RNA polymerase variants and their applications

Abstract

The present application provides a class of T7 RNA polymerase variants and applications thereof, wherein the amino acid sequence of the T7 RNA polymerase variant comprises 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. The variant has improved catalytic activity relative to the wild-type T7 RNA polymerase, and can increase the capping rate of mRNA products during in vitro transcription (co-transcription capping). In addition, the present application also provides a method for preparing RNA using such variants, and by using the method to prepare RNA molecules, more capped mRNA can be obtained.

Description

Technical Field

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

[0002] The 5' end of complete eukaryotic mRNA contains a 7-methylguanosine (m7G) cap, which is formed in vivo through the catalysis of RNA triphosphatase, mRNA guanylate transferase, mRNA methyltransferase, and mRNA nucleoside 2'-oxymethyltransferase. This 5' cap structure participates in preventing mRNA degradation by exonucleases, reducing mRNA immunogenicity, regulating mRNA half-life, and regulating translation. In the process of preparing mRNA through in vitro transcription (IVT), researchers often add cap analogs to mimic the 5' cap structure of eukaryotic mRNA.

[0003] The 5' cap structure of mRNA has a significant impact on mRNA stability, translation efficiency, and immunogenicity. With ongoing research, the importance of cap analogs has become increasingly prominent, leading to the continuous development of novel cap analogs for use in mRNA vaccines and therapeutic RNAs. Currently, cap analogs have evolved to the third generation. First-generation cap analogs, due to the presence of two free 3'-OH groups, exhibit reverse incorporation, making them rarely seen on the market. The most common commercially available cap analogs are the second-generation ARCA cap analog (Equation 1) and the third-generation cap analog (Equation 2), whose main structural formulas are shown in the examples below.

[0004]

[0005] Formula 1

[0006]

[0007] Formula 2

[0008] ARCA cap analogs are modified cap analogs in which the 3'-OH group near m7G is replaced with -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. Third-generation cap analogs, such as CleanCap AG, can form a Cap 1 structure, significantly improving the capping efficiency compared to second-generation cap analogs. However, this requires replacing the wild-type promoter with 5'-TAATACGACTCACTATAGG-3'. Whether this promoter replacement will introduce safety issues or new impurities remains to be proven. Even with a wild-type promoter, the capping efficiency remains low, and uncapped RNA products not only waste raw materials but also require subsequent column purification, increasing production costs. Therefore, modifying T7 RNA polymerase can improve cap analog utilization without requiring wild-type promoter replacement, which is of great significance for the economical production of mRNA and drug safety. Summary of the Invention Invention Overview

[0010] In a first aspect, this application provides a class of RNA polymerase variants whose amino acid sequence contains a mutation relative to SEQ ID NO: 1 at at least one amino acid selected from the positions R34, K172, Y178, R386, D388, Q435, N437 or D438, wherein the mutation type may be either substitution or deletion.

[0011] Secondly, this application provides one or more biological materials selected from the following:

[0012] 1) A polynucleotide molecule encoding an RNA polymerase variant;

[0013] 2) Expression vectors containing polynucleotide molecules as described in 1);

[0014] 3) Host cells containing the polynucleotide molecules described in 1), or host cells containing the expression vectors described in 2).

[0015] Thirdly, this application provides a method for preparing the aforementioned RNA polymerase variant.

[0016] Fourthly, this application provides a composition comprising at least one RNA polymerase variant as described in this application.

[0017] Fifthly, this application provides a kit comprising at least one RNA polymerase variant as described in this application.

[0018] Sixthly, this application also provides the use of the above-mentioned RNA polymerase variants, compositions or kits in in vitro transcription.

[0019] Seventhly, this application also provides a method for preparing RNA or capped RNA. Invention Details

[0021] RNA polymerase variants

[0022] The RNA polymerase variant provided in this application is a phage T7 RNA polymerase (T7 RNAP) variant, the amino acid sequence of which, relative to SEQ ID NO: 1, contains at least one mutation selected from the following amino acid sites: R34, K172, Y178, R386, D388, Q435, N437 or D438, wherein the mutation type is selected from substitution or deletion.

