RNA polymerase variant, preparation method thereof and application of RNA polymerase variant in RNA synthesis

Abstract

The invention provides the RNA polymerase variant and the preparation method thereof, the RNA polymerase variant is applied to in-vitro transcription, an RNA product with low dsRNA pollution and high integrity can be obtained, meanwhile, the utilization efficiency of cap analogues in a co-transcription capping reaction system can be improved, and the cost is saved. In addition, the invention further provides a method for generating RNA through in-vitro transcription.

Description

Technical Field

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

[0002] 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, allowing for rapid global supply.

[0003] 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.

[0004] 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

[0005] This application provides an RNA polymerase variant, a method for its preparation, and its application in RNA synthesis. In a first aspect, this application provides an RNA polymerase variant whose amino acid sequence, relative to SEQ ID NO: 1, includes mutations at at least one, at least two, at least three, at least four, at least five, or at least six amino acids selected from positions D130, N171, K172, R173, Y178, R298, Y385, K387, D388, or F880, wherein the mutation type is selected from deletion or substitution, and the amino acid sequence of the variant has at least 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 1. In some embodiments, the variant includes mutations at one, two, three, four, five, or six amino acids selected from positions D130, N171, K172, R173, Y178, R298, Y385, K387, D388, or F880; wherein the mutation type is selected from deletion or substitution. In some embodiments, the mutation type is a deletion (denoted by DEL, e.g., DEL5, indicating an amino acid deletion at position 5). In some embodiments, the mutation type is a substitution (e.g., K5A, indicating a lysine mutation to alanine at position 5). In some embodiments, the mutation type includes both deletions and substitutions, i.e., deletions at some positions and substitutions at others.

[0006] In some embodiments, the RNA polymerase variant provided in this application has an amino acid mutation relative to SEQ ID NO: 1, wherein the mutated amino acid site is selected from N171, K172, R173, Y178, R298, Y385, K387, D388, or F880, and the mutation type is substitution or deletion. In some embodiments, the amino acid sequence of the variant has an amino acid mutation relative to SEQ ID NO: 1, wherein the mutation is selected from: (1) Substitution at position N171, wherein the substituted amino acid is G; or (2) Substitution or deletion at position K172, wherein the substituted amino acid may be selected from A, G, E, D, H, Y, S, W, P, I, M, V, F, T, C, N, L; or (3) Substitution or deletion at position R173, wherein the substituted amino acid may be selected from A, C, G, E, D, H, Y, S, W, P, N, Q; or (4) Substitution at position Y178, wherein the substituted amino acid is H; or (5) Substitution at position R298, wherein the substituted amino acid is A; or (6) Substitution at position Y385, wherein the substituted amino acid may be selected from A, E, or D; or (7) Substitution at position K387, wherein the substituted amino acid may be selected from A, Y, S, Q; or (8) Substitution at position D388, wherein the substituted amino acid may be selected from A, G, L; or (9) Substitution at position F880, wherein the substituted amino acid may be selected from A, G, W.

