RNA polymerase variants and uses thereof

Abstract

The invention provides a T7RNA polymerase variant and application thereof, the amino acid sequence of the T7RNA polymerase variant comprises substitution or deletion of at least one of the following amino acid sites relative to SEQ ID NO: 1: R34, K172, Y178, R386, D388, Q435, N437 and D438. Compared with wild type T7RNA polymerase, the variant has improved catalytic activity, and can improve the capping rate of mRNA products in the in vitro transcription (co-transcription capping) process. In addition, the invention also provides a method for preparing RNA by using the variant, and more capped mRNA can be obtained by adopting the method for preparing RNA molecules.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and particularly to RNA polymerase variants, methods for their preparation, and their applications in RNA synthesis. Background Art

[0002] The 5'-end of a complete eukaryotic mRNA has a 7-methylguanosine (m7G) cap, which is formed in vivo through the catalysis of RNA triphosphatase, mRNA guanylyltransferase, mRNA methyltransferase, and mRNA nucleoside 2'-O-methyltransferase. This 5'-cap structure participates in processes such as preventing mRNA degradation by exonucleases, reducing the immunogenicity of mRNA, regulating the mRNA half-life, and controlling translation. During the in vitro transcription (IVT) process for preparing mRNA, researchers usually add cap analogs during the preparation process to mimic the 5'-cap structure of eukaryotic cell mRNA.

[0003] The cap structure at the 5'-end of mRNA has a significant impact on aspects such as the stability, translation efficiency, and immunogenicity of mRNA. With the continuous in-depth research, the importance of cap analogs has become increasingly prominent. Therefore, people have continuously developed new types of cap analogs for use in mRNA vaccines and therapeutic RNAs. Now, cap analogs have developed to the third generation. The first-generation cap analogs are rarely seen on the market because they have two free 3'-OH groups, which cause the cap analogs to be incorporated in the reverse direction. The commonly seen ones on the market mainly include the second-generation ARCA cap analog (Formula 1) and the third-generation cap analogs (such as Formula 2), and their main structural formulas are exemplified as follows:

[0004]

[0005] The ARCA cap analog is a modified cap analog in which the 3'-OH group near m7G is replaced by -OCH3. Due to this substitution, RNA polymerase can only initiate transcription using the remaining hydroxyl group, forcing the ARCA cap to be incorporated in the forward direction. The third-generation cap analog, such as CleanCap AG, can form a Cap 1 structure, showing a significant improvement in the capping rate compared to the second-generation cap analog. However, it requires the replacement of the wild-type promoter, from the original 5'-TAATACGACTCACTATAGG-3' to 5'-TAATACGACTCACTATAAG-3'. Whether the replacement of the promoter will bring safety issues and the generation of new impurities remains to be proven. If the wild-type promoter is used, the capping rate is still low. The uncapped RNA products not only cause waste of raw materials but also need to be removed by column purification later, increasing the production cost. Therefore, if the T7 RNA polymerase is modified, the utilization rate of the cap analog can be improved without the replacement of the wild-type promoter, which is of great significance for the economical production of mRNA and drug safety. Summary of the Invention

[0007] In a first aspect, the present application provides a class of RNA polymerase variants, the amino acid sequence of which contains mutations of at least one amino acid selected from the positions of R34, K172, Y178, R386, D388, Q435, N437 or D438 relative to SEQ ID NO: 1, and the mutation types can be selected from substitution or deletion.

[0008] In a second aspect, the present application provides one or more of the following biological materials:

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

[0010] 2) An expression vector containing the polynucleotide molecule as described in 1);

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

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

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

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

[0015] In a sixth aspect, the present application further provides the application of the above RNA polymerase variant, composition or kit in in vitro transcription.

[0016] In a seventh aspect, the present application also provides a method for preparing RNA or capped RNA. Detailed Description of the Invention

[0018] RNA Polymerase Variant

[0019] The RNA polymerase variant provided by the present application is a bacteriophage T7 RNA polymerase (T7 RNAP) variant, and the amino acid sequence of the variant contains at least one mutation selected from the following amino acid sites relative to SEQ ID NO: 1: R34, K172, Y178, R386, D388, Q435, N437, or D438, and the mutation type is selected from substitution or deletion.

[0020] In some embodiments, the substitution at the R34 site of the variant is A.

[0021] In some embodiments, the mutation type at the K172 site of the variant is deletion.