[0023] In some implementations, the variant is replaced with A at the R34 site.

[0024] In some implementations, the mutation type at the K172 site of the variant is deletion.

[0025] In some implementations, the substitution at the Y178 site of the variant may be either H or D.

[0026] In some implementations, the substitution at the R386 site of the variant may be selected from: C, W, M, A, I, or F.

[0027] In some implementations, the variant is replaced with K at the D388 site.

[0028] In some implementations, the substitution at the Q435 site of the variant may be selected from A, H, or T.

[0029] In some implementations, the substitution at the N437 site of the variant may be either F or T.

[0030] In some implementations, the substitution at the D438 site of the variant can be selected from: T, L, I, P or V.

[0031] In some embodiments, the amino acid sequence of the variant, compared to SEQ ID NO: 1, includes any of the following substitutions or groups of substitutions: 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.

[0032] In some embodiments, the RNA polymerase variants provided in this application have an amino acid sequence that is at least 97%, at least 98%, at least 99%, or higher sequence identical to any of the sequences shown in SEQ ID NO: 2-22. In some embodiments, the amino acid sequence of the variant is as shown in any of SEQ ID NO: 2-22.

[0033] Polynucleotide molecules

[0034] The polynucleotide molecules provided in this application encode any of the RNA polymerase variants described in this application. In some embodiments, the polynucleotide molecules are as shown in any of SEQ ID NO: 23-44.

[0035] The polynucleotide molecule described in this application may have various modifications in its coding region, as long as the amino acid sequence of the variant does not change with the degeneracy of the codon or with the preferred codon in the organism expressing the variant.

[0036] expression carrier

[0037] The expression vectors provided in this application refer to linear or circular DNA molecules, typically containing elements such as multiple cloning sites, resistance genes, and replication origin sites. In some embodiments, the expression vector described in this application is pQE-80L.

[0038] In some embodiments, the vector described in this application comprises a multinucleotide molecule encoding a variant of the RNA polymerase described in this application. In some further embodiments, the vector also comprises one or more regulatory sequences (such as enhancer, promoter, and terminator sequences) operatively linked to the multinucleotide molecule encoding the variant.

[0039] host cells

[0040] The host cell provided in this application can be any cell that is favorable for the expression of the variants of this application, that is, any cell that is susceptible after being transformed, transfected or transduced with the expression vector of this application, and includes any cell progeny that is different from the parent cell due to mutations that occur during replication.

[0041] In some embodiments, the host cell described in this application comprises the above-described polynucleotide molecule or the expression vector.

[0042] In some embodiments, the host cell is a prokaryotic cell, which may be selected from Gram-positive or Gram-negative bacteria. In some embodiments, the host cell is a Gram-positive bacterium, including but not limited to: *Bacillus*, *Clostridium*, *Enterococcus*, *Bacillus aeruginosa*, *Lactobacillus*, *Lactococcus*, *Bacillus cereus*, *Staphylococcus*, *Streptococcus*, and *Streptomyces*. In some embodiments, the host cell is a Gram-negative bacterium, including but not limited to: *Campylobacter*, *Escherichia coli*, *Flavobacterium*, *Fusobacterium*, *Helicobacter*, *Selenobacter*, *Neisseria*, *Pseudomonas*, *Salmonella*, and *Ureaplasma*.

[0043] Methods for preparing variants

[0044] This application provides a method for preparing the above-mentioned RNA polymerase variant, comprising: (1) culturing the host cell described in this application under conditions suitable for the expression of the variant; and (2) recovering the variant.

[0045] In some embodiments, the method for recovering variants can be a method known in the art, such as centrifugation, filtration, treatment with a crystalline protein precipitant (salting out), extraction, ultrasonic disruption, ultrafiltration, dialysis, various chromatographic methods such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC, and combinations thereof.

[0046] In some embodiments, the preparation method further includes a purification step of the variant, which can be a method known in the art, such as chromatography (e.g., ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography), ammonium sulfate precipitation, etc.