[0007] In some embodiments, the RNA polymerase variant provided in this application has an amino acid sequence relative to SEQ ID NO: 1 containing mutations of two, three, four, or five amino acids at positions selected from D130, K172, R173, Y178, R298, Y385, K387, D388, or F880, and the amino acid sequence of the variant has at least 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with SEQ ID NO: 1. In some embodiments, the variant comprises: (1) a mutation at position K172, and further comprises one, two, three, or four amino acid mutations selected from positions D130, R173, Y178, R298, Y385, K387, D388, or F880, the mutation type being substitution or deletion; or (2) a mutation at position R173, and further comprises one, two, or three amino acid mutations selected from positions D130, Y178, R298, K387, or D388, the mutation type being substitution or deletion; or (3) two or three amino acid mutations at positions R298, Y385, K387, or D388, the mutation type being substitution or deletion. In some embodiments, the amino acid sequence of the variant is relative to SEQ ID NO: 1, possessing a combination of mutation points selected from any of the following: K172+R173, D130+K172, K172+K387, K172+F880, K172+D388, K172+R298, D130+R173, R173+Y178, R173+D388, R173+R298, K387+R298, Y385+ R298, D388+R298, Y385+K387, Y385+D388, K387+D388, K172+R173+Y385, K172+R1 73+D388, K172+R173+K387, K172+R173+F880, K172+R173+Y178, D130+K172+R173, D130+K172+Y178, D130+K172+K387, D130+R173+D388, K172+Y178+D388, D130+K1 72+D388, K172+K387+R298, Y385+K387+D388, K172+R173+Y385+F880, K172+R173+ D388+F880, K172+R173+Y178+D388, D130+K172+R173+D388, D130+K172+R173+Y1 78. D130+R173+Y178+K387, D130+K172+Y178+D388, D130+K172+R173+Y178+D388.In some embodiments, the mutation type at position D130 of the variant is further selected as D130E. In some embodiments, the mutation type at position K172 of the variant can be selected from DEL172, K172A, or K172G. In some embodiments, the mutation type at position R173 of the variant can be selected from DEL173, R173A, R173G, or R173C. In some embodiments, the mutation type at position Y178 of the variant can be selected from Y178H or Y178P. In some embodiments, the mutation type at position R298 of the variant is R298A. In some embodiments, the mutation type at position Y385 of the variant is Y385A. In some embodiments, the mutation type at position K387 of the variant can be selected from K387S, K387Y, or K387G. In some embodiments, the mutation type at the D388 position of the variant may be selected from D388Y, D388A, or D388G. In some embodiments, the mutation type at the F880 position of the variant may be selected from F880A or F880Y.

[0008] In some embodiments, the variant includes a mutation at position K172, and also includes mutations of one, two, three, or four amino acids selected from positions D130, R173, Y178, R298, Y385, K387, D388, or F880, the mutation type being substitution or deletion, and the amino acid sequence of the variant has at least 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with SEQ ID NO: 1. In some embodiments, the variant comprises a mutation group selected from any of the following: K172+R173, D130+K172, K172+K387, K172+F880, K172+D388, K172+R298, K172+R173+Y385, K172+R173+D388, K172+R173+K387, K172+R173+F880, K172+R173+Y178, D130+K172+R173, D130+K172+Y178, D130+K172+Y178, D130+K1 72+K387, K172+Y178+D388, D130+K172+D388, K172+K387+R298, K172+R173+Y385+F880, K172+R173+D388+F880, K172+R173+Y178+D388, D130+K172+R173+D388, D130+K172+R173+Y178, D130+K172+Y178+D388, D130+K172+R173+Y178+D388. In some embodiments, further, the mutation type at position D130 of the variant is D130E. In some embodiments, the mutation type at position K172 of the variant is further selected from DEL172, K172A, or K172G. In some embodiments, the mutation type at position R173 of the variant is further selected from DEL173, R173A, R173G, or R173C. In some embodiments, the mutation type at position Y178 of the variant is Y178H. The mutation type at position R298 of the variant is R298A. In some embodiments, the mutation type at position Y385 of the variant is Y385A. In some embodiments, the mutation type at position K387 of the variant is further selected from K387S, K387Y, or K387G. In some embodiments, the mutation type at position D388 of the variant is further selected from D388Y, D388A, or D388G. In some implementations, the mutation type at the F880 position of the variant can be selected from F880A or F880Y.

[0009] In some embodiments, the variant includes a mutation at position R173 and also includes a mutation of one, two, or three amino acids selected from positions D130, Y178, R298, K387, D388, or F880, wherein the mutation type is substitution or deletion, and the amino acid sequence of the variant has at least 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with SEQ ID NO: 1. In some embodiments, the variant includes a mutation group selected from any of the following: D130+R173, R173+Y178, R173+D388, R173+R298, D130+R173+D388, D130+R173+Y178+K387. In some embodiments, further, the mutation type at position D130 of the variant is D130E. In some embodiments, the mutation type at position R173 of the variant is further selected from DEL173, R173A, R173G, or R173C. In some embodiments, the mutation type at position Y178 of the variant is further selected from Y178H or Y178P. In some embodiments, the mutation type at position R298 of the variant is further selected from R298A. In some embodiments, the mutation type at position K387 of the variant is further selected from K387Y. In some embodiments, the mutation type at position D388 of the variant is further selected from D388Y or D388G.

