RNA polymerase variants and uses thereof

Abstract

The application provides a T7 RNA polymerase variant and application thereof, wherein the amino acid sequence of the T7 RNA polymerase variant comprises at least one substitution or deletion of an amino acid site selected from R34, K172, Y178, R386, D388, Q435, N437, D438, relative to SEQ ID NO: 1. The variant has improved catalytic activity relative to wild-type T7 RNA polymerase, and can improve the capping rate of mRNA products in the process of in vitro transcription (cotranscriptional capping). In addition, the application also provides a method for preparing RNA by using the variant, and more capped mRNA can be obtained by using the method for preparing RNA molecules.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to RNA polymerase variants, methods for preparing the same and their use in RNA synthesis. BACKGROUND

[0002] The 5' end of intact mRNA of eukaryotes has a 7-methylguanosine (m7G) cap, which is formed in vivo by RNA triphosphatase, mRNA guanosine transferase, mRNA methyltransferase, mRNA nucleoside 2'-oxymethyltransferase catalysis. The 5' cap structure is involved in preventing mRNA degradation by exonucleases, reducing mRNA immunogenicity, regulating mRNA half-life, and regulating translation. In the process of in vitro transcription (IVT) to prepare mRNA, researchers usually add cap analogs during the preparation process to simulate the 5' cap structure of eukaryotic mRNA.

[0003] The cap structure at the 5' end of mRNA has a significant impact on the stability, translation efficiency and immunogenicity of mRNA. With the deepening of research, the importance of cap analogs is increasingly prominent, and therefore, new types of cap analogs are continuously developed for application in mRNA vaccines and therapeutic RNA. Today, cap analogs have developed to the third generation. The first generation of cap analogs is rarely seen on the market due to the presence of two free 3'-OH, which leads to reverse incorporation of the cap analog. The commonly seen cap analogs on the market are mainly the second generation ARCA cap analog (Formula 1) and the third generation cap analog (such as Formula 2), the main structural formula of which is shown in the examples:

[0004]

[0005] ARCA cap analog is a modified cap analog, whose 3'-OH group near m7G is replaced by -OCH3. Due to the replacement, RNA polymerase can only initiate transcription by 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, which has significantly improved capping rate compared to the second generation cap analog, but requires replacement of the wild-type promoter from 5'-TAATACGACTCACTATAGG-3' to 5'-TAATACGACTCACTATAAG-3'. Whether the replacement of the promoter will cause safety problems and new impurities needs to be proved. If the wild-type promoter is used, the capping rate is still low, and the RNA product that fails to be capped successfully not only causes waste of raw materials, but also needs to be removed by column purification in the later stage, increasing the production cost. Therefore, if the T7 RNA polymerase is modified, the utilization rate of the cap analog can be improved without replacing the wild-type promoter, which is of great significance in the economic production of mRNA and drug safety. SUMMARY

[0006] In a first aspect, the present application provides a RNA polymerase variant, whose amino acid sequence comprises at least one mutation of an amino acid at a position selected from R34, K172, Y178, R386, D388, Q435, N437 or D438 relative to SEQ ID NO: 1, and the type of mutation can be selected from substitution or deletion.

[0007] In a second aspect, the present application provides one or more biological materials selected from the following:

[0008] 1) a polynucleotide molecule encoding a RNA polymerase variant;

[0009] 2) an expression vector comprising the polynucleotide molecule as described in 1);

[0010] 3) a host cell comprising the polynucleotide molecule as described in 1), or a host cell comprising the expression vector as described in 2).

[0011] In a third aspect, the present application provides a method for preparing the above-mentioned RNA polymerase variant.

[0012] In a fourth aspect, the present application provides a composition comprising at least one RNA polymerase variant as described herein.

[0013] In a fifth aspect, the present application provides a kit comprising at least one RNA polymerase variant as described herein.

