RNA polymerase variants and uses thereof

Abstract

The invention provides an RNA (Ribonucleic Acid) polymerase variant and a preparation method thereof, and an RNA product with low dsRNA pollution can be obtained by applying the RNA polymerase variant disclosed by the invention to in-vitro transcription. In addition, the invention further provides a method for preparing RNA through in-vitro transcription.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to RNA polymerase variants, their preparation methods, and their applications in RNA synthesis. Background Technology

[0002] Before Paxlovid (a combination of nematriberi and ritonavir tablets) was available (it received emergency approval from the FDA on November 22, 2021), vaccines were the most effective line of defense against infection. In this massive global fight against the pandemic, billions of people have been vaccinated with COVID-19 vaccines such as BNT162b2 (mRNA vaccine) developed by BioNTech and Pfizer, mRNA-1273 (mRNA vaccine) developed by Moderna, or AZD1222 (adenovirus vector vaccine) developed by AstraZeneca. Among these, RNA vaccines have the highest protective efficacy. mRNA vaccines have made a significant contribution to this fight by preventing infection, reducing the rate of severe illness, and curbing the spread of the epidemic.

[0003] mRNA vaccines have a relatively short development cycle, enabling the rapid development of new candidate vaccines to address viral mutations. Through a dual mechanism of humoral and T-cell immunity, they exhibit strong immunogenicity and significant effects. Furthermore, their production process is simple, facilitating efficient research and development and large-scale production, thus enabling rapid and efficient global supply for combating pandemics like COVID-19.

[0004] According to the latest news, in addition to mRNA, research on circular RNA (circRNA)-related drugs has also made breakthrough progress. Orna Therapeutics has developed in vivo cell therapy products using circRNA, and a research report presented at the 2022 American Society for Gene and Cell Therapy (ASGCT) Annual Meeting has demonstrated its great application potential in other fields such as cancer treatment.

[0005] RNA has played a significant role in the development of vaccines and other drugs, but the removal of some impurities in the actual production process still requires further research. Among them, double-stranded RNA (dsRNA) impurities can cause strong immunogenicity (Goubau et al., 2014; Kato et al., 2006; Mu et al., 2018). Therefore, there is an urgent need to develop effective methods to reduce double-stranded RNA impurities. Summary of the Invention

[0006] In a first aspect, the present invention provides a class of RNA polymerase variants whose amino acid sequence, compared with SEQ ID NO: 1, contains at least one mutation selected from the following amino acid sites: R50, F55, I154, L170, K179, or F182, wherein the mutation type is selected from substitution or deletion.

[0007] In a second aspect, the present invention provides a class of biological materials selected from one or more of the following: 1) The polynucleotide molecule encoding the above variants; 2) Expression vectors containing polynucleotide molecules as described in 1); 3) Host cells containing the polynucleotide molecules described in 1), or host cells containing the expression vectors described in 2).

[0008] Thirdly, the present invention provides a method for preparing the above-mentioned RNA polymerase variant.

[0009] Fourthly, the present invention provides a composition comprising at least one RNA polymerase variant as described herein.

[0010] Fifthly, the present invention provides a kit comprising at least one RNA polymerase variant as described herein.

[0011] In a sixth aspect, the present invention provides the application of the above-mentioned RNA polymerase variants, compositions or kits in the in vitro transcription preparation of RNA.

[0012] In a seventh aspect, the present invention also provides a method for preparing RNA. Invention Details RNA polymerase variants The RNA polymerase variant provided by this invention contains at least one mutation selected from the following amino acid sites compared with the amino acid sequence SEQ ID NO: 1 of wild-type T7 RNA polymerase: R50, F55, I154, L170, K179 or F182; the mutation type can be selected from substitution or deletion.

[0014] In some embodiments, the amino acid sequence of the variant has a mutation of one amino acid relative to SEQ ID NO: 1, wherein the mutated amino acid site is selected from R50 or I154. In some embodiments, the mutation at the R50 site is R50A or R50G, and the mutation at the I154 site is I154D.