[0047] Composition

[0048] The composition provided in this application comprises at least one RNA polymerase variant described in this application.

[0049] The composition described in this application can be a composition for storing RNA polymerase variants. In some embodiments, in addition to the aforementioned RNA polymerase variants, the composition described in this application may optionally contain: buffer components (such as Tris base, Tris-HCl, HEPES, MOPS), salts (such as NaCl), enzyme inhibitors (such as EDTA), reducing agents (such as DTT), surfactants (such as Triton X-100), stabilizers (such as glycerol), and other components. In some embodiments, the composition for storing RNA polymerase variants described in this application comprises: RNA polymerase variant, Tris-HCl, NaCl, EDTA, DTT, Triton X-100, and glycerol.

[0050] The compositions of this application may also be in vitro transcription reaction compositions. In some embodiments, the compositions, in addition to the RNA polymerase variants described above, further comprise one or more in vitro transcription reaction reagents (e.g., buffer components, modified or unmodified nucleoside triphosphates, RNase inhibitors, pyrophosphatase, magnesium ions, water, etc.). In some embodiments, the compositions further comprise a DNA template. In some embodiments, the compositions further comprise a cap analogue.

[0051] In some embodiments, the in vitro transcription reaction composition of this application comprises: an RNA polymerase variant, a buffer component, a modified or unmodified nucleoside triphosphate, an RNase inhibitor, a pyrophosphatase, magnesium ions, water, and a cap analog. In some embodiments, the in vitro transcription reaction composition of this application comprises: an RNA polymerase variant, a buffer component, a modified or unmodified nucleoside triphosphate, an RNase inhibitor, a pyrophosphatase, magnesium ions, water, a cap analog, and a DNA template.

[0052] Reagent test kit

[0053] The kit provided in this application contains at least one RNA polymerase variant described in this application.

[0054] In some embodiments, the kit may also contain one or more in vitro transcription reaction reagents (e.g., buffer components, modified or unmodified nucleoside triphosphates, RNase inhibitors, pyrophosphatase, magnesium ions, water, etc.). In some embodiments, the kit also contains a cap analogue. In some embodiments, each component in the kit (if applicable) may be provided in liquid form (e.g., in solution) or in solid form (e.g., dry powder).

[0055] The kit described in this application may include one or more containers containing one or more components described in this application and optional instructions for use.

[0056] Applications or uses

[0057] This application provides the use of the above-mentioned RNA polymerase variants, compositions, or kits in in vitro transcription.

[0058] This application also provides the use of the above-mentioned RNA polymerase variants, compositions, or kits in various methods, including but not limited to RNA preparation, RNA probe preparation, RNA vaccine preparation, and protein preparation.

[0059] Methods for preparing RNA

[0060] This application provides a method for preparing RNA. In some embodiments, the method includes contacting a DNA template, modified or unmodified nucleoside triphosphates, with at least one RNA polymerase variant described in this application, incubating in an in vitro transcription reaction system to obtain a target RNA product. In some embodiments, the RNA product may be dsRNA, ssRNA, mRNA, siRNA, miRNA, piRNA, shRNA, or gRNA.

[0061] This application also provides a method for preparing capped mRNA. In some embodiments, the method includes contacting a DNA template, a modified or unmodified nucleoside triphosphate, a capping analogue, and at least one RNA polymerase variant described in this application, incubating in an in vitro transcription reaction system to obtain the target product.

[0062] Suitable in vitro transcription reaction systems and incubation conditions for generating RNA products or capped mRNA products are well known in the art. Those skilled in the art can determine appropriate reaction system pH, reaction temperature, reaction time, salt concentration, or whether to add exogenous cofactors, taking into account the optimal activity of RNA polymerase. In some embodiments, the in vitro transcription reaction system described in this application includes in vitro transcription reaction reagents: one or more buffer components, modified or unmodified nucleoside triphosphates, RNase inhibitors, pyrophosphatase, magnesium ions, water, etc. In some embodiments, the incubation temperature in the incubation step described in this application is 30-50°C, preferably 37°C. In some embodiments, the incubation time in the incubation step described in this application is 20-240 min, preferably 60 min.

