RNA polymerase variants

Abstract

The present application provides a class of RNA polymerase variants, which have improved catalytic activity relative to wild-type T7 RNA polymerase, and can improve the capping rate of mRNA products in the in vitro co-transcription capping process. In addition, the present application also provides a method for preparing RNA using such variants, and using the method for preparing RNA molecules, more capped mRNA can be obtained.

Description

Technical Field

[0001] This invention 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'-oxomethyltransferase. 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 below:

[0004]

[0005] 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 CleanCapAG, 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. If the wild-type promoter is used, the capping efficiency is still 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 the utilization rate of cap analogs without requiring wild-type promoter replacement, which is of great significance for the economical production of mRNA and drug safety. Invention Overview

[0006] In a first aspect, the present invention provides a class of RNA polymerase variants having an amino acid sequence that is at least 95% identical to that of SEQ ID NO: 1, and containing a mutation of at least one amino acid selected from positions K387, D388 or K389, wherein the mutation type may be either substitution or deletion.

[0007] In some embodiments, the amino acid sequence of the variant includes a substitution at position K387 compared to SEQ ID NO: 1. In some embodiments, the amino acid sequence of the variant includes a substitution at position D388 compared to SEQ ID NO: 1. In some embodiments, the amino acid sequence of the variant includes a substitution at position K389 compared to SEQ ID NO: 1.

[0008] In a second aspect, the present invention provides a composition comprising at least one of the above-described RNA polymerase variants.

[0009] Thirdly, the present invention provides a kit comprising at least one of the above-described RNA polymerase variants.

[0010] Fourthly, the present invention also provides the use of RNA polymerase variants, compositions or kits in in vitro transcription.

[0011] Fifthly, the present invention provides a method for preparing RNA or capped mRNA.

[0012] In a sixth aspect, the present invention provides a polynucleotide molecule encoding the above-mentioned RNA polymerase variant; an expression vector comprising the polynucleotide molecule; and a host cell comprising the polynucleotide molecule or the vector.

[0013] In a seventh aspect, the present invention also provides a method for preparing the above-mentioned RNA polymerase variant. Invention Details

[0015] RNA polymerase variants

[0016] The RNA polymerase variant provided by this invention is a phage T7 RNA polymerase (T7 RNAP) variant, whose amino acid sequence has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the amino acid sequence of wild-type T7 RNAP SEQ ID NO: 1.

[0017] In some embodiments, the amino acid sequence of the variant, relative to SEQ ID NO: 1, contains at least one mutation selected from the following amino acid sites: K387, D388, or K389, wherein the mutation type is selected from substitution or deletion.

[0018] In some embodiments, the amino acid sequence of the variant contains a substitution selected from the following amino acid sites relative to SEQ ID NO: 1: K387, D388, or K389.

[0019] In some embodiments, the substituted amino acid at the K387 site of the variant may be selected from: Q, G, N, H or S.

[0020] In some embodiments, the variant has a substituted amino acid, K, at the D388 site.

[0021] In some embodiments, the substituted amino acid at the K389 site of the variant may be selected from: W, I, V, L, F, T, Q, H, S, N, C, D, E, A, G, M, P, or Y.

[0022] In some embodiments, the RNA polymerase variants provided by the present invention 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: 2-25. In some embodiments, the amino acid sequence of the variant is as shown in any of SEQ ID NO: 2-25.

[0023] Polynucleotide molecules

[0024] The polynucleotide molecule provided by this invention encodes any of the RNA polymerase variants described herein. In some embodiments, the polynucleotide molecule is as shown in any of SEQ ID NO: 27-50.

[0025] The polynucleotide molecule described in this invention 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.

[0026] expression carrier

[0027] The expression vector provided by this invention refers to a linear or circular DNA molecule, which typically contains elements such as multiple cloning sites, resistance genes, and replication initiation sites. In some embodiments, the expression vector described in this invention is pQE-80L.

[0028] In some embodiments, the vector of the present invention comprises a polynucleotide molecule encoding a variant of the RNA polymerase of the present invention. In some further embodiments, the vector also comprises one or more regulatory sequences (such as enhancer, promoter, and terminator sequences) operatively linked to the polynucleotide molecule encoding the variant.

[0029] host cells

[0030] The host cell provided by this invention can be any cell that is favorable for the expression of the variants of this invention, that is, any cell that is susceptible after being transformed, transfected or transduced with the expression vector of this invention, and includes any cell progeny that is different from the parent cell due to mutations that occur during replication.

