA products, etc., compared to those produced using wild-type RNA polymerase.

[0056] In some embodiments, compared to using wild-type RNA polymerase, the capped mRNA products prepared using the methods described herein can increase the utilization rate of the cap analog, wherein the capping efficiency of the obtained mRNA products can be increased by at least 5%, at least 10%, at least 15%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29% or 30%.

[0057] Hat analogues

[0058] The cap analog used in the method for preparing a capped mRNA product or the cap analog in an in vitro transcription reaction composition disclosed in the present invention refers to a molecule that can be complementary to a nucleotide molecule on a DNA template at the transcription start site.

[0059] 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 dinucleotide cap. In some embodiments, the cap analog is preferably an ARCA cap.

[0060] In vitro transcription reaction reagents

[0061] 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.

[0062] In some embodiments, the buffer component can be selected from one or more of: phosphate buffer, Tris buffer, MOPS buffer, HEPES buffer, citrate buffer, acetate buffer, malate buffer, MES buffer, histidine buffer, PIPES buffer, bis-tris buffer and ethanolamine buffer.

[0063] In some embodiments, the nucleoside triphosphates can be selected from modified or unmodified nucleoside triphosphates (including their analogs). In some embodiments, the nucleoside triphosphates can be selected from unmodified ATP, GTP, CTP, and UTP. In some embodiments, the nucleoside triphosphates can be selected from modified nucleoside triphosphates, and the types of modifications 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 nucleoside triphosphates with labels (the labels can be biotin, fluorescent substances, digoxigenin, radioactive elements, etc.).

[0064] 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 reagent.

[0065] Other implementation options:

[0066] 1. An RNA polymerase variant, wherein the amino acid sequence thereof comprises at least one mutation selected from the following amino acid positions relative to SEQ ID NO: 1: G436 or D438, wherein the mutation type is selected from substitution or deletion.

[0067] 2. A variant according to item 1, wherein:

[0068] (1) the substitution at position G436 is selected from W, H, I, L, N, V, E, A, D, T;

[0069] (2) The substitution at position D438 is selected from E, M, and W.

[0070] 3. The variant according to item 1, wherein the amino acid sequence comprises any one of the mutations selected from the group consisting of G436W, G436H, G436I, G436L, G436N, G436V, G436E, G436A, G436D, G436T, D438E, D438M, D438W, and R394L+G436H relative to SEQ ID NO: 1.

[0071] 4. The variant according to item 1, whose amino acid sequence is shown in any one of SEQ ID NOs: 3-16.

[0072] 5. A polynucleotide molecule encoding the variant according to any one of items 1 to 4.

[0073] 6. An expression vector comprising the polynucleotide molecule according to item 5.

[0074] 7. A host cell comprising the polynucleotide molecule according to item 5 or the expression vector according to item 6.

[0075] 8. A method for preparing the variant according to any one of items 1 to 4, comprising:

[0076] (1) culturing the host cell described in item 7 under conditions suitable for expression of the variant; and

[0077] (2) Recycling variants.

[0078] 9. A composition comprising the variant according to any one of items 1 to 4.

[0079] 10. The composition as described in item 9, further comprising a cap analog.

[0080] 11. The composition as described in item 9 or 10, further comprising a DNA template.

[0081] 12. A kit comprising the variant according to any one of items 1 to 4.

[0082] 13. Use of the variant described in any one of items 1 to 4, the composition described in any one of items 9 to 11, or the kit described in item 12 in in vitro transcription.

[0083] 14. 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 items 1 to 4, and incubating the mixture in an in vitro transcription reaction system to obtain a target RNA product.

[0084] 15. A method for preparing capped mRNA, comprising contacting a DNA template, modified or unmodified nucleoside triphosphates, and a cap analog with the RNA polymerase variant 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.

[0085] 16. The method according to item 15, wherein the cap analog is a dinucleotide cap, preferably an ARCA cap.

[0086] Beneficial effects

[0087] The present disclosure provides a class of RNA polymerase variants and a method for preparing the same. By modifying wild-type T7 RNA polymerase, RNA polymerase variants with higher catalytic efficiency are obtained. Such variants can be used to increase the capping rate of mRNA products during in vitro transcription, thereby obtaining more capped mRNA products. BRIEF DESCRIPTION OF THE DRAWINGS

[0088] Figure 1 Schematic diagram of the construction of recombinant plasmid;

[0089] Figure 2 The effect of RNA polymerase and its variants on mRNA capping rate. DETAILED DESCRIPTION

[0090] The technical solutions of the present application are further described below with reference to specific examples. However, the following examples are merely examples of the present application and do not represent or limit the scope of protection of the present application. The scope of protection of the present application shall be subject to the claims. In the following examples, unless otherwise specified, all reagents and consumables used were purchased from common suppliers in the field, and the experimental methods and technical means used were conventional methods and means in the field.

[0091] In the present disclosure, the unit enzyme activity (U) is defined as the enzyme activity that can be converted into 1 nmol [ 3 The amount of enzyme required to incorporate [H]ATP into the acid-insoluble precipitate was defined as 1 activity unit.