[0010] In some embodiments, the variant comprises a mutation of any two or three amino acids selected from positions R298, Y385, K387, or D388, wherein the mutation type is substitution or deletion, and the amino acid sequence of the variant has at least 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with SEQ ID NO: 1. In some embodiments, the variant comprises a mutation group selected from any of the following: K387+R298, Y385+R298, D388+R298, Y385+K387, Y385+D388, K387+D388, Y385+K387+D388. In some embodiments, further, the mutation type at position R298 is R298A. In some embodiments, further, the substitution at position Y385 is Y385A. In some embodiments, the substitution at position K387 of the variant may be selected from K387S or K387Y. In some embodiments, the substitution at position D388 of the variant may be selected from D388A or D388G.

[0011] In some embodiments, the amino acid sequence of the variant described in this application has at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or higher sequence identity with any amino acid sequence selected from SEQ ID NO: 2-141. In some embodiments, the amino acid sequence of the variant described in this application is selected from SEQ ID NO: 2-141.

[0012] Secondly, this application provides a method for preparing an RNA polymerase variant, comprising producing at least one RNA polymerase variant described in this application in a host cell. In some embodiments, the host cell contains an expression vector carrying a nucleotide sequence corresponding to the RNA polymerase variant described in this application. In some embodiments, various modifications may be present in the coding region of the polynucleotide sequence, as long as the amino acid sequence of the variant does not change with codon degeneracy or with the preferred codon in the organism expressing the variant. In some embodiments, the nucleotide sequence is selected from SEQ ID NO: 143-282.

[0013] Thirdly, this application provides a method for reducing the generation of dsRNA impurities during in vitro transcription to prepare RNA, comprising contacting a DNA template with one or more RNA polymerase variants described in this application and incubating in an in vitro transcription system.

[0014] Fourthly, this application provides a method for improving the integrity of in vitro transcribed RNA products, comprising contacting a DNA template with one or more RNA polymerase variants described in this application and incubating in an in vitro transcription system.

[0015] Fifthly, this application provides a method for generating RNA, comprising contacting a DNA template with one or more RNA polymerase variants described in this application under conditions that lead to the generation of RNA transcripts, and incubating in an in vitro transcription system.

[0016] In some embodiments, the RNA prepared using the method described in this application may be coding RNA or non-coding RNA, including but not limited to mRNA, siRNA, gRNA, saRNA, dsRNA, ssRNA, miRNA, piRNA, shRNA, etc. In some embodiments, the RNA product is mRNA. In some embodiments, the RNA product is saRNA.

[0017] In some embodiments, the DNA template length is selected from 1000-13000 bp. In some embodiments, the DNA template length is selected from 8000-13000 bp. In some embodiments, the DNA template length is selected from 10000-13000 bp.

[0018] In some embodiments, compared with RNA produced using wild-type RNA polymerase (SEQ ID NO: 1), the residual amount of dsRNA impurities after purification is reduced by at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or higher. In some embodiments, the residual dsRNA impurity rate (dsRNA residual amount / total RNA amount) after purification of RNA produced using the method described in this application is less than 0.04%, less than 0.03%, less than 0.02%, less than 0.01%, less than 0.005%, less than 0.004%, less than 0.003%, less than 0.002%, less than 0.001%, less than 0.0005%, less than 0.0003%, or less than 0.0001%.

[0019] In some embodiments, the mRNA produced using the method described in this application, after purification, exhibits an improvement in the integrity of the mRNA product by at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15% compared to that produced using wild-type T7 RNA polymerase (SEQ ID NO: 1).

[0020] In some embodiments, the saRNA produced using the method described in this application, after purification, exhibits an improvement in the integrity of the saRNA product by at least about 3%, about 5%, about 10%, about 12%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, or about 30% compared to that produced using wild-type T7 RNA polymerase.