[0015] In some embodiments, the amino acid sequence of the variant, relative to SEQ ID NO: 1, comprises: (1) A mutation at the R50 site, and further comprising a mutation at any one amino acid selected from the I154, L170, K179, or F182 sites, wherein the mutation type is selected from substitution or deletion, and the mutation at the R50 site is R50A; or (2) A mutation at the F55 site, and further comprising a mutation of any one, two, or three amino acids selected from the sites I154, N165, K172, R173, H176, Y178P, K179, K387, K389, K740, K741, P742, G753, or Q754, wherein the mutation type is selected from substitution or deletion, wherein the mutation at the F55 site is selected from F55M, F55L, F55P, F55G, or F55N; or (3) A mutation at the I154 site, and further comprising a mutation at any one of the following amino acids selected from the R34, R50, R52, F55, L59, G572, or K610 sites, wherein the mutation type is selected from substitution or deletion, wherein the mutation at the I154 site is I154D; or (4) A mutation at the L170 site, and further comprising a mutation of any one, two, or three amino acids selected from the R34, R50, R52, L59, A69, K71, T101, A262, N289, R386, K389, I479, I482, I581, or E607 sites, wherein the mutation type is selected from substitution or deletion, wherein the mutation at the L170 site is selected from L170E or L170D; or (5) A mutation at the K179 site, and further comprising a mutation at any one of the amino acids selected from the R34, R50, F55, or L59 sites, wherein the mutation type is selected from substitution or deletion, wherein the mutation at the K179 site is K179D; or (6) A mutation at the F182 site, and also includes a mutation at any one of the amino acids selected from the R50, R52 or L59 sites, the mutation type being selected from substitution or deletion, wherein the mutation at the F182 site is F182E.

[0016] In some embodiments, the amino acid sequence of the variant contains a mutation selected from any of the following sites relative to SEQ ID NO: 1: (1) R50A+I154D, R50A+L170D, R50A+K179D, R50A+L170E or R50A+F182E; or (2) K172A+R173G+K387Y+F55M, K172A+R173G+K387Y+F55L, F55L+I154D, F55L+K 179D, K741A+F55P, P742G+F55P, Q754V+F55P, Y178P+F55P, Y178P+F55G, K740A+ F55P, Y178D+F55G, Y178G+F55P, Y178G+F55G, Y178H+F55P, Y178H+F55G, N165D+F55P, N165D+F55G, H176D+F55P, H176D+F55G, Y178D+F55P or F55N+G753A+K389A; or (3) I154D+G572R, R34A+I154D, R52A+I154D, L59A+I154D or I154D+K610I; or (4) L170D+A262V+I479T+E607G, K71E+L170D, L170D+A262V, R34A+L170D, R52A+L170D, L59A+L170D, L59A+L170E, L170D+K389A, L170D+N289A, L170D+I482V, L170D+A69S, L170D+R386M or T101I+L170D+I581T; or (5) R34A+K179D, L59A+K179D; or (6) L59A+F182E, R52A+F182E.

[0017] In some embodiments, the amino acid sequence of the variant is as shown in any one of SEQ ID NO: 3, SEQ ID NO: 9-10 or SEQ ID NO: 14-61.

[0018] biomaterials This invention provides a polynucleotide encoding an RNA polymerase variant. Due to codon degeneracy or codon bias in host cells expressing the polypeptide, the polynucleotide sequence can be any polynucleotide sequence encoding the variant without altering the amino acid sequence. In some embodiments, the polynucleotide sequence encoding the RNA polymerase variant of this invention may be selected from any of the sequences shown in SEQ ID NO: 64, SEQ ID NO: 70-71, or SEQ ID NO: 75-122.

[0019] The expression vector provided by this invention comprises a multinucleotide molecule encoding a variant of the RNA polymerase of this invention. In some embodiments, the expression vector further comprises one or more regulatory sequences, including but not limited to enhancers, promoters, leader peptide sequences, signal peptide sequences, and terminator sequences; wherein the regulatory sequences are operatively linked to the multinucleotide molecule encoding the variant.

[0020] In some embodiments, the expression vector may be a linear or circular DNA molecule, typically containing elements such as a multiple cloning site, an antibiotic resistance gene, and a replication initiation site. In some embodiments, the expression vector of the present invention is preferably pQE-80L.

[0021] The host cell provided by this invention refers to any cell that is advantageous for the expression of the variants of this invention, that is, any cell that is susceptible after being transformed, transfected or transduced by the expression vector described in this invention, and includes any daughter cells that are different from the parent cells due to mutations that occur during replication.

[0022] In some embodiments, the host cell is a prokaryotic cell, selectable 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*. In some embodiments, the host cell is *Escherichia coli* BL21(DE3).