[0022] In some embodiments, the substitution at the Y178 site of the variant is selected from: H or D.

[0023] In some embodiments, the substitution at the R386 site of the variant is selected from: C, W, M, A, I, or F.

[0024] In some embodiments, the substitution at the D388 site of the variant is K.

[0025] In some embodiments, the substitution at the Q435 site of the variant is selected from: A, H, or T.

[0026] In some embodiments, the substitution at the N437 site of the variant is selected from: F or T.

[0027] In some embodiments, the substitution at the D438 site of the variant is selected from: T, L, I, P, or V.

[0028] In some embodiments, the amino acid sequence of the variant contains 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.

[0029] In some embodiments, the RNA polymerase variants provided by the present application have at least 97%, at least 98%, at least 99% or higher sequence identity compared to the sequences shown in any of SEQ ID NOs: 2-22. In some embodiments, the amino acid sequence of the variant is as shown in any of SEQ ID NOs: 2-22.

[0030] Polynucleotide molecule

[0031] The polynucleotide molecules provided by the present application encode any of the RNA polymerase variants described herein. In some embodiments, the polynucleotide molecule is as shown in any of SEQ ID NOs: 23-44.

[0032] Among them, various modifications may exist in the coding region of the polynucleotide molecules described in the present application, as long as the variant amino acid sequences of the present application do not change due to codon degeneracy or do not change with the preferred codons in the organisms expressing the variants.

[0033] Expression vector

[0034] The expression vectors provided by the present application refer to linear or circular DNA molecules, which usually contain elements such as multiple cloning sites, resistance genes, replication origins, etc. In some embodiments, the expression vector described in the present application is pQE-80L.

[0035] In some embodiments, the vectors described in the present application contain polynucleotide molecules encoding the RNA polymerase variants of the present application. In some embodiments, further, the vector further contains one or more regulatory sequences (such as enhancer, promoter and terminator sequences, etc.), which are operably linked to the polynucleotide molecule encoding the variant.

[0036] Host cell

[0037] The host cells provided by the present application can be any cells that are beneficial to the expression of the variants of the present application, that is, any cells that are susceptible after being transformed, transfected or transduced with the expression vectors of the present application, covering any cell progeny that are different from the parental cells due to mutations occurring during the replication process.

[0038] In some embodiments, the host cells described in the present application contain the above polynucleotide molecules or the expression vectors.

[0039] In some embodiments, the host cell is a prokaryotic cell, optionally 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, Oceanobacillus, 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, Pelobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

[0040] Method for preparing variant

[0041] The present application provides a method for preparing the above-mentioned RNA polymerase variant, comprising: (1) culturing the host cell of the present application under conditions suitable for the expression of the variant; and (2) recovering the variant.

[0042] 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 crystalline protein precipitant (salting-out method), extraction, sonication, ultrafiltration, dialysis, various chromatographies such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC, and combinations of the above methods.

[0043] In some embodiments, the preparation method further includes a step of purifying the variant, and the purification step can be a method well-known in the art, such as chromatography (such as ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography), ammonium sulfate precipitation method, etc.

[0044] Composition

[0045] The composition provided by the present application comprises at least one RNA polymerase variant described in the present application.

[0046] The composition described in the present application can be a composition for storing the RNA polymerase variant. In some embodiments, the composition described in the present application, in addition to the above-mentioned RNA polymerase variant, may optionally comprise: 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 the RNA polymerase variant described in the present application comprises: RNA polymerase variant, Tris-HCl, NaCl, EDTA, DTT, Triton X-100, and glycerol.

[0047] The composition of the present application can also be an in vitro transcription reaction composition. In some embodiments, in addition to the above-mentioned RNA polymerase variants, the composition further comprises one or more in vitro transcription reaction reagents (such as buffer components, modified or unmodified nucleoside triphosphates, RNase inhibitors, pyrophosphatases, magnesium ions, water, etc.). In some embodiments, the composition further comprises a DNA template. In some embodiments, the composition further comprises a cap analog.

[0048] In some embodiments, the in vitro transcription reaction composition described in the present application comprises: an RNA polymerase variant, buffer components, modified or unmodified nucleoside triphosphates, RNase inhibitors, pyrophosphatases, magnesium ions, water, and a cap analog. In some embodiments, the in vitro transcription reaction composition described in the present application comprises: an RNA polymerase variant, buffer components, modified or unmodified nucleoside triphosphates, RNase inhibitors, pyrophosphatases, magnesium ions, water, a cap analog, and a DNA template.