[0021] Sixthly, this application provides a method for generating capped RNA through in vitro transcription, comprising incubating a DNA template with one or more RNA polymerase variants and cap analogs described in this application in an in vitro transcription reaction system.

[0022] In some embodiments, the cap analog is a dinucleotide cap, a trinucleotide cap, or a tetranucleotide cap. In some embodiments, the cap analog is a trinucleotide cap. In some embodiments, the trinucleotide cap is selected from GAA, GAC, GAG, GAU, GCA, GCC, GCG, GCU, GGA, GGC, GGG, GGU, GUA, GUC, GUG, and GUU. In some embodiments, the trinucleotide cap is selected from m 7 GpppApA, m 7 GpppApC, m 7 GpppApG, m 7 GpppApU, m 7 GpppCpA、m 7 GpppCpC, m 7 GpppCpG、m 7 GpppCpU, m 7 GpppGpA, m 7 GpppGpC, m 7 GpppGpG, m 7 GpppGpU, m 7 GpppUpA, m 7 GpppUpC, m 7 GpppUpG, and m 7 GpppUpU. In some embodiments, the trinucleotide cap is selected from m 7 G 3′ OMe pppApA、m 7 G 3′OMe pppApC, m 7 G 3′OMe pppApG, m 7 G 3′OMe pppApU, m 7 G 3′OMe pppCpA、m 7 G 3′OMe pppCpC、m 7 G 3′OMe pppCpG, m 7 G 3′OMe pppCpU、m 7 G 3′OMe pppGpA, m 7 G 3′OMe pppGpC, m 7 G 3′OMe pppGpG, m 7 G 3′ OMe pppGpU, m 7 G 3′OMepppUpA、m 7 G 3′OMe pppUpC、m 7 G 3′OMe pppUpG, and m 7 G 3′OMe pppUpU. In some embodiments, the trinucleotide cap is selected from m 7 G 3′OMe pppA 2′OMe pA, m 7 G 3′OMe pppA 2′OMe pC, m 7 G 3′OMe pppA 2′OMe pG, m 7 G 3′OMe pppA 2′OMe pU, m 7 G 3′OMe pppC 2′OMe pA, m 7 G 3′OMe pppC 2′OMe pC, m 7 G 3′OMe pppC 2′OMe pG, m 7 G 3′ OMe pppC 2′OMe pU, m 7 G 3′OMe pppG 2′OMe pA, m 7 G 3′OMe pppG 2′OMe pC, m 7 G 3′OMe pppG 2′OMe pG, m 7 G 3′OMe pppG 2′ OMe pU, m 7 G 3′OMe pppU 2′OMe pA, m 7 G 3′OMe pppU 2′OMe pC, m 7 G 3′OMe pppU 2′OMe pG, and m 7 G 3′OMe pppU 2′OMe pU. In some embodiments, the trinucleotide cap is selected from m 7 GpppA 2′OMe pA, m 7 GpppA2′OMe pC, m 7 GpppA 2′OMe pG, m 7 GpppA 2′OMe pU, m 7 GpppC 2′OMe pA, m 7 GpppC 2′OMe pC, m 7 GpppC 2′OMe pG, m 7 GpppC 2′OMe pU, m 7 GpppG 2′ OMe pA, m 7 GpppG 2′OMe pC, m 7 GpppG 2′OMe pG, m 7 GpppG 2′OMe pU, m 7 GpppU 2′OMe pA, m 7 GpppU 2′OMe pC, m 7 GpppU 2′OMe pG, and m 7 GpppU 2′OMe pU. In some embodiments, the trinucleotide cap is preferably m7GpppA2′OMepG.

[0023] In some embodiments, the in vitro transcriptional capping reaction using the polymerase variant described in this application increases the capping rate of the mRNA product to 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% compared to using wild-type T7 RNA polymerase (SEQ ID NO: 1). In some embodiments, the capping rate of the mRNA product can be increased to 100%.