[0023] Preparation methods of RNA polymerase variants The method for preparing RNA polymerase variants provided by the present invention includes 1) culturing the host cells described in the present invention under suitable variant expression conditions; and 2) recovering the variant.

[0024] 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.

[0025] 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), ammonium sulfate precipitation, etc.

[0026] Composition The composition provided by the present invention comprises at least one RNA polymerase variant described in the present invention.

[0027] 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 contain: RNA polymerase variants, Tris-HCl, NaCl, EDTA, DTT, Triton X-100, and glycerol.

[0028] In some embodiments, the composition further comprises template DNA. In some embodiments, the composition further comprises at least one in vitro transcription component, which may be selected from one or more buffer components, modified or unmodified nucleoside triphosphates, RNase inhibitors, inorganic pyrophosphatases, magnesium ions, etc.

[0029] Reagent test kit The kit provided by this invention contains at least one RNA polymerase variant as described in this invention.

[0030] In some embodiments, the kit may further comprise one or more in vitro transcription reaction reagents, such as buffer components, modified or unmodified nucleoside triphosphates, RNase inhibitors, pyrophosphatase, magnesium ions, water, etc. In some embodiments, the kit further comprises a cap analog, which may be selected from unmethylated cap analogs, dimethylated cap analogs, trimethylated cap analogs, dimethylated symmetrical cap analogs, or anti-reverse cap analogs. In some embodiments, the kit further comprises at least one in vitro transcription component, which may be selected from one or more buffer components, modified or unmodified nucleoside triphosphates, RNase inhibitors, inorganic pyrophosphatase, magnesium ions, etc. In one embodiment, the in vitro transcription system components may be selected from commercially available RNA in vitro transcription reagents.

[0031] application This invention provides the application of the above-described RNA polymerase variants, compositions, or kits in the in vitro transcription preparation of RNA. This invention also provides the application of at least one variant as described herein in reducing the generation of dsRNA impurities in the in vitro transcription preparation of RNA.

[0032] In some embodiments, the in vitro transcription preparation of RNA includes contacting a DNA template, a modified or unmodified nucleoside triphosphate, with at least one RNA polymerase variant described in this invention, incubating in an in vitro transcription reaction system to obtain the target product.

[0033] Preparation method This invention provides a method for preparing RNA, comprising contacting a DNA template, a modified or unmodified nucleoside triphosphate with at least one RNA polymerase variant described in this invention, incubating in an in vitro transcription reaction system to obtain the target product.

[0034] Suitable in vitro transcription reaction systems and incubation conditions for generating RNA 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 step of the present invention uses an incubation time of 20–240 min, preferably 60 min.

[0035] In some embodiments, the RNA product prepared using the method of the present invention has higher yield, and / or higher integrity, and / or less dsRNA impurity content, and / or more capped mRNA product compared to that prepared using wild-type RNA polymerase (SEQ ID NO: 1).

[0036] In some embodiments, the relative residual amount of dsRNA impurities (the ratio of residual dsRNA in the variant group to residual dsRNA in the WT group) of the RNA product prepared using the method described in this invention is less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 2%, less than 1%, or lower, compared to the RNA product prepared using wild-type RNA polymerase.

[0037] In some embodiments, the in vitro transcription reaction system includes one or more buffer components. In some embodiments, the buffer component may be selected from Tris-HCl, Hepes, citric acid, or commercially available buffer components. In some embodiments, the in vitro transcription buffer system also includes an RNase inhibitor, inorganic pyrophosphatase, and magnesium ions. In some embodiments, the in vitro transcription buffer system also includes water (e.g., DEPC-water, RNase-free water, DNase-free water, sterile purified water, deionized water, distilled water, etc.). In some embodiments, the in vitro transcription buffer system also includes a cap analogue, which may be selected from unmethylated cap analogues, dimethylated cap analogues, trimethylated cap analogues, dimethylated symmetrical cap analogues, or anti-reverse cap analogues.

[0038] Other implementation plans RNA polymerase variants, the amino acid sequence of which, relative to SEQ ID NO: 1, includes at least one substitution or deletion selected from the following amino acid sites: R34, R50, R52, F55, L59, A69, K71, T101, I154, N165, L170, K172, R173, H176, Y178, K179, F182, A262D, N289, R386, K387, K389, I479, I482, I581, G572, E607, K610, K740, K741, P742, G753, Q754.