[0063] In some embodiments, RNA products or capped mRNA products prepared using the methods described in this application have higher yields, and / or higher integrity, and / or lower dsRNA impurity content, and / or more capped mRNA products compared to those prepared using wild-type RNA polymerase.

[0064] In some embodiments, the capped mRNA product prepared using the method described in this application can improve the utilization rate of the capped analog compared to using wild-type RNA polymerase, wherein the capping rate of the obtained mRNA product 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%.

[0065] Hat-like items

[0066] The cap analogues used in the methods for preparing capped mRNA products or in in vitro transcription reaction compositions described in this application refer to molecules that are complementary to nucleotide molecules on the DNA template at the transcription start site.

[0067] In some embodiments, the cap analogue may be selected from a dinucleotide cap, a trinucleotide cap, or a tetranucleotide cap. In some embodiments, the cap analogue is a trinucleotide cap, which may be selected from GAA, GAC, GAG, GAU, GCA, GCC, GCG, GCU, GGA, GGC, GGG, GGU, GUA, GUC, GUG, and GUU. In some embodiments, the trinucleotide cap may be selected from m7GpppApA, m7GpppApC, m7GpppApG, m7GpppApU, m7GpppCpA, m7GpppCpC, m7GpppCpG, m7GpppCpU, m7GpppGpA, m7GpppGpC, m7GpppGpG, m7GpppGpU, m7GpppUpA, m7GppppUpC, m7GppppUpG, and m7GppppUpU. In some embodiments, the trinucleotide cap may be selected from m7G3′OMepppApA, m7G3′OMepppApC, m7G3′OMepppApG, m7G3′OMepppApU, m7G3′OMepppCpA, m7G3′OMepppCpC, m7G3′OMepppCpG, m7G3′OMepppCpU, m7G3′OMepppGpA, m7G3′OMepppGpC, m7G3′OMepppGpG, m7G3′OMepppGpU, m7G3′OMepppUpA, m7G3′OMepppUpC, m7G3′OMepppUpG, and m7G3′OMepppUpU. In some embodiments, the trinucleotide cap may be 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 may be selected from m7GpppA2′OMepA, m7GpppA2′OMepC, m7GpppA2′OMepG, m7GpppA2′OMepU, m7GpppC2′OMepA, m7GpppC2′OMepC, m7GpppC2′OMepG, m7GpppC2′OMepU, m7GpppG2′OMepA, m7GpppG2′OMepC, m7GpppG2′OMepG, m7GpppG2′OMepU, m7GpppU2′OMepA, m7GpppU2′OMepC, m7GpppU2′OMepG, and m7GpppU2′OMepU.

[0068] In some embodiments, the hat analogue described in this application is preferably m7GpppA2′OMepG.

[0069] In some embodiments, when using a trinucleotide-capped GAG (such as m7GpppA2′OMepG) to prepare capped mRNA products, or in in vitro transcription reaction compositions containing such a capped analogue, 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 capped analogue are complementary to the +1 site of the antisense strand of the DNA template molecule (such as...). Figure 2 ).

[0070] In vitro transcription reaction reagents

[0071] The in vitro transcription reaction reagent described in this application includes buffer components, nucleoside triphosphates, RNase inhibitors, inorganic pyrophosphatase, magnesium ions, and water (such as DEPC-water, RNase-free water, DNase-free water, sterile purified water, deionized water, distilled water, etc.).

[0072] In some embodiments, the buffering component may be selected from one or more of the following: phosphate buffer, Tris buffer, MOPS buffer, HEPES buffer, citrate buffer, acetate buffer, malate buffer, MES buffer, histidine buffer, PIPES buffer, bis-tris buffer, and ethanolamine buffer.