[0014] In a sixth aspect, the present application also provides the use of the above-mentioned RNA polymerase variant, composition or kit in in vitro transcription.

[0015] In a seventh aspect, the present application also provides a method for preparing RNA or capped RNA. DETAILED DESCRIPTION

[0017] RNA polymerase variant

[0018] The RNA polymerase variant provided by the present application is a bacteriophage T7 RNA polymerase (T7 RNAP) variant, the amino acid sequence of which comprises at least one mutation selected from the following amino acid positions: R34, K172, Y178, R386, D388, Q435, N437 or D438, relative to SEQ ID NO: 1, and the type of mutation is selected from substitution or deletion.

[0019] In some embodiments, the substitution of the variant at the R34 position is A.

[0020] In some embodiments, the type of mutation of the variant at the K172 position is deletion.

[0021] In some embodiments, the substitution of the variant at the Y178 position can be selected from: H or D.

[0022] In some embodiments, the substitution of the variant at the R386 position can be selected from: C, W, M, A, I or F.

[0023] In some embodiments, the substitution of the variant at the D388 position is K.

[0024] In some embodiments, the substitution of the variant at the Q435 position can be selected from: A, H or T.

[0025] In some embodiments, the substitution of the variant at the N437 position can be selected from: F or T.

[0026] In some embodiments, the substitution of the variant at the D438 position can be selected from: T, L, I, P or V.

[0027] In some embodiments, the amino acid sequence of the variant comprises any of the following substitutions or substitution groups, relative 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.

[0028] In some embodiments, the RNA polymerase variant provided herein has an amino acid sequence that has at least 97%, at least 98%, at least 99% or more sequence identity to the sequence set forth in any one of SEQ ID NOs: 2-22. In some embodiments, the variant has an amino acid sequence set forth in any one of SEQ ID NOs: 2-22.

[0029] Polynucleotide molecule

[0030] The polynucleotide molecule provided herein encodes any one of the RNA polymerase variants described herein. In some embodiments, the polynucleotide molecule is set forth in any one of SEQ ID NOs: 23-44.

[0031] In some embodiments, the polynucleotide molecule provided herein can have various modifications in the coding region, as long as the amino acid sequence of the variant is not changed with the degeneracy of the codon or the preferred codon in the organism expressing the variant.

[0032] Expression vector

[0033] The expression vector provided herein refers to a linear or circular DNA molecule, which typically contains elements such as a multiple cloning site, a resistance gene, a replication initiation site, etc. In some embodiments, the expression vector provided herein is pQE-80L.

[0034] In some embodiments, the vector provided herein contains a polynucleotide molecule encoding the RNA polymerase variant provided herein. In some embodiments, further, the vector also contains one or more regulatory sequences (such as enhancer, promoter and terminator sequences, etc.) operably linked to the polynucleotide molecule encoding the variant.

[0035] Host cell

[0036] The host cell provided herein can be any cell that is susceptible to transformation, transfection or transduction with a polynucleotide molecule or an expression vector provided herein, and which can then express the variant. The cell can be any progeny of the parent cell that has a genotypic identity with the parent cell that can be determined using a polynucleotide molecule provided herein.

[0037] In some embodiments, the host cell provided herein contains the polynucleotide molecule or the expression vector described above.

[0038] In some embodiments, the host cell is a prokaryotic cell, which can be selected from a gram-positive bacterium or a gram-negative bacterium. In some embodiments, the host cell is a gram-positive bacterium, 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 bacterium, including but not limited to: Campylobacter, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacter, Limnobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

[0039] Methods of making variants

[0040] The present application provides methods of making the above-described RNA polymerase variants, comprising: (1) culturing the host cell described herein under conditions suitable for expression of the variant; and (2) recovering the variant.

[0041] In some embodiments, the method of recovering the variant can be a method known in the art, such as centrifugation, filtration, treatment with a crystallization protein precipitant (salting-out), extraction, sonication, ultrafiltration, dialysis, various chromatographic methods such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC, and combinations thereof.