[0049] Kit

[0050] The kit provided by the present application comprises at least one RNA polymerase variant described in the present application.

[0051] In some embodiments, the kit may further comprise one or more in vitro transcription reaction reagents (such as buffer components, modified or unmodified nucleoside triphosphates, RNase inhibitors, pyrophosphatases, magnesium ions, water, etc.). In some embodiments, the kit further comprises a cap analog. In some embodiments, each component (if applicable) in the kit can be provided in liquid form (such as in solution) or in solid form (such as dry powder).

[0052] The kit described in the present application may include one or more containers for accommodating one or more components described in the present application and optionally instruction materials.

[0053] Application or use

[0054] The present application provides the use of the above-mentioned RNA polymerase variant, composition, or kit in in vitro transcription.

[0055] The present application also provides the use of the above-mentioned RNA polymerase variant, composition, or kit 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.

[0056] Method for preparing RNA

[0057] The present application provides a method for preparing RNA. In some embodiments, the method comprises contacting a DNA template, modified or unmodified nucleoside triphosphates with at least one RNA polymerase variant described in the present application, 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.

[0058] The present application 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 with at least one RNA polymerase variant described in the present application, and incubating in an in vitro transcription reaction system to obtain a target product.

[0059] In vitro transcription reaction systems and incubation conditions suitable for generating RNA products or capped mRNA products are well known in the art. Those of ordinary skill in the art can determine appropriate reaction system pH values, reaction temperatures, reaction times, salt concentrations, or whether to add exogenous cofactors, etc. considering the optimal activity of RNA polymerase. In some embodiments, the in vitro transcription reaction system described in the present application contains in vitro transcription reaction reagents: one or more buffer components, modified or unmodified nucleoside triphosphates, RNase inhibitors, pyrophosphatases, magnesium ions, water, etc. In some embodiments, in the incubation step described in the present application, the incubation temperature is 30-50°C, preferably 37°C. In some embodiments, in the incubation step described in the present application, the incubation time is 20-240 min, preferably 60 min.

[0060] In some embodiments, the RNA products or capped mRNA products prepared by the method described in the present application have higher yields, and / or higher integrity, and / or lower dsRNA impurity content, and / or more capped mRNA products, etc. compared with those prepared using wild-type RNA polymerase.

[0061] In some embodiments, the utilization rate of the cap analog can be increased for the capped mRNA products prepared by the method described in the present application compared with using wild-type RNA polymerase, and the capping rate of the obtained mRNA products can be increased to 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 100%.

[0062] Cap analog

[0063] The cap analog in the method for preparing capped mRNA products described in this application or the cap analog in the in vitro transcription reaction composition refers to a molecule having a molecule that can be complementary to the nucleotide molecule on the DNA template at the transcription start site.

[0064] In some embodiments, the cap analog is selected from a dinucleotide cap, a trinucleotide cap, or a tetranucleotide cap. In some embodiments, the cap analog is a trinucleotide cap and is 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′OMepppC2′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.

[0065] In some embodiments, the cap analog of the present application is preferably m7GpppA2′OMepG.

[0066] In some embodiments, when preparing a capped mRNA product using the trinucleotide cap GAG (such as m7GpppA2′OMepG), or in an in vitro transcription reaction composition containing this cap analog, the first nucleotide at the +1 site of the DNA template molecule (sense strand) is G, and the second nucleotide at the +2 site is G. In some embodiments, the nucleotide residues in the m7GpppA2′OMepG cap analog can be complementary base-paired with the +1 site of the antisense strand of the DNA template molecule (such as Figure 2 ).

[0067] In vitro transcription reaction reagents

[0068] The in vitro transcription reaction reagents as described in the present application include buffer components, nucleoside triphosphates, RNase inhibitors, inorganic pyrophosphatases, magnesium ions, water (such as DEPC-water, RNase-free water, DNase-free water, sterile purified water, deionized water, distilled water, etc.), and the like.

[0069] In some embodiments, the buffer component is 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.