[0024] In a seventh aspect, this application provides a method for performing in vitro transcription, comprising contacting a DNA template with one or more RNA polymerase variants described in this application under conditions that lead to the generation of RNA transcripts, and incubating in an in vitro transcription system. In some embodiments, the method includes the steps of: 1) providing a DNA template containing a T7 promoter functionally linked to a target nucleotide sequence to be transcribed; 2) contacting the DNA template from step 1) with one or more RNA polymerase variants described in this application; and 3) incubating the DNA template and the RNA polymerase variants in an in vitro transcription system.

[0025] In some embodiments, the incubation temperature in step 3) is 30-50°C, preferably 37°C. In some embodiments, the incubation time in step 3) is 20-240 min, preferably 60 min.

[0026] The in vitro transcription system described in this application contains nucleoside triphosphates and buffer components.

[0027] In some embodiments, the nucleoside triphosphate may be selected from modified or unmodified nucleoside triphosphates (including analogues thereof). In some embodiments, the nucleoside triphosphate may be selected from unmodified ATP, GTP, CTP, and UTP. In some embodiments, the nucleoside triphosphate may be selected from modified nucleoside triphosphates, including but 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 (labels may be biotin, fluorescent substances, digoxigenin, radioactive elements, etc.).

[0028] In some embodiments, the in vitro transcription system also includes RNase inhibitors, inorganic pyrophosphatase, and magnesium ions. In some embodiments, the in vitro transcription system further includes DEPC water.

[0029] Eighthly, this application provides a composition or kit comprising one or more RNA polymerase variants described in this application.

[0030] In one embodiment, the composition or kit further comprises one or more buffer components. In one embodiment, the composition or kit further comprises one or more in vitro transcription system components, wherein the in vitro transcription components are selected from nucleoside triphosphates, RNase inhibitors, inorganic pyrophosphatases, magnesium ions, etc. In one embodiment, the in vitro transcription system components are selected from commercially available mRNA in vitro transcription reagents.

[0031] In some embodiments, the composition or kit further comprises a cap analog. In some embodiments, the cap analog is a dinucleotide cap, a trinucleotide cap, or a tetranucleotide cap. In some embodiments, the cap analog is a trinucleotide cap. In some embodiments, the trinucleotide cap is selected from GAA, GAC, GAG, GAU, GCA, GCC, GCG, GCU, GGA, GGC, GGG, GGU, GUA, GUC, GUG, and GUU. In some embodiments, the trinucleotide cap is preferably m7GpppA2′OMepG.

[0032] Ninthly, this application provides a composition comprising RNA and a pharmaceutically acceptable excipient, wherein the RNA is produced by the in vitro transcription method described in this application. In some embodiments, the RNA product is unmodified. In some embodiments, the RNA product is chemically modified.

[0033] Other implementation plans: 1. An RNA polymerase variant having an amino acid sequence relative to SEQ ID NO:1 containing one, two, three, four, or five amino acid mutations at positions selected from D130, N171, K172, R173, Y178, R298, Y385, K387, D388, or F880, said mutation being a substitution or deletion, and said variant having an amino acid sequence identity of at least 97%, at least 98%, or at least 99% with SEQ ID NO:1.

[0034] 2. The variant as described in item 1, wherein the amino acid sequence of the variant has a mutation of one amino acid relative to SEQ ID NO: 1, wherein the position of the mutated amino acid is selected from N171, K172, R173, Y178, R298, Y385, K387, D388 or F880.

[0035] 3. The variant as described in item 1, wherein the amino acid sequence of the variant relative to SEQ ID NO: 1 comprises: (1) A mutation at position K172, and further comprising mutations of any one, two, three, or four amino acids selected from positions D130, R173, Y178, R298, Y385, K387, D388, or F880, wherein the mutation type is substitution or deletion; or (2) A mutation at the K173 site, and further comprising a mutation of one, two, or three amino acids selected from the D130, Y178, R298, K387, or D388 sites, wherein the mutation type is substitution or deletion; or (3) A mutation of any two or three amino acids at the positions of R298, Y385, K387 or D388, wherein the mutation type is substitution.