[0042] In some embodiments, the method of making further comprises a step of purifying 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, and the like.

[0043] Compositions

[0044] The present application provides compositions comprising at least one RNA polymerase variant described herein.

[0045] The compositions described herein can be a composition for storing the RNA polymerase variant. In some embodiments, the compositions described herein can optionally comprise, in addition to the RNA polymerase variant described above, a buffering component (e.g., Tris base, Tris-HCl, HEPES, MOPS), a salt (e.g., NaCl), an enzyme inhibitor (e.g., EDTA), a reducing agent (e.g., DTT), a surfactant (e.g., Triton X-100), a stabilizer (e.g., glycerol), and the like. In some embodiments, the composition for storing the RNA polymerase variant described herein comprises: an RNA polymerase variant, Tris-HCl, NaCl, EDTA, DTT, Triton X-100, and glycerol.

[0046] The compositions of the present application can also be in vitro transcription reaction compositions. In some embodiments, the compositions 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 addition to the RNA polymerase variants described above. In some embodiments, the compositions further comprise a DNA template. In some embodiments, the compositions further comprise a cap analog.

[0047] In some embodiments, the in vitro transcription reaction compositions described herein comprise: an RNA polymerase variant, buffer components, modified or unmodified nucleoside triphosphates, RNase inhibitors, pyrophosphatase, magnesium ions, water, and a cap analog. In some embodiments, the in vitro transcription reaction compositions described herein comprise: an RNA polymerase variant, buffer components, modified or unmodified nucleoside triphosphates, RNase inhibitors, pyrophosphatase, magnesium ions, water, a cap analog, and a DNA template.

[0048] Kits

[0049] The kits provided herein comprise at least one RNA polymerase variant described herein.

[0050] In some embodiments, the kits can 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 kits further comprise a cap analog. In some embodiments, each component of the kit (if applicable) can be provided in a liquid form (e.g., in solution) or in a solid form (e.g., a dry powder).

[0051] The kits described herein can comprise one or more containers comprising one or more components described herein and optionally instructions for use.

[0052] Uses or methods of use

[0053] The present application provides uses of the RNA polymerase variants, compositions, or kits described above in in vitro transcription.

[0054] The present application also provides uses of the RNA polymerase variants, compositions, or kits described above 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.

[0055] Methods of making RNA

[0056] 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 herein, incubating in an in vitro transcription reaction system, and obtaining a target RNA product. In some embodiments, the RNA product can be dsRNA, ssRNA, mRNA, siRNA, miRNA, piRNA, shRNA, or gRNA.

[0057] 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 herein, incubating in an in vitro transcription reaction system, and obtaining a target product.

[0058] The in vitro transcription reaction system and incubation conditions suitable for generating RNA products or capped mRNA products are well known in the art, and one of ordinary skill in the art can determine the appropriate reaction system pH, reaction temperature, reaction time, salt concentration, or whether to add exogenous cofactors, etc. in consideration of the optimal activity of the RNA polymerase. In some embodiments, the in vitro transcription reaction system described herein comprises in vitro transcription reaction reagents: one or more buffer components, modified or unmodified nucleoside triphosphates, RNase inhibitors, pyrophosphatase, magnesium ions, water, etc. In some embodiments, the incubation temperature in the incubation step described herein is 30-50°C, preferably 37°C. In some embodiments, the incubation time in the incubation step described herein is 20-240 min, preferably 60 min.

[0059] In some embodiments, the RNA product or capped mRNA product prepared by the method described herein has higher yield, and / or has higher integrity, and / or has less dsRNA impurity content, and / or more capped mRNA product, etc. compared to the use of wild-type RNA polymerase.

[0060] In some embodiments, the capped mRNA product prepared by the method described herein has improved utilization of cap analogs compared to the use of wild-type RNA polymerase, wherein the capping rate of the obtained mRNA product can be improved 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%.