[0070] In some embodiments, the nucleoside triphosphate may be selected from modified or unmodified nucleoside triphosphates (including their analogs). In some embodiments, the nucleoside triphosphate may be selected from unmodified ATP, GTP, CTP, UTP. In some embodiments, the nucleoside triphosphate may 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), nucleoside triphosphates with labels (the label may be biotin, fluorescent substances, digoxin, radioactive elements, etc.).

[0071] In some embodiments, the in vitro transcription reaction reagent described in the present application may be selected from any commercially available in vitro transcription reagent for RNA.

[0072] Other embodiments:

[0073] 1. An RNA polymerase variant, whose amino acid sequence contains at least one substitution or deletion selected from the following amino acid sites relative to SEQ ID NO: 1: R34, K172, Y178, R386, D388, Q435, N437, D438.

[0074] 2. The variant according to item 1, wherein:

[0075] (1) The substitution at the R386 position is selected from C or W;

[0076] (2) The substitution at the D388 position is K;

[0077] (3) The substitution at the Q435 position is selected from A, H, T;

[0078] (4) The substitution at the N437 position is selected from F, T;

[0079] (5) The substitution at the D438 position is selected from T, L, I, P, V;

[0080] (6) The substitution at the R34 position is selected from A;

[0081] (7) The mutation at the K172 position is a deletion;

[0082] (8) The substitution at the Y178 position is selected from H, D.

[0083] 3. The variant according to the first item, wherein its amino acid sequence contains a mutation at any site selected from the following with respect to SEQ ID NO: 1: 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, R386C+D438P.

[0084] 4. The variant according to item 1, wherein the amino acid sequence of the variant is as shown in any one of SEQ ID NOs: 2-22.

[0085] 5. A polynucleotide molecule encoding the variant according to any one of items 1-4.

[0086] 6. An expression vector comprising the polynucleotide molecule according to item 5.

[0087] 7. A host cell comprising the polynucleotide molecule according to item 5, or the expression vector according to item 6.

[0088] 8. A method for preparing the variant according to any one of items 1-4, comprising:

[0089] (1) Culturing the host cell according to item 7; and

[0090] (2) Recovering the variant.

[0091] 9. A composition comprising the variant according to any one of items 1-4.

[0092] 10. The composition according to item 9, further comprising a cap analog.

[0093] 11. The composition according to item 9 or 10, further comprising a DNA template.

[0094] 12. A kit comprising the variant according to any one of items 1-4.

[0095] 13. Use of the variant according to any one of items 1-4, the composition according to any one of items 9-11, or the kit according to item 12 in in vitro transcription.

[0096] 14. 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 items 1-4, incubating in an in vitro transcription reaction system to obtain a target RNA product.

[0097] 15. A method for preparing capped mRNA, which comprises contacting a DNA template, modified or unmodified nucleoside triphosphates, a cap analogue with any of the RNA polymerase variants described in items 1-4, and incubating in an in vitro transcription reaction system to obtain a target product.

[0098] 16. The method according to item 15, wherein the cap analogue is a trinucleotide cap, preferably m7GpppA2′OMepG.

[0099] 17. In the composition according to item 11 or any of the methods according to items 14-16, the DNA template has 2′-deoxyguanosine residues at positions +1 and +2.

[0100] 18. Use of any of the variants according to items 1-4, any of the compositions according to items 9-11, or the kit according to item 12 in the transcription of a DNA template having 2′-deoxyguanosine residues at positions +1 and +2.

[0101] Beneficial effects

[0102] The present application provides a class of RNA polymerase variants and a preparation method thereof. By modifying wild-type T7 RNA polymerase, RNA polymerase variants with high catalytic efficiency are obtained. Using such variants, the capping rate of mRNA products in the in vitro transcription process can be increased, and more capped mRNA products can be obtained. In addition, the present application also provides a method for preparing capped mRNA. Using this method, the yield of capped mRNA products can be increased, while the dosage of cap analogues can be reduced, avoiding waste of raw materials and saving production costs, which is of great significance for the economical production of RNA. Description of the drawings

[0103] Figure 1 It is a schematic diagram of a double-stranded DNA template;

[0104] Figure 2 It is the complementary pairing of a cap analogue and a DNA template;

[0105] Figure 3 It is a schematic diagram of the construction of a recombinant plasmid;

[0106] Figure 4 It is the effect of RNA polymerase and its variants (R386W, Q435A, Q435H, Q435T, N437F, N437T, D438T, D438L, D438I, D438P, D438V, R386A+R34A, R386M+R34A, R386M+Y178H, R386W+N437T, R386I+Del172, R386F+Y178D) on the capping rate of mRNA;

[0107] Figure 5 The effects of RNA polymerase and its variants (WT, R386C, R386W, D388K, R386C+D438P, R386W+N437T, D388K+D438P, N437T, D438P) on the mRNA capping rate. Specific implementation manners

[0108] In the embodiments of the present application, the unit enzyme activity (U) is defined as the amount of enzyme required to incorporate 1 nmol 3 of [³H]ATP into acid-insoluble precipitate within 1 hour under the conditions of 37°C and pH 8.0, which is defined as 1 active unit.