[0092] Example 1: Preparation of RNA polymerase variants

[0093] The RNA polymerases shown in Table 1 were synthesized by DNA sequence (SEQ ID NO: 17-32) and PCR amplified. The amplified ... E. coli BL21 (DE3), spread on LB plate containing ampicillin resistance, put in 37 ℃ incubator overnight, extract and sequence the single colony, and finally obtain the recombinant engineered bacteria containing the target gene. E. coli The recombinant strain was inoculated into LB medium for overnight activation culture, and then inoculated into fermentation broth (LB medium) at 1% v / v. Culture was continued to OD600 of 0.6-0.8 after adding IPTG at a final concentration of 0.5 mol / L for 4-6 h. The strain was collected by centrifugation at 12,000 rpm and 4°C. The collected strain was washed with 0.2 M PBS buffer (pH 7.0) to obtain bacterial cells. After ultrasonic disruption, the RNA polymerase stock solution was purified by affinity chromatography (His trap HP, 29-0510-21, Cytiva). The correspondence between RNA polymerase variants and amino acid sequences is shown in Tables 1-1 to 1-4:

[0094] Table 1-1

[0095]

[0096] Table 1-2

[0097]

[0098] Table 1-3

[0099]

[0100] Table 1-4

[0101]

[0102] Example 2: Preparation of mRNA by in vitro transcription reaction

[0103] Dilute the enzyme stock solution 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. Prepare a 20 μL MIX solution according to the reaction system in Table 2 and transfer it to an Eppendorf tube. Transfer the MIX solution to an eight-well strip, mix thoroughly, and centrifuge. Place the eight-well strip in a PCR instrument and incubate at 37°C for 1 hour. Then, add 36 μL of magnetic beads (Vazyme, Cat. No. N412), mix thoroughly, and incubate at room temperature for 2-5 minutes. Purify the mRNA by placing the mixture on a magnetic rack and transferring it to an RNase-free centrifuge tube to obtain purified mRNA.

[0104] Table 2: Reaction system ratio

[0105]

[0106] Example 3: Capping rate detection

[0107] After pre-treatment with the mRNA Capping Rate Detection Kit (Vazyme, Catalog No.: DD3510-01), the capping rate of the mRNA product was detected by LC-MS:

[0108] (1) The purified mRNA in Example 2 was combined with the probe. The reaction system is shown in Table 3, and the reaction conditions are shown in Table 4.

[0109] Table 3: Reaction system

[0110]

[0111] Table 4: Reaction conditions

[0112]

[0113] (2) RNase H digestion: Prepare the enzyme digestion reaction system according to Table 5, vortex thoroughly to mix, and place in a PCR instrument for reaction at 25°C for 20 min.

[0114] Table 5: Reaction system

[0115]

[0116] (3) SA magnetic bead binding:

[0117] ① Magnetic bead washing: Take 9 μL SA magnetic beads (Tian Di Ren He, Cat. No.: SM017005) into a centrifuge tube and place it on a magnetic rack. After the solution becomes clear, use a pipette to discard the supernatant; remove the centrifuge tube from the magnetic rack, add 200 μL RNase-free H2O to rinse, place it on a magnetic rack, after the solution becomes clear, use a pipette to discard the supernatant, and add 200 μL RNase-free H2O to repeat the rinse.

[0118] ② Reaction conditions: Remove the centrifuge tube from the magnetic stand, add the enzyme-digested product to the SA magnetic beads (solid), pipette 20-30 times to mix thoroughly, place on a roller and incubate at room temperature for 30 minutes to allow the magnetic beads and enzyme-digested product to fully combine.

[0119] (4) Rinse and wash:

[0120] ① Place the product from the previous step on a magnetic stand for 2-3 minutes until the solution becomes clear, then discard the supernatant with a pipette;

[0121] ② Add 200 μL of rinse solution to rinse, taking care not to blow away the magnetic beads, let it stand for 0.5-1 min, and discard the supernatant with a pipette;

[0122] ③Repeat step ②;

[0123] ④ Remove the centrifuge tube from the magnetic stand, add 30 μL of elution buffer, and pipette 10-20 times to mix evenly to evenly disperse the magnetic beads and fully elute them;

[0124] ⑤ Place the tube in a PCR instrument and react at 85°C for 3 minutes. Immediately place the tube on a magnetic rack. After the solution has clarified (0.5-1 minute), transfer the supernatant to a new centrifuge tube. The supernatant is the desired product.

[0125] ⑥ The above product was sent to the Thermo scientific Vanquish Flex-Qrbitrap Exploris120 to detect 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; chromatographic column: Nano ChromCore C18 3 μm, 4.6*100mm;; ion mode: negative ion; scan mode: full scan; scan range: 600-3000). The capping rate calculation formula is:

[0126] mRNA capping rate (%) = (capped mRNA / (capped mRNA + uncapped mRNA)) ×100%.

[0127] Test results are shown in Figure 2 The T7 RNA polymerase variants in Example 1 can all effectively improve the capping rate of mRNA products, among which the variant G436W has the most significant improvement efficiency, which can significantly increase the capping rate from 64.65% of the original wild type to >90%.

Claims

1. RNA polymerase variant, characterized in that The amino acid sequence of the variant comprises a mutation at position G436 relative to SEQ ID NO:

1. The amino acid sequence of the variant is shown in any one of SEQ ID NOs: 3-12 and 16.

2. Biomaterial, characterized in that The biological material is selected from one or more of the following: 1) A polynucleotide molecule encoding the RNA polymerase variant according to claim 1; 2) an expression vector comprising the polynucleotide molecule described in 1); 3) A host cell comprising the polynucleotide molecule described in 1), or a host cell comprising the expression vector described in 2).

3. The method for preparing the variant according to claim 1, characterized in that: include: (1) Cultivating the host cell as claimed in claim 2 under conditions suitable for expression of the variant; and (2) Recycling variants.

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

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

6. Use of the variant according to claim 1, the composition according to claim 4, or the kit according to claim 5 in in vitro transcription.

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

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

9. The method according to claim 8, wherein The cap analog is a dinucleotide cap.

10. The method according to claim 9, wherein The dinucleotide cap is an ARCA cap.

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