[0039] The variant described in item 1, wherein: (1) The substitution at site R34 is A; (2) The substitution at the R50 site is A or G; (3) The substitution at the R52 site is A; (4) The substitution at the F55 site is selected from M, L, P, G, and N; (5) The substitution at the L59 site is A; (6) The substitution at site A69 is selected from S; (7) The substitution at the K71 site is E; (8) Substitution I at the T101 site; (9) The substitution at site I154 is D; (10) The substitution at the N165 site is D; (11) The substitution at the L170 site is selected from D and E; (12) The substitution at site K172 is A; (13) The substitution at site R173 is G; (14) The substitution at the H176 site is D; (15) The substitution at the Y178 site is selected from D, G, H, and P; (16) The substitution at the K179 site is D; (17) The substitution at the F182 site is E; (18) The substitution at site A262 is V; (19) The substitution at site N289 is A; (20) The substitution at site R386 is M; (21) The substitution at site K387 is Y; (22) The substitution at site K389 is A; (23) The substitution at site I479 is T; (24) The substitution at site I482 is V; (25) The substitution at site I581 is T; (26) The substitution at site G572 is R; (27) The substitution at site E607 is G; (28) The substitution at the K610 site is I; (29) The substitution at site K740 is A; (30) The substitution at site K741 is A; (31) The substitution at site P742 is G; (32) The substitution at the G753 site is A; (33) The substitution at the Q754 site is V.

[0040] 3. The variant as described in item 1, wherein the amino acid sequence of the variant, relative to SEQ ID NO: 1, contains a mutation selected from any of the following sites: R50A, R50G, I154D, R50A+I154D, R50A+L170D, R50A+K179D, R50A+L170E, R50A+F182E, K172A+R173G+K387Y+F55M, K172A+R173G+K387Y+F55L, F55L+I154D, F55L+K17 9D, K741A+F55P, P742G+F55P, Q754V+F55P, Y178P+F55P, Y178P+F55G, K740A+F55P, Y178D+F5 5G, Y178G+F55P, Y178G+F55G, Y178H+F55P, Y178H+F55G, N165D+F55P, N165D+F55G, H176D+F55 P, H176D+F55G, Y178D+F55P, F55N+G753A+K389A, I154D+G572R, R34A+I154D, R52A+I154D, L5 9A+I154D, I154D+K610I, L170D+A262V+I479T+E607G, K71E+L170D, L170D+A262V, R34A+L170D , R52A+L170D, L59A+L170D, L59A+L170E, L170D+K389A, L170D+N289A, L170D+I482V, L170D+A 69S, L170D+R386M, T101I+L170D+I581T, R34A+K179D, L59A+K179D, L59A+F182E, R52A+F182E.

[0041] 4. The variant as described in item 1, wherein the amino acid sequence of the variant is shown in any one of SEQ ID NO: 3, SEQ ID NO: 9-10 or SEQ ID NO: 14-61.

[0042] 5. Biological materials, selected from one or more of the following: 1) A polynucleotide molecule encoding an RNA polymerase variant as described in any of items 1-4; 2) Expression vectors containing polynucleotide molecules as described in 1); 3) A host cell containing a polynucleotide molecule as described in 1), or a host cell containing an expression vector as described in 2).

[0043] 6. The method for preparing the variant according to any one of claims 1-4, characterized in that it comprises: (1) Culturing the host cells as described in claim 5; and (2) Recycle variants.

[0044] 7. A composition comprising any of the variants described in items 1-4.

[0045] 8. A kit containing any of the variants described in items 1-4.

[0046] 9. Use of any variant described in items 1-4, the composition described in item 7, or the kit described in item 8 in in vitro transcription.

[0047] 10. Applications of any variant described in items 1-4, the composition described in item 7, or the kit described in item 8 to reduce the generation of dsRNA impurities during in vitro transcription to prepare RNA.

[0048] 11. As in the application of item 10, the RNA preparation process includes contacting a DNA template with any of the variants described in item -4 and incubating it in an in vitro transcription system.

[0049] 12. A method for preparing RNA, the method comprising contacting a DNA template, a modified or unmodified nucleoside triphosphate, and an RNA polymerase variant, incubating in an in vitro transcription reaction system to obtain a target RNA product; wherein the amino acid sequence of the variant is as shown in any one of SEQ ID NO: 3, SEQ ID NO: 9-10, or SEQ ID NO: 14-61. Attached Figure Description

[0050] Figure 1 This is a schematic diagram of the construction of recombinant plasmids.