[0031] In some embodiments, the host cell of the present invention comprises the above-described polynucleotide molecule or the expression vector.

[0032] 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*.

[0033] Methods for preparing variants

[0034] The present invention provides a method for preparing the above-mentioned RNA polymerase variant, comprising: (1) culturing the host cell described herein under conditions suitable for the expression of the variant; and (2) recovering the variant.

[0035] 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.

[0036] 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, chromatographic focusing, and size exclusion chromatography), isoelectric point focusing electrophoresis, ammonium sulfate precipitation, SDS-PAGE, etc.

[0037] Composition

[0038] The composition provided by the present invention comprises at least one RNA polymerase variant described in the present invention.

[0039] The compositions described in this invention can be compositions for storing RNA polymerase variants. In some embodiments, in addition to the aforementioned RNA polymerase variants, the compositions of this invention 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 compositions of this invention for storing RNA polymerase variants comprise: RNA polymerase variant, Tris-HCl, NaCl, EDTA, DTT, Triton X-100, and glycerol.

[0040] The compositions of the present invention can 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.

[0041] In some embodiments, the in vitro transcription reaction composition of the present invention comprises: 1) 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 2) a DNA template.

[0042] Reagent test kit

[0043] The kit provided by this invention comprises at least one RNA polymerase variant described herein. 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 also comprises a cap analogue. 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).

[0044] The kit described in this invention may include one or more containers containing one or more components described in this invention and optionally instructions for use.

[0045] Applications or uses

[0046] This invention provides the application of the above-mentioned RNA polymerase variants, compositions, or kits in in vitro transcription.

[0047] The present invention also provides the use of the above-mentioned RNA polymerase variants, compositions, or kits in a variety of methods, including but not limited to RNA preparation, RNA probe preparation, RNA vaccine preparation, and protein preparation.

[0048] Methods for preparing RNA

[0049] This invention provides a method for RNA preparation. 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 invention, 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.

[0050] This invention 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 invention, incubating in an in vitro transcription reaction system to obtain the target product.

[0051] 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 of the present invention 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 step of the present invention uses an incubation temperature of 30–50°C, preferably 37°C. In some embodiments, the incubation time of the incubation step of the present invention is 20–240 min, preferably 60 min.

[0052] In some embodiments, RNA products or capped mRNA products prepared using the methods described in this invention 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.

[0053] In some embodiments, the capped mRNA product prepared using the method of the present invention 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%.

[0054] Hat-like items

[0055] The cap analogue used in the method for preparing capped mRNA products or in the in vitro transcription reaction composition described in this invention refers to a molecule that is complementary to a nucleotide molecule on the DNA template at the transcription start site.

[0056] 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.

[0057] In some embodiments, the hat analogue described in this invention is preferably m7GpppA2′OMepG.

[0058] 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 is C, and the second nucleotide at the +2 site is C (such as...). Figure 1 In some implementations, the nucleotide residues in the m7GpppA2′OMepG cap analogue can be complementary to the +1 or +2 site of the DNA template molecule (e.g., Figure 2 ).

[0059] In vitro transcription reaction reagents

[0060] The in vitro transcription reaction reagent described in this invention includes buffer components, nucleoside triphosphates, RNase inhibitors, inorganic pyrophosphatase, magnesium ions, and water (e.g., DEPC-water, RNase-free water, DNase-free water, sterile purified water, deionized water, distilled water, etc.).

[0061] 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.

[0062] In some embodiments, the nucleoside triphosphate may be selected from modified or unmodified nucleoside triphosphates (including analogues). 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, a fluorescent substance, digoxigenin, a radioactive element, etc.).

[0063] In some embodiments, the in vitro transcription reaction reagent of the present invention may be selected from any commercially available RNA in vitro transcription reagent.

[0064] Other implementation plans:

[0065] 1. An RNA polymerase variant whose amino acid sequence contains at least one substitution selected from the following amino acid sites relative to SEQ ID NO: 1: K387, D388 or K389.

[0066] 2. A variant as described in item 1, wherein:

[0067] (1) The substitution at position K387 can be selected from Q, G, N, H or S;

[0068] (2) Replace K at position D388;

[0069] (3) The substitution at position K389 can be selected from W, I, V, L, F, T, Q, H, S, N, C, D, E, A, G, M, P or Y.