[0073] In some embodiments, the nucleoside triphosphate may be selected from modified or unmodified nucleoside triphosphates (including analogues thereof). In some embodiments, the nucleoside triphosphate may be selected from unmodified ATP, GTP, CTP, or UTP. In some embodiments, the nucleoside triphosphate may be selected from modified nucleoside triphosphates, and the modification types on the nucleoside include, but are not limited to, m1A (N1-methyladenosine), m6A (N6-methyladenosine), m5C (5-methylcytidine), 5moU (5-methoxyuridine), ψ (pseudouridine), m1ψ (N1-methyl-pseudouridine), and labeled nucleoside triphosphates (the label may be biotin, fluorescent substances, digoxigenin, radioactive elements, etc.).

[0074] In some embodiments, the in vitro transcription reaction reagent described in this application may be selected from any commercially available RNA in vitro transcription reagent.

[0075] Other implementation plans:

[0076] 1. An RNA polymerase variant whose amino acid sequence relative to SEQ ID NO: 1 includes at least one substitution or deletion selected from the following amino acid sites: R34, K172, Y178, R386, D388, Q435, N437, D438.

[0077] 2. A variant as described in item 1, wherein:

[0078] (1) The substitution at position R386 is selected from C or W;

[0079] (2) The substitution at position D388 is K;

[0080] (3) The substitution at position Q435 is selected from A, H, and T;

[0081] (4) The substitution at position N437 is selected from F and T;

[0082] (5) The substitution at position D438 is selected from T, L, I, P, and V;

[0083] (6) The substitution at position R34 is selected from A;

[0084] (7) The mutation at position K172 is a deletion;

[0085] (8) The substitution at position Y178 is selected from H and D.

[0086] 3. The variant described in item 1, wherein the amino acid sequence relative to SEQ ID NO: 1 contains any mutation selected from the following sites: 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.

[0087] 4. The variants described in item 1, wherein the amino acid sequences of the variants are shown in any of SEQ ID NO: 2-22.

[0088] 5. A polynucleotide molecule that encodes a variant as described in any of items 1-4.

[0089] 6. An expression vector comprising a polynucleotide molecule as described in item 5.

[0090] 7. A host cell containing a polynucleotide molecule as described in item 5, or an expression vector as described in item 6.

[0091] 8. A method for preparing any of the variants described in items 1-4, comprising:

[0092] (1) Culturing host cells as described in item 7; and

[0093] (2) Recycle variants.

[0094] 9. A composition comprising any of the variants described in items 1-4.

[0095] 10. The composition as described in item 9, further comprising a hat analogue.

[0096] 11. The composition as described in item 9 or 10, further comprising a DNA template.

[0097] 12. A kit containing any of the variants described in items 1-4.

[0098] 13. Use of any variant described in items 1-4, any composition described in items 9-11, or the kit described in item 12 in in vitro transcription.

[0099] 14. A method for preparing RNA, comprising contacting a DNA template, a modified or unmodified nucleoside triphosphate, with any of the RNA polymerase variants described in items 1-4, incubating in an in vitro transcription reaction system to obtain a target RNA product.

[0100] 15. A method for preparing capped mRNA, comprising contacting a DNA template, a modified or unmodified nucleoside triphosphate, a cap analogue, and any of the RNA polymerase variants described in items 1-4, incubating in an in vitro transcription reaction system to obtain the target product.

[0101] 16. The method as described in item 15, wherein the cap analogue is a trinucleotide cap, preferably m7GpppA2′OMepG.

[0102] 17. In the composition of claim 11 or the method of any of claims 14-16, the DNA template positions +1 and +2 are 2'-deoxyguanosine residues.

[0103] 18. Use of any variant described in items 1-4, any composition described in items 9-11, or the kit described in item 12, in the transcription of DNA templates containing 2'-deoxyguanosine residues at template positions +1 and +2.