[0051] Beneficial effects The T7 RNA polymerase variants of this invention exhibit high catalytic efficiency. Compared to wild-type T7 RNA polymerase, the addition of T7 RNA polymerase mutants to the in vitro transcription reaction system significantly reduces the production of double-stranded dsRNA contaminants. In particular, the mutant combinations K172A+R173G+K387Y+F55M, K172A+R173G+K387Y+F55L, R34A+L170D, R50A+L170D, R50A+K179D, R50A+L170E, R50A+F182E, H176D+F55P, H176D+F55G, L59A+L170D, L59A+L170E, L59A+K179D, Y178D+F55P, and L59A+F182E can reduce dsRNA levels to below 0.5% of the wild-type. Detailed Implementation

[0052] 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.

[0053] In this embodiment of the invention, enzyme activity is defined as: the ability of 1 nmol [ to produce an enzyme activity] within 1 hour at 37°C and pH 8.0. 3 The amount of enzyme required to incorporate H]ATP into an acid-insoluble precipitate is defined as one active unit.

[0054] Example 1: Preparation of RNA polymerase variants The RNA polymerases and their variants shown in Table 1 were synthesized using DNA sequences (SEQ ID NO: 62-122) 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. After culturing 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 5°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.

[0055] WT is a wild-type T7 RNA polymerase, and its amino acid sequence is as follows: (SEQ ID NO: 1) The correspondences between RNA polymerases and their variants and amino acid sequences are shown in Tables 1-1 to 1-3: Table 1-1: Mutation sites and corresponding amino acid sequence numbers of wild-type and variant RNA polymerases

[0056] Table 1-2

[0057] Table 1-3

[0058] Example 2: Generation of dsRNA impurities during in vitro transcription 2.1: Unmodified NTP (1) The enzyme stock solution obtained in Example 1 was diluted with storage buffer (Vazyme, catalog number: DD4101) to an enzyme activity of 300 U / μL. The reaction components (20 μL) in Table 2 were loaded into an octet and mixed well, and centrifuged. The octet was placed on a PCR instrument and reacted at 37°C for 1 h. Then, 36 μL of magnetic beads (Vazyme, catalog number: N412) were added, mixed well, and incubated at room temperature for 2-5 min. The mixture was 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. (2) The content of dsRNA impurities was tested using a dsRNA detection kit (Vazyme, catalog number: DD3509).

[0059] Table 2: Reaction System Proportions

[0060] Table 3-1: dsRNA detection results

[0061] Table 3-2

[0062] Table 3-3

[0063] The dsRNA detection results are shown in Tables 3-1 to 3-3. Compared with the WT group, the polymerase variants in Example 1 can effectively reduce the generation of dsRNA impurities during in vitro transcription. Among them, the mutant combinations K172A+R173G+K387Y+F55M, K172A+R173G+K387Y+F55L, R34A+L170D, R50A+L170D, R50A+K179D, R50A+L170E, R50A+F182E, H176D+F55P, H176D+F55G, L59A+L170D, L59A+K179D, L59A+L170E, Y178D+F55P, and L59A+F182E can reduce dsRNA to below 0.5% of the wild type.

Claims

1. An RNA polymerase variant, characterized in that, 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: R50, F55, I154, L170, K179, or F182, wherein the mutation type is selected from substitution or deletion.

2. The variant as claimed in claim 1, characterized in that, The amino acid sequence of the variant has a mutation of one amino acid relative to SEQ ID NO: 1, wherein the mutated amino acid is selected from R50 or I154.