[0070] 3. The variant described in item 1, wherein the amino acid sequence has at least 99% or higher sequence identity compared with any of the sequences described in SEQ ID NO: 2-25.

[0071] 4. A polynucleotide molecule that encodes a variant as described in any of items 1-3.

[0072] 5. An expression vector containing a polynucleotide molecule as described in item 4.

[0073] 6. A host cell containing a polynucleotide molecule as described in item 4, or an expression vector as described in item 5.

[0074] 7. A method for preparing any of the variants described in items 1-3, comprising:

[0075] (1) Culture host cells as described in section 6 under conditions suitable for variant expression; and

[0076] (2) Recycle variants.

[0077] 8. A composition comprising any of the variants described in items 1-3.

[0078] 9. The composition as described in item 8, further comprising a hat analogue.

[0079] 10. The composition as described in item 8 or 9, further comprising a DNA template.

[0080] 11. A kit containing any of the variants described in items 1-3.

[0081] 12. Use of any variant described in items 1-3, any composition described in items 8-10, or the kit described in item 11 in in vitro transcription.

[0082] 13. 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 1-3, incubating in an in vitro transcription reaction system to obtain a target RNA product.

[0083] 14. A method for preparing capped mRNA, comprising contacting a DNA template, a modified or unmodified nucleoside triphosphate, a capping analogue, with any of the RNA polymerase variants described in items 1-3, incubating in an in vitro transcription reaction system to obtain the target product.

[0084] 15. The method as described in item 14, wherein the cap analog is a trinucleotide cap, preferably m7GpppA2′OMepG.

[0085] Beneficial effects

[0086] This invention provides a class of RNA polymerase variants. 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 invention provides a method for preparing capped mRNA. This method can increase the yield of capped mRNA products while reducing the amount of cap analogues used, avoiding waste of raw materials and saving production costs, which is of great significance for the economical production of RNA. Brief description of the attached diagram

[0087] Figure 1 A schematic diagram of a double-stranded DNA template;

[0088] Figure 2This is a complementary pairing between the cap-like analogue and the DNA template;

[0089] Figure 3 This is a schematic diagram illustrating the construction of recombinant plasmids;

[0090] Figure 4 The effect of RNA polymerase and its variants on mRNA capping rate. Detailed Implementation

[0091] The technical solution of the present invention will be further illustrated below with reference to specific embodiments. However, the following embodiments are merely examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims. In the following embodiments, unless otherwise specified, the reagents and consumables used are purchased from ordinary suppliers in the art, and the experimental methods and techniques used are conventional methods and techniques in the art.

[0092] In this embodiment of the invention, enzyme activity is defined as the ability of 1 nmol of enzyme to produce an enzyme activity at 37°C and pH 8.0 for 1 hour. 3 The amount of enzyme required to incorporate H]ATP into an acid-insoluble precipitate is defined as one active unit.

[0093] Example 1: Preparation of RNA polymerase variants

[0094] The RNA polymerases and their variants shown in Table 1 were synthesized using DNA sequences (SEQ ID NO: 26-50) and then amplified by PCR. The DNA was then introduced into the BseRI and HindIII restriction sites of the expression vector pQE-80L to obtain a recombinant expression vector. This constructed vector was then introduced into *E. coli* BL21(DE3) via chemical transformation. The vector was plated on LB agar plates containing ampicillin and incubated overnight at 37°C. Single colonies were then subjected to plasmid extraction and sequencing to obtain recombinant engineered bacteria containing the target gene. The successfully sequenced *E. coli* recombinant strains were inoculated into LB medium for overnight activation culture, followed by inoculation into the fermentation broth (LB medium) at 1–5% v / v and cultured until OD500. The 600 value was 0.6-0.8. After adding IPTG to a final concentration of 0.5 mol / L and culturing for another 4-6 hours, the strain was collected by centrifugation at 12000 rpm and 4℃. The collected strain was washed with 0.2 M PBS buffer at pH 7.0 to obtain the bacterial cells. After sonication, affinity chromatography was performed to purify the RNA polymerase stock solution.

[0095] The correspondence between RNA polymerase variants and amino acid sequences is shown in Table 1:

[0096] Table 1: Correspondence between T7 wild-type polymerase and its variants and amino acids

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104] Example 2: Preparation of mRNA by in vitro transcription reaction

[0105] 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 300 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.