[0104] Beneficial effects

[0105] This application provides a class of RNA polymerase variants and their preparation methods. By modifying wild-type T7 RNA polymerase, RNA polymerase variants with higher catalytic efficiency are obtained. Using these variants, the capping rate of mRNA products during in vitro transcription can be increased, resulting in more capped mRNA products. Furthermore, this application also provides a method for preparing capped mRNA, which can increase the yield of capped mRNA products while reducing the amount of cap analogues used, avoiding raw material waste and saving production costs. This is of great significance for the economical production of RNA. Attached Figure Description

[0106] Figure 1 A schematic diagram of a double-stranded DNA template;

[0107] Figure 2 This is a complementary pairing between the cap-like analogue and the DNA template;

[0108] Figure 3 This is a schematic diagram illustrating the construction of recombinant plasmids;

[0109] Figure 4 The effects of RNA polymerases and their 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 mRNA capping efficiency;

[0110] Figure 5 The effects of RNA polymerase and its variants (WT, R386C, R386W, D388K, R386C+D438P, R386W+N437T, D388K+D438P, N437T, D438P) on mRNA capping rate. Detailed Implementation

[0111] In this application embodiment, unit enzyme activity (U) is defined as the amount of enzyme that can be activated by 1 nmol of enzyme at 37°C and pH 8.0 within 1 hour. 3 The amount of enzyme required to incorporate H]ATP into an acid-insoluble precipitate is defined as one active unit.

[0112] Example 1: Preparation of RNA polymerase variants

[0113] The RNA polymerases shown in Tables 1-1 to 1-6 were synthesized using DNA sequences (SEQ ID NO: 23-44) and then amplified by PCR. The resulting DNA was then introduced into the BseRI and HindIII restriction sites of the expression vector pQE-80L to obtain a recombinant expression vector. The constructed vector was then introduced into the DNA via chemical transformation. E. coli In BL21(DE3), the bacteria were plated on LB agar plates containing ampicillin and incubated overnight at 37°C. Single colonies that grew were then subjected to plasmid extraction and sequencing to obtain recombinant engineered bacteria containing the target gene. Successfully sequenced colonies were then... E. coli After overnight activation culture in LB medium, the recombinant strain was inoculated into the fermentation broth (LB medium) at a concentration of 1-5% v / v and cultured until the OD 600 value reached 0.6-0.8. IPTG was added to a final concentration of 0.5 mol / L and cultured for another 4-6 h. The strain was then collected by centrifugation at 12000 rpm and 4°C. The collected strain was washed with 0.2 M PBS buffer (pH 7.0) to obtain the bacterial cells. After sonication, affinity chromatography was performed to purify the RNA polymerase stock solution.

[0114] The correspondences between RNA polymerase variants and amino acid sequences are shown in Tables 1-1 to 1-6:

[0115] Table 1-1:

[0116]

[0117] Table 1-2:

[0118]

[0119] Table 1-3:

[0120]

[0121] Table 1-4:

[0122]

[0123] Table 1-5:

[0124]

[0125] Table 1-6:

[0126]

[0127] Example 2: Preparation of mRNA by in vitro transcription reaction

[0128] The enzyme stock solution was diluted with storage buffer (50 mM Tris-HCl (25℃, pH 7.9), 100 mM NaCl, 0.1 mM EDTA, 2 mM DTT, 0.1% Triton X-100, 50% Glycerol) to an enzyme activity of 400 U / μL. A MIX solution was prepared according to the reaction system (20 μL) in Table 2 and transferred to an EP tube. The MIX solution was then transferred to an octet, mixed, and centrifuged. The octet was placed on a PCR instrument and reacted at 37℃ for 1 h. 36 μL of magnetic beads (Vazyme, catalog number: N412) was added, mixed, and incubated at room temperature for 2–5 min. The mixture was then placed on a magnetic rack to purify the mRNA. After purification, the mRNA was transferred to an RNase-free centrifuge tube to obtain the purified mRNA.

[0129] Table 2: Reaction System Proportions

[0130]

[0131] Example 3: Capping Rate Detection

[0132] After pretreatment using the mRNA Capping Rate Detection Kit (Vazyme, catalog number: DD3510-01), the capping rate of the mRNA product was detected by LC-MS.

[0133] (1) The purified mRNA from Example 2 was bound to the probe. The reaction system is shown in Table 3 and the reaction conditions are shown in Table 4.