[0109] Example 1 Preparation of RNA polymerase variants

[0110] The RNA polymerases shown in Table 1-1 to Table 1-6 were subjected to DNA sequence synthesis (SEQ ID NO: 23-44), followed by PCR amplification. Then, the recombinant expression vector was obtained by introducing it into the BseRI and HindIII restriction enzyme sites of the expression vector pQE-80L. The constructed vector was introduced into E. coli BL21(DE3) by chemical transformation technology, spread on an LB plate containing ampicillin resistance, and placed in an incubator at 37°C overnight. The grown single colonies were subjected to plasmid extraction and sequencing, and finally, the recombinant engineering bacteria containing the target gene were obtained. The successfully sequenced E. coli recombinant strain was inoculated into LB medium for overnight activation culture, and then inoculated into the fermentation broth (LB medium) at 1-5% V / V. After culturing until the OD600 value reached 0.6-0.8, IPTG with a final concentration of 0.5 mol / L was added and the culture was continued for 4-6 h. Then, the strain was collected by centrifugation at 12,000 rpm and 4°C. The collected strain was washed with a PBS buffer solution with a pH value of 7.0 and a concentration of 0.2 M to obtain the bacterial cells. After ultrasonic disruption, affinity chromatography purification was performed to obtain the crude RNA polymerase solution.

[0111] The corresponding relationship between RNA polymerase variants and amino acid sequences is shown in Table 1-1 to Table 1-6:

[0112] Table 1:

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119] Example 2 Preparation of mRNA by in vitro transcription reaction

[0120] The stock enzyme 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. The MIX solution was prepared in an EP tube according to the reaction system (20 μL) in Table 2, transferred to an eight-strip tube, mixed well, and centrifuged; the eight-strip tube was placed on a PCR instrument and reacted at 37 °C for 1 h, then 36 μL of magnetic beads (Vazyme, product number: N412) was added, mixed well, and incubated at room temperature for 2 - 5 min; the mixture was placed on a magnetic stand to purify the mRNA, and after purification, it was transferred to an RNase-free centrifuge tube to obtain the purified mRNA.

[0121] Table 2: Reaction system ratio

[0122]

[0123] Example 3 Capping rate detection

[0124] After pretreatment with an mRNA Capping Rate detection kit (Vazyme, product number: DD3510-01), the capping rate of the mRNA product was detected by LC-MS:

[0125] (1) The purified mRNA in Example 2 was bound to the probe, and the reaction system is shown in Table 3 and the reaction conditions are shown in Table 4;

[0126] Table 3: Reaction system

[0127]

[0128] Table 4: Reaction conditions

[0129] Temperature Time 95℃ 2 mins 70～16℃ -0.1℃ / s, ≈180 cycles 16℃ ∞

[0130] (2) RNase H digestion: Prepare the reaction digestion system according to Table 5, vortex and mix well, then place it in a PCR instrument and react at 25 °C for 20 min;

[0131] Table 5: Reaction system

[0132] Component Volume Previous step product 21 μL RNase H Reaction Buffer (10×) 3 μL RNase H (5 U / μl) 3 μL <![CDATA[RNase-free H2O]]> 3 μL Total system 30 μL

[0133] (3) SA magnetic bead binding:

[0134] ① Magnetic bead washing: Take 9 μL of SA magnetic beads (Cat. No.: SM017005) into a centrifuge tube, place it on a magnetic stand. After the solution becomes clear, aspirate and discard the supernatant with a pipette; Remove the centrifuge tube from the magnetic stand, add 200 μL of RNase-free H2O for rinsing, place it on the magnetic stand, aspirate and discard the supernatant with a pipette after the solution becomes clear, and then add 200 μL of RNase-free H2O to repeat the rinsing once.