[0036] 4. The variant as described in section 3, wherein the variant comprises a mutation group selected from any of the following: K172+R173, D130+K172, K172+K387, K172+F880, K172+D388, K172+R298, D130+R173, R173+Y178, R173+D388, R173+R298, K387+R2 98. Y385+R298, D388+R298, Y385+K387, Y385+D388, K387+D388, K172+R173+Y385, K 172+R173+D388, K172+R173+K387, K172+R173+F880, K172+R173+Y178, D130+K172+ R173, D130+K172+Y178, D130+K172+K387, D130+R173+D388, K172+Y178+D388, D130 +K172+D388, K172+K387+R298, Y385+K387+D388, K172+R173+Y385+F880, K172+R17 3+D388+F880, K172+R173+Y178+D388, D130+K172+R173+D388, D130+K172+R173+Y178, D130+R173+Y178+K387, D130+K172+Y178+D388, or D130+K172+R173+Y178+D388.

[0037] 5. A variant as described in item 2, wherein, 1) The mutation type at position N171 is N171G; 2) The mutation type at position K172 is selected from DEL172, K172A, K172H, K172R, K172Y, K172S, K172E, K172D, K172W, K172F, K172I, K172M, K172V, K172P, K172T, K172C, K172N, K172Q, K172G, or K172L; 3) The mutation type at position R173 is selected from DEL173, R173A, R173H, R173R, R173Y, R173S, R173E, R173D, R173W, R173F, R173I, R173M, R173V, R173P, R173T, R173C, R173N, R173Q, R173G, or R173L; 4) The mutation type at position Y178 is Y178H; 5) The mutation type at position R298 is R298A; 6) The mutation type at the Y385 position is selected from Y385A, Y385D, or Y385E; 7) The mutation type at position K387 is selected from K387Q, K387Y, K387S, or K387A; 8) The mutation type at position D388 is selected from D388A, D388G, or D388L; and 9) The mutation type at the F880 position is selected from F880A, F880G or F880W.

[0038] 6. A variant as described in item 3 or 4, wherein, 1) The mutation type at position D130 is D130E; 2) The mutation type at the K172 position can be selected from DEL172, K172A, or K172G; 3) The mutation type at the R173 position can be selected from DEL173, R173A, R173G or R173C; 4) The mutation type at position Y178 can be selected from Y178H or Y178P; 5) The mutation type at position R298 is R298A; 6) The mutation type at position Y385 is Y385A; 7) The mutation type at the K387 position can be selected from K387S, K387Y, or K387G; 8) The mutation type at position D388 can be selected from D388Y, D388A, or D388G; and 9) The mutation type at the F880 position can be selected from F880A or F880Y.

[0039] 7. The variant as described in item 1, wherein the amino acid sequence of the variant is shown in any of SEQ ID NO: 2-141.

[0040] 8. A nucleotide sequence that encodes a variant as described in any of items 1-7.

[0041] 9. A method for preparing an RNA polymerase variant, comprising producing at least one RNA polymerase variant as described in items 1-7 in a host cell; wherein the host cell contains an expression vector carrying a nucleotide sequence as described in item 8.

[0042] 10. A method for reducing the generation of dsRNA impurities during in vitro transcription to prepare RNA, comprising contacting a DNA template with a variant as shown in any of items 1-7 and incubating it in an in vitro transcription system.

[0043] 11. A method for improving the integrity of in vitro transcribed RNA products, comprising contacting a DNA template with a variant as described in any of items 1-7 and incubating it in an in vitro transcription system.

[0044] 12. The use of at least one variant of items 1-7 or the method described in items 10-11 in the in vitro transcription of RNA.

[0045] 13. A method for generating RNA by in vitro transcription, comprising contacting a DNA template with at least one variant as described in items 1-7 and incubating it in an in vitro transcription system.

[0046] 14. A method for generating capped RNA by in vitro transcription, comprising contacting a DNA template with at least one variant and cap analog as described in items 1-7, and incubating in an in vitro transcription reaction system.

[0047] 15. A composition or kit comprising at least one RNA polymerase variant as described in any of items 1-7 and at least one buffer component.

[0048] 16. The composition or kit described in item 15 further comprises a cap analogue.