[0134] Table 3: Reaction System

[0135]

[0136] Table 4: Reaction Conditions

[0137]

[0138] (2) RNase H digestion: Prepare the reaction digestion system according to Table 5, vortex thoroughly to mix evenly, and place it in a PCR instrument. React at 25℃ for 20 min.

[0139] Table 5: Reaction System

[0140]

[0141] (3) SA magnetic bead bonding:

[0142] ① Magnetic bead cleaning: Take 9 μL of SA magnetic beads (Cat.No.:SM017005) into a centrifuge tube, place it on a magnetic rack, and wait for the solution to become clear. Then, use a pipette to remove the supernatant. Remove the centrifuge tube from the magnetic rack, add 200 μL of RNase-free H2O to rinse, place it on the magnetic rack, and wait for the solution to become clear. Then, use a pipette to remove the supernatant, and add another 200 μL of RNase-free H2O to rinse once more.

[0143] ②Reaction conditions: Remove the centrifuge tube from the magnetic rack, add the enzyme digestion product to the SA magnetic beads (solid), pipette and mix thoroughly 20-30 times, place on a tumbler and incubate at room temperature for 30 minutes to allow the magnetic beads to fully combine with the enzyme digestion product.

[0144] (4) Rinsing and elution:

[0145] ① Place the product from the previous step on a magnetic rack for 2-3 minutes until the solution becomes clear, then use a pipette to remove the supernatant;

[0146] ② Add 200 μL of rinsing solution to rinse, being careful not to blow away the magnetic beads, let stand for 0.5~1 min, and then use a pipette to remove the supernatant;

[0147] ③ Repeat step ②;

[0148] ④ Remove the centrifuge tube from the magnetic rack, add 30 μL of elution buffer, and mix thoroughly by pipetting 10-20 times to ensure even dispersion of the magnetic beads and complete elution;

[0149] ⑤ Place it in a PCR instrument and react at 85℃ for 3 min. Immediately after that, place it on a magnetic rack. After the solution becomes clear (0.5~1 min), aspirate the supernatant into a new centrifuge tube. The supernatant is the desired product.

[0150] ⑥ The above product was sent to a Thermo Scientific Vanquish Flex-Qrbitrap Exploris 120 chromatographic instrument for capping rate determination (mobile phase: Phase A: 2% hexafluoroisopropanol-1% N'N-diisopropylethylamine aqueous solution; Phase B: 2% hexafluoroisopropanol-1% N'N-diisopropylethylamine methanol solution; column: Nano ChromCore C18 3μm, 4.6*100mm; ion mode: negative ion; scan mode: full scan; scan range: 600-3000). Capping rate calculation formula:

[0151] mRNA capping rate (%) = (capped mRNA / (capped mRNA + uncapped mRNA)) ×100%.

[0152] Test results are shown Figure 4 When the ratio of cap analog to raw material NTP is as low as 0.33:1, the T7 RNA polymerase variants in Example 1 can all significantly improve the capping rate of mRNA products, with R386W+N437T increasing the capping rate to 100%.

[0153] Example 4: Preparation of mRNA by in vitro transcription reaction

[0154] mRNA was prepared by in vitro transcription according to Example 2 above, and the amount of cap analog added was further reduced to 0.24 μL (as shown in Table 6). The other reaction conditions were the same, and the capping rate was further detected according to Example 3.

[0155] Table 6: Reaction System Proportions

[0156]

[0157] The results are as follows Figure 5 As shown, compared with the wild-type T7 RNAP, all mutation sites effectively improved the capping rate. Among them, the T7 RNAP superposition mutations R386C+D438P, R386W+N437T, and D388K+D438P all significantly improved the capping rate based on the single site. Specifically, the capping rate of D388K+D438P increased to 95.5% compared with the 84% capping rate of D388K and the 68.5% capping rate of D438P, with an improvement of >10%.

Claims

1. An RNA polymerase variant, characterized in that The amino acid sequence of the variant comprises a substitution at position R386 relative to SEQ ID NO:

1. The amino acid sequence of the variant is shown in any one of SEQ ID NO: 2, 15-20 or 22.

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) Recycling 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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