[0061] Cap analogs

[0062] The cap analog in the method for preparing a capped mRNA product or the cap analog in the in vitro transcription reaction composition described in the present application refers to a molecule having complementarity to a nucleotide molecule on a DNA template at a transcription initiation site.

[0063] In some embodiments, the cap analog can be selected from a di-nucleotide cap, a tri-nucleotide cap, or a tetra-nucleotide cap. In some embodiments, the cap analog is a tri-nucleotide cap, which can be selected from GAA, GAC, GAG, GAU, GCA, GCC, GCG, GCU, GGA, GGC, GGG, GGU, GUA, GUC, GUG, and GUU. In some embodiments, the tri-nucleotide cap can be selected from m7GpppApA, m7GpppApC, m7GpppApG, m7GpppApU, m7GpppCpA, m7GpppCpC, m7GpppCpG, m7GpppCpU, m7GpppGpA, m7GpppGpC, m7GpppGpG, m7GpppGpU, m7GpppUpA, m7GpppUpC, m7GpppUpG, and m7GpppUpU. In some embodiments, the tri-nucleotide cap can be selected from m7G3ΌMepppApA, m7G3ΌMepppApC, m7G3ΌMepppApG, m7G3ΌMepppApU, m7G3ΌMepppCpA, m7G3ΌMepppCpC, m7G3ΌMepppCpG, m7G3ΌMepppCpU, m7G3ΌMepppGpA, m7G3ΌMepppGpC, m7G3ΌMepppGpG, m7G3ΌMepppGpU, m7G3ΌMepppUpA, m7G3ΌMepppUpC, m7G3ΌMepppUpG, and m7G3ΌMepppUpU. In some embodiments, the tri-nucleotide cap can be selected from m7G3ΌMepppA2ΌMepA, m7G3ΌMepppA2ΌMepC, m7G3ΌMepppA2ΌMepG, m7G3ΌMepppA2ΌMepU, m7G3ΌMepppC2ΌMepA, m7G3ΌMepppC2ΌMepC, m7G3ΌMepppC2ΌMepG, m7G3ΌMepppC2ΌMepU, m7G3ΌMepppG2ΌMepA, m7G3ΌMepppG2ΌMepC, m7G3ΌMepppG2ΌMepG, m7G3ΌMepppG2ΌMepU, m7G3ΌMepppU2ΌMepA, m7G3ΌMepppU2ΌMepC, m7G3ΌMepppU2ΌMepG, and m7G3ΌMepppU2ΌMepU.In some embodiments, the trinucleotide cap can be selected from the group consisting of 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.

[0064] In some embodiments, the cap analog described herein is preferably m7GpppA2'OMepG.

[0065] In some embodiments, when a trinucleotide cap GAG (e.g., m7GpppA2'OMepG) is used to prepare a capped mRNA product, or in an in vitro transcription reaction composition containing the cap analog, the first nucleotide at the +1 position of the DNA template molecule (sense strand) is G, and the second nucleotide at the +2 position is G. In some embodiments, the nucleotide residues in the m7GpppA2'OMepG cap analog can be complementary to the +1 position of the anti-sense strand of the DNA template molecule (e.g., Figure 2 ).

[0066] In vitro transcription reaction reagents

[0067] The in vitro transcription reaction reagents described herein include buffer components, nucleoside triphosphates, RNAse inhibitors, inorganic pyrophosphatase, magnesium ions, water (e.g., DEPC-water, RNase-free water, DNase-free water, sterile purified water, deionized water, distilled water, etc.), and the like.

[0068] In some embodiments, the buffer components can be selected from one or more of the following: phosphate buffers, Tris buffers, MOPS buffers, HEPES buffers, citrate buffers, acetate buffers, malate buffers, MES buffers, histidine buffers, PIPES buffers, bis-tris buffers, and ethanolamine buffers.