[0106] Table 2: Reaction System Proportions

[0107]

[0108]

[0109] Example 3: Capping Rate Detection

[0110] After pretreatment using the mRNA Capping Rate Detection Kit (Vazyme, catalog number: DD3510-01), the capping rate of the mRNA product was detected by MS.

[0111] (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.

[0112] Table 3: Reaction System

[0113]

[0114] Table 4: Reaction Conditions

[0115]

[0116]

[0117] (2) RNase H digestion: Prepare the digestion reaction system according to Table 5, vortex thoroughly to mix evenly, and place in a PCR instrument. React at 25℃ for 20 min.

[0118] Table 5: Reaction System

[0119] Components 25 pmol volume Previous product 21μL RNaseHReactionBuffer(10×) 3μL RNaseH (5 U / μl) 3μL <![CDATA[RNase-freeH2O]]> 3μL Overall system 30μL

[0120] (3) SA magnetic bead bonding:

[0121] ① Magnetic bead cleaning: Take 9 μL of SA magnetic beads 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.

[0122] 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.

[0123] (4) Rinsing and elution:

[0124] 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;

[0125] ② Add 200 μL of rinsing solution to rinse, being careful not to blow away the magnetic beads, let stand for 0.5 to 1 minute, and then use a pipette to remove the supernatant;

[0126] ③ Repeat step ②;

[0127] 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;

[0128] ⑤ Place it in a PCR instrument and react at 85℃ for 3 minutes. Immediately after that, place it on a magnetic rack. After the solution becomes clear (0.5-1 minute), aspirate the supernatant into a new centrifuge tube. The supernatant is the desired product.

[0129] ⑥ The above product was sent to a mass spectrometer to detect the capping rate. The capping rate was calculated using the following formula:

[0130] mRNA capping rate (%)=(capped mRNA / (capped mRNA+uncapped mRNA))×100%.

[0131] Test results are shown Figure 4 When the ratio of cap analog to raw material NTP is as low as 0.5:1, the T7 RNA polymerase variants in Example 1 can all significantly improve the capping rate of mRNA products (compared to the WT group); among them, variants K387G, K387N, K387H, K387S, D388K, K389I, K389F, K389Q, K389S, K389N, K389D, K389E, K389A, K389G, K389M and K389P can increase the capping rate to over 95%.

[0132] Example 4: Detection of product integrity

[0133] (1) Dilute the enzyme stock solution (variant K389M) with storage buffer (50mM Tris-HCl (25℃, pH 7.9), 100mM NaCl, 0.1mM EDTA, 2mM DTT, 0.1% Triton X-100, 50% Glycerol) to an enzyme activity of 300 U / μL. Prepare the MIX solution according to the reaction system (20 μL) in Table 6, transfer the MIX solution to an octet, mix well, and centrifuge; place the octet on a PCR instrument and react at 37℃ for 1 h, then add 36 μL of magnetic beads (Vazyme, catalog number: N412), mix well, and incubate at room temperature for 2–5 min; place the mixture on a magnetic rack to purify the mRNA, and transfer the purified mRNA to an RNase-free centrifuge tube;

[0134] (2) The integrity of mRNA (intact RNA product / total RNA product) was detected by capillary electrophoresis using the Qsep400 fully automated nucleic acid analyzer.

[0135] Table 6: Reaction System Proportions

[0136]

[0137]

[0138] The results showed that, compared with wild-type T7 RNA polymerase (integrity of 83.13±0.34%), the K389M variant could improve the integrity of RNA products to 86.82±1.5%.

Claims

1. An RNA polymerase variant, characterized in that, The amino acid sequence of the variant is shown in SEQ ID NO:

7.

2. A polynucleotide molecule, characterized in that, The polynucleotide molecule encodes the variant as described in claim 1.

3. An expression carrier, characterized in that, The expression vector comprises the polynucleotide molecule as described in claim 2.

4. A host cell, characterized in that, The host cell contains the polynucleotide molecule as described in claim 2 or the expression vector as described in claim 3.

5. The method for preparing the variant as described in claim 1, characterized in that, The method includes: (1) Culturing the host cells as described in claim 4; and (2) Recycle variants.

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

7. A reagent kit, characterized in that, The kit comprises the variant as described in claim 1.

8. The use of the variant of claim 1, the composition of claim 6, or the kit of claim 7 in in vitro transcription.

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

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

11. The method as described in claim 10, characterized in that, The cap analogue is a trinucleotide cap.

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

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