[0135] ② Reaction conditions: Remove the centrifuge tube from the magnetic stand, add the digested product to the SA magnetic beads (solid), pipette 20 - 30 times to mix well, place it on a tumbling instrument and incubate at room temperature for 30 min to allow the magnetic beads to fully bind to the digested product.

[0136] (4) Rinsing and elution:

[0137] ① Place the product from the previous step on the magnetic stand for 2 - 3 min. After the solution becomes clear, aspirate and discard the supernatant with a pipette;

[0138] ② Add 200 μL of wash buffer for rinsing, be careful not to disperse the magnetic beads, let it stand for 0.5 - 1 min, and aspirate and discard the supernatant with a pipette;

[0139] ③ Repeat step ②;

[0140] ④ Remove the centrifuge tube from the magnetic stand, add 30 μL of elution buffer, pipette 10 - 20 times to mix well, disperse the magnetic beads evenly, and elute fully;

[0141] ⑤ Place it in a PCR instrument, react at 85 °C for 3 min and then immediately place it on the magnetic stand. After the solution becomes clear (0.5 - 1 min), aspirate the supernatant into a new centrifuge tube. The supernatant is the required product;

[0142] ⑥ Send the above product to 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 3μm, 4.6*100mm;; ion mode: negative ion; scan mode: Full scan; scan range: 600 - 3000), the capping rate calculation formula:

[0143] mRNA capping rate (%) = (capped mRNA / (capped mRNA + uncapped mRNA)) × 100%.

[0144] The test results are shown inFigure 4 When the ratio of the cap analog to the raw material NTP was as low as 0.33:1, all the T7 RNA polymerase variants in Example 1 could well improve the capping rate of the mRNA product, and R386W+N437T increased the capping rate to 100%.

[0145] Preparation of mRNA by in vitro transcription reaction in Example 4

[0146] Refer to Example 2 above for the preparation of mRNA by in vitro transcription, and further reduce the addition amount of the cap analog to 0.24 μL (specifically shown in Table 6), with the remaining reaction conditions being the same, and further refer to Example 3 for the capping rate detection. Table 6: Reaction system ratio

[0147]

[0148]

[0149] The results are as Figure 5 shown. Compared with the T7 RNAP wild type, all mutation sites effectively increased the capping rate. Among them, the T7 RNAP double mutations R386C+D438P, R386W+N437T, and D388K+D438P were further significantly increased on the basis of single sites. Among them, D388K+D438P was increased from the capping rate of 84% of the single site D388K and 68.5% of the single site D438P to 95.5%, with an increase amplitude > 10%.

Claims

1. An RNA polymerase variant, characterized in that The amino acid sequence of the variant comprises a substitution at position D438 relative to SEQ ID NO:

1. The amino acid sequence of the variant is shown in any one of SEQ ID NO: 10-14 or 21.

2. A polynucleotide molecule, characterized in that The polynucleotide molecule encodes the variant according to claim 1.

3. An expression vector, characterized in that Comprising the polynucleotide molecule as claimed in claim 2.

4. A host cell, characterized in that Comprising the polynucleotide molecule as claimed in claim 2 or the expression vector as claimed in claim 3.

5. The method for preparing the variant according to claim 1, characterized in that: include: (1) culturing the host cell according to claim 4; and (2) Recover variants.

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

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

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

9. A method for preparing RNA, characterized in that: The method comprises contacting a DNA template, modified or unmodified nucleoside triphosphates with the RNA polymerase variant of claim 1, and incubating them in an in vitro transcription reaction system to obtain a target RNA product.

10. A method for preparing capped mRNA, characterized in that: The method comprises contacting a DNA template, modified or unmodified nucleoside triphosphates, a cap analog and the RNA polymerase variant of claim 1, incubating them in an in vitro transcription reaction system, and obtaining a target product.

11. The method according to claim 10, characterized in that The cap analog is a trinucleotide cap.

12. The method according to claim 11, characterized in that The trinucleotide cap is m7GpppA2′OMepG.

13. The method according to any one of claims 9 to 12, characterized in that: Positions +1 and +2 of the DNA template are 2'-deoxyguanosine residues.

14. Use of the variant of claim 1, the composition of claim 6, or the kit of claim 7 for transcription of a DNA template having 2'-deoxyguanosine residues at template positions +1 and +2.

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