[0069] In some embodiments, the nucleoside triphosphates can be selected from modified or unmodified nucleoside triphosphates (including analogs thereof). In some embodiments, the nucleoside triphosphates can be selected from unmodified ATP, GTP, CTP, UTP. In some embodiments, the nucleoside triphosphates can be selected from modified nucleoside triphosphates, the types of modification on the nucleoside include but are not limited to m1A (N1-methyladenosine), m6A (N6-methyladenosine), m5C (5-methylcytidine), 5moU (5-methoxyuridine), ψ (pseudo-uridine), m1ψ (N1-methyl-pseudo-uridine), nucleoside triphosphates with labels (the labels can be biotin, fluorescent substances, digoxin, radioactive elements, etc.).

[0070] In some embodiments, the in vitro transcription reaction reagents described in the present application can be selected from any commercially available RNA in vitro transcription reagents.

[0071] Other embodiments:

[0072] 1. A variant of RNA polymerase, whose amino acid sequence comprises at least one substitution or deletion at an amino acid position selected from R34, K172, Y178, R386, D388, Q435, N437, D438, relative to SEQ ID NO: 1.

[0073] 2. The variant of item 1, wherein:

[0074] (1) the substitution at the R386 position is selected from C or W;

[0075] (2) the substitution at the D388 position is K;

[0076] (3) the substitution at the Q435 position is selected from A, H, T;

[0077] (4) the substitution at the N437 position is selected from F, T;

[0078] (5) the substitution at the D438 position is selected from T, L, I, P, V;

[0079] (6) the substitution at the R34 position is selected from A;

[0080] (7) the mutation at the K172 position is a deletion;

[0081] (8) the substitution at the Y178 position is selected from H, D.

[0082] 3. The variant of item 1, which amino acid sequence comprises any mutation selected from the group consisting of 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, relative to SEQ ID NO: 1.

[0083] 4. The variant of item 1, which variant has an amino acid sequence as set forth in any one of SEQ ID NOs: 2-22.

[0084] 5. A polynucleotide molecule encoding the variant of any one of items 1-4.

[0085] 6. An expression vector comprising the polynucleotide molecule of item 5.

[0086] 7. A host cell comprising the polynucleotide molecule of item 5, or the expression vector of item 6.

[0087] 8. A method of making the variant of any one of items 1-4, comprising:

[0088] (1) culturing the host cell of item 7; and

[0089] (2) recovering the variant.

[0090] 9. A composition comprising the variant of any one of items 1-4.

[0091] 10. The composition of item 9, further comprising a cap analog.

[0092] 11. The composition of item 9 or item 10, further comprising a DNA template.

[0093] 12. A kit comprising the variant of any one of items 1-4.

[0094] 13. Use of the variant of any one of items 1-4, the composition of any one of items 9-11, or the kit of item 12 in in vitro transcription.

[0095] 14. A method of making RNA, comprising contacting a DNA template, modified or unmodified nucleoside triphosphates with the RNA polymerase variant of any one of items 1-4, incubating in an in vitro transcription reaction system, and obtaining a target RNA product.

[0096] 15. A method for preparing capped mRNA, comprising contacting a DNA template, modified or unmodified nucleoside triphosphates, a cap analog with the RNA polymerase variant of any one of items 1-4, incubating in an in vitro transcription reaction system, and obtaining the target product.

[0097] 16. The method of item 15, wherein the cap analog is a trinucleotide cap, preferably m7GpppA2'OMepG.

[0098] 17. The composition of item 11 or the method of any one of items 14-16, wherein the DNA template comprises 2'-deoxyguanosine residues at positions +1 and +2.

[0099] 18. Use of the variant of any one of items 1-4, the composition of any one of items 9-11, or the kit of item 12 in the transcription of a DNA template comprising 2'-deoxyguanosine residues at positions +1 and +2.