3. The variant as claimed in claim 1, characterized in that, The amino acid sequence of the variant, relative to SEQ ID NO: 1, includes: (1) A mutation at the R50 site, and further comprising a mutation at any one amino acid selected from the I154, L170, K179, or F182 sites, wherein the mutation type is selected from substitution or deletion, wherein the mutation at the R50 site is R50A; or (2) A mutation at the F55 site, and further comprising a mutation of any one, two, or three amino acids selected from the sites I154, N165, K172, R173, H176, Y178P, K179, K387, K389, K740, K741, P742, G753, or Q754, wherein the mutation type is selected from substitution or deletion, wherein the mutation at the F55 site is selected from F55M, F55L, F55P, F55G, or F55N; or (3) A mutation at the I154 site, and further comprising a mutation at any one of the following amino acids selected from the R34, R50, R52, F55, L59, G572, or K610 sites, wherein the mutation type is selected from substitution or deletion, wherein the mutation at the I154 site is I154D; or (4) A mutation at the L170 site, and further comprising a mutation of any one, two, or three amino acids selected from the R34, R50, R52, L59, A69, K71, T101, A262, N289, R386, K389, I479, I482, I581, or E607 sites, wherein the mutation type is selected from substitution or deletion, and the mutation at the L170 site is selected from L170E or L170D; or (5) A mutation at the K179 site, and further comprising a mutation at any one of the amino acids selected from the R34, R50, F55, or L59 sites, wherein the mutation type is selected from substitution or deletion, and the mutation at the K179 site is K179D; or (6) A mutation at the F182 site, and also includes a mutation at any one of the amino acids selected from the R50, R52 or L59 sites, the mutation type being selected from substitution or deletion, wherein the mutation at the F182 site is F182E.

4. The variant as claimed in claim 3, characterized in that, The amino acid sequence of the variant, relative to SEQ ID NO: 1, contains a mutation selected from any of the following sites: (1) R50A+I154D, R50A+L170D, R50A+K179D, R50A+L170E or R50A+F182E; or (2) K172A+R173G+K387Y+F55M, K172A+R173G+K387Y+F55L, F55L+I154D, F55L+K 179D, K741A+F55P, P742G+F55P, Q754V+F55P, Y178P+F55P, Y178P+F55G, K740A+ F55P, Y178D+F55G, Y178G+F55P, Y178G+F55G, Y178H+F55P, Y178H+F55G, N165D+F55P, N165D+F55G, H176D+F55P, H176D+F55G, Y178D+F55P or F55N+G753A+K389A; or (3) I154D+G572R, R34A+I154D, R52A+I154D, L59A+I154D or I154D+K610I; or (4) L170D+A262V+I479T+E607G, K71E+L170D, L170D+A262V, R34A+L170D, R52A+L170D, L59A+L170D, L59A+L170E, L170D+K389A, L170D+N289A, L170D+I482V, L170D+A69S, L170D+R386M or T101I+L170D+I581T; or (5) R34A+K179D, L59A+K179D; or (6) L59A+F182E, R52A+F182E.

5. The variant as claimed in claim 2, characterized in that, The mutation at the R50 site is selected from R50A or R50G, and the mutation at the I154 site is I154D.

6. The variant as claimed in claim 1, characterized in that, The amino acid sequences of the variants are shown in any one of SEQ ID NO: 3, SEQ ID NO: 9-10 or SEQ ID NO: 14-61.

7. A biomaterial, characterized in that, The biomaterial is selected from one or more of the following: 1) Encoding a polynucleotide molecule that encodes any of the RNA polymerase variants as described in claims 1-6; 2) Expression vectors containing polynucleotide molecules as described in 1); 3) A host cell containing a polynucleotide molecule as described in 1), or a host cell containing an expression vector as described in 2).

8. The method for preparing the variant according to any one of claims 1-6, characterized in that, include: (1) Culturing the host cells as described in claim 7; and (2) Recycle variants.

9. A composition, characterized in that, It includes the variants as described in any one of claims 1-6.

10. A reagent kit, characterized in that, It includes the variants as described in any one of claims 1-6.

11. The use of any variant of claims 1-6, the composition of claim 9, or the kit of claim 10 in in vitro transcription.

12. The application of any variant of claims 1-6, the composition of claim 9, or the kit of claim 10 in reducing the generation of dsRNA impurities during in vitro transcription to prepare RNA.

13. The application of claim 12, wherein the RNA preparation process comprises contacting a DNA template with a variant as described in any one of claims 1-6 and incubating it in an in vitro transcription system.

14. A method for preparing RNA, characterized in that, The method includes contacting a DNA template, a modified or unmodified nucleoside triphosphate, and an RNA polymerase variant, incubating them in an in vitro transcription reaction system to obtain a target RNA product; wherein the amino acid sequence of the polymerase variant is shown in any one of SEQ ID NO: 3, SEQ ID NO: 9-10, or SEQ ID NO: 14-61.

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