[0100] Advantages

[0101] The present application provides a class of RNA polymerase variants and methods for preparing the same. By modifying the wild-type T7 RNA polymerase, the present application provides RNA polymerase variants with higher catalytic efficiency. The use of the variants can improve the capping rate of mRNA products in the process of in vitro transcription, and more capped mRNA products can be obtained. In addition, the present application also provides a method for preparing capped mRNA. The use of the method can improve the yield of capped mRNA products, reduce the amount of cap analogs, avoid the waste of raw materials, save production costs, and is of great significance for the economic production of RNA. BRIEF DESCRIPTION OF DRAWINGS

[0102] Figure 1 Figure 1 is a schematic diagram of a double-stranded DNA template;

[0103] Figure 2 Figure 3 is a schematic diagram of the complementary pairing of a cap analog and a DNA template;

[0104] Figure 3 Figure 4 is a schematic diagram of the construction of a recombinant plasmid;

[0105] Figure 4 Figure 5 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.

[0106] 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

[0107] In this application embodiment, unit enzyme activity (U) is defined as the amount of enzyme that, under conditions of 37°C and pH 8.0, produces 1 nmol of enzyme activity within 1 hour. 3 The amount of enzyme required to incorporate H]ATP into an acid-insoluble precipitate is defined as one active unit.

[0108] Example 1: Preparation of RNA polymerase variants

[0109] 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 E. coli BL21(DE3) using chemical transformation technology. The vector was plated on LB agar plates containing ampicillin and incubated overnight at 37°C. The resulting single colonies were subjected to plasmid extraction and sequencing to obtain the recombinant engineered bacteria containing the target gene. The successfully sequenced recombinant E. coli strain was inoculated into LB medium for overnight activation culture, and then inoculated into fermentation broth (LB medium) at 1-5% v / v. The culture was continued until the OD 600 value reached 0.6-0.8. IPTG was added to a final concentration of 0.5 mol / L, and the culture was continued for 4-6 h. The strain was 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 bacterial cells. After sonication, affinity chromatography was performed to purify the RNA polymerase stock solution.

[0110] The correspondence between RNA polymerase variants and amino acid sequences is shown in Tables 1-1 to 1-6:

[0111] Table 1:

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118] Example 2 Preparation of mRNA by in vitro transcription reaction

[0119] The enzyme stock 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 according to the reaction system (20 μL) in Table 2 into an EP tube, and the MIX solution was transferred to an eight-row array, mixed, and centrifuged; the eight-row array was placed on a PCR instrument at 37°C for 1 h, then 36 μL of magnetic beads (Vazyme, Catalog No: N412) were added, mixed, 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.

[0120] Table 2: Reaction system ratio

[0121]

[0122] Example 3 Detection of capping rate

[0123] After pretreatment with the mRNA Capping Rate Detection Kit (Vazyme, Catalog No: DD3510-01), the capping rate of the mRNA product was detected by LC-MS:

[0124] (1) The purified mRNA in Example 2 was combined with the probe, and the reaction system was as shown in Table 3, and the reaction conditions were as shown in Table 4;

[0125] Table 3: Reaction system

[0126]

[0127] Table 4: Reaction conditions

[0128] Temperature Time 95℃ 2 mins 70～16℃ -0.1°C / s, ~180 cycles 16℃ ∞

[0129] (2) RNase H enzyme digestion: the reaction enzyme digestion reaction system was prepared according to Table 5, and after being mixed uniformly by vortexing, it was placed in a PCR instrument and reacted at 25°C for 20 min;

[0130] Table 5: Reaction system

[0131] Component Volume Product from previous step 21 μL RNase H Reaction Buffer (10x) 3 μL RNase H (5 U / μl) 3 μL RNase-free H2O 3 μL Total system 30 μL

[0132] (3) SA magnetic bead binding:

[0133] ① Magnetic bead washing: Take 9 μL SA magnetic beads (Cat. No.: SM017005) into a centrifuge tube, place it on a magnetic stand, and when the solution is clear, use a pipette to discard the supernatant; remove the centrifuge tube from the magnetic stand, add 200 μL RNase-free H2O, and place it on the magnetic stand. When the solution is clear, use a pipette to discard the supernatant, and add 200 μL RNase-free H2O and repeat the rinse once.

[0134] ② Reaction conditions: Remove the centrifuge tube from the magnetic stand, add the enzyme digestion product to the SA magnetic beads (solid), and use a pipette to blow and beat 20-30 times to mix evenly. Incubate at room temperature on a rolling instrument for 30 min to allow the magnetic beads to fully bind with the enzyme digestion product.

[0135] (4) Rinse and elute:

[0136] ① Place the product from the previous step on a magnetic stand for 2-3 min, and when the solution is clear, use a pipette to discard the supernatant;

[0137] ② Add 200 μL rinse solution, taking care not to blow the magnetic beads apart, and let it stand for 0.5-1 min. Use a pipette to discard the supernatant;

[0138] ③ Repeat step ②;

[0139] ④ Remove the centrifuge tube from the magnetic stand, add 30 uL elution solution, and use a pipette to mix evenly by blowing and beating 10-20 times to disperse the magnetic beads evenly and elute completely;

[0140] ⑤ Place it in a PCR instrument and react at 85℃ for 3 min, then place it on a magnetic stand. When the solution is clear (0.5-1 min), transfer the supernatant to a new centrifuge tube. The supernatant is the desired product;

[0141] ⑥ The above product was sent to Thermo scientific Vanquish Flex-Qrbitrap Exploris120 for detection of capping rate (mobile phase: A phase: 2% hexafluoroisopropanol-1% N'N-diisopropylethylamine aqueous solution, B phase: 2% hexafluoroisopropanol-1% N'N-diisopropylethylamine methanol solution; chromatographic column: Nano ChromCore C183 μm, 4.6*100 mm; ion mode: negative ion; scan mode: Full scan; scan range: 600-3000), and the capping rate calculation formula:

[0142] mRNA capping rate (%) = (capped mRNA / (capped mRNA + uncapped mRNA)) x 100%.

[0143] The detection results are shown inFigure 4 At a low ratio of cap analog to NTP of 0.33:1, the T7 RNA polymerase variants in Example 1 can all improve the capping rate of mRNA product well, and R386W+N437T improves the capping rate to 100%.

[0144] Example 4 Preparation of mRNA by in vitro transcription reaction

[0145] The mRNA was prepared by in vitro transcription according to Example 2, and the amount of cap analog was further reduced to 0.24 μL (as shown in Table 6), and the rest of the reaction conditions were the same, and the capping rate was further detected according to Example 3. Table 6: Reaction system ratio

[0146]

[0147]

[0148] The results are shown in Figure 5 Relative to the wild type T7 RNAP, all the mutation sites effectively improve the capping rate, and the superposition mutations R386C+D438P, R386W+N437T, D388K+D438P of T7 RNAP further significantly improve the capping rate on a unit point basis, and D388K+D438P improves the capping rate to 95.5% from 84% of D388K and 68.5% of D438P, with an improvement of >10%.

Claims

1. An RNA polymerase variant, characterized in that, The amino acid sequence of the variant is substituted at position Q435 relative to SEQ ID NO: 1, and the amino acid sequence of the variant is shown in any of SEQ ID NO: 5-7.

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, It contains the polynucleotide molecule as described in claim 2.

4. A host cell, characterized in that, It comprises 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, include: (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.

13. The method according to any one of claims 9-12, characterized in that, The DNA template is located at positions +1 and +2, which contain 2'-deoxyguanosine residues.

14. The use of the variant of claim 1, the composition of claim 6, or the kit of claim 7 in the transcription of DNA templates containing 2'-deoxyguanosine residues at template positions +1 and +2.

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