[0049] This application has the following beneficial effects: The RNA polymerase variant described in this application exhibits high catalytic efficiency. Its use in mRNA synthesis reduces the generation of double-stranded RNA contaminants. Compared to existing reported mRNA preparation methods, it produces the lowest level of double-stranded RNA contaminants, making it a safe, green, and low-double-stranded RNA contaminant method for mRNA preparation. This method significantly reduces the potential for immunogenicity caused by double-stranded RNA impurities. Furthermore, some RNA polymerase variants provided in this application can improve the integrity of RNA in in vitro transcription products; the variant DEL172+K387S can also improve the utilization rate of cap analogs in the co-transcriptional capping reaction, saving costs. Attached Figure Description

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

[0051] In this application embodiment, enzyme activity is defined as the amount of enzyme required to generate 1 μmol of product or convert 1 μmol of substrate within 1 minute under specific reaction conditions.

[0052] 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: 142-282), then amplified by PCR. The resulting DNA was then introduced into the BseRI and HindIII restriction sites of the expression vector pQE-80L to obtain a recombinant expression vector. The constructed vector was then introduced into the target vector via chemical transformation. E. coli In BL21(DE3), the bacteria were plated on LB agar plates containing ampicillin and incubated overnight at 37°C. Single colonies that grew were then subjected to plasmid extraction and sequencing. The resulting recombinant engineered bacteria containing the target gene were successfully sequenced. E. coli After overnight activation culture in LB medium, the recombinant strain was inoculated into the fermentation broth (LB medium) at a concentration of 1-5% v / v and cultured until the OD600 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.

[0053] WT is a wild-type T7 RNA polymerase variant, and its amino acid sequence is as follows: MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDILESDFAFA (SEQ ID NO: 1) The RNA polymerase variants and their mutation sites are shown in Table 1: Table 1: Mutation sites of RNA polymerase variants and the corresponding amino acid sequence numbers

[0054]

[0055] 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 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 (Vazyme, catalog number: N412). 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).

[0056] Table 2: Reaction System Proportions

[0057] The dsRNA detection results are shown in Table 3. Compared with the WT group, most polymerase variants in Example 1 can effectively reduce the generation of dsRNA impurities during in vitro transcription. Among them, the variants of DEL172-173+Y385A, DEL172-173+D388A, and DEL172-173+D388G can reduce the residual amount of dsRNA by 99%.

[0058] Table 3: dsRNA Residual Status

[0059] 2.2 Modified NTP (m1ψ) (1) The stock solutions of RNA polymerase variants DEL172-173+Y385A, DEL172-173+D388A and DEL172-173+D388G were diluted with storage buffer (Vazyme, catalog number: DD4101) to an enzyme activity of 300 U / μL. The reaction components (20 μL) in Table 4 were loaded into an octet and mixed well, and centrifuged. The octet was placed on a PCR instrument and reacted at 37℃ for 1 h. Then, 36 μL of magnetic beads were added and mixed well, and incubated at room temperature for 2-5 min. The mixture was placed on a magnetic rack to purify the mRNA (Vazyme, catalog number: N412). 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).

[0060] Table 4: Reaction System Proportions

[0061] The dsRNA detection results showed that the in vitro transcription reaction involving enzyme variants DEL172-173+D388A and DEL172-173+D388G could reduce the residual rate of dsRNA (dsRNA residual amount / total RNA amount) to 0.0003%; the in vitro transcription reaction involving enzyme variant DEL172-173+Y385A could reduce the residual rate of dsRNA to 0.0001%.

[0062] Example 3: Comparison of the integrity of in vitro transcribed RNA products 3.1 Unmodified NTP (Template SEQ ID NO: 283) (1) Obtain the purified mRNA by referring to step (1) in 2.1 of Example 2; (2) The integrity of mRNA (intact RNA product / total RNA) was detected by capillary electrophoresis using the Qsep400 fully automated nucleic acid analyzer.

[0063] The test results are shown in Tables 5-1 and 5-2.

[0064] 3.2 Modified NTP (m1ψ, template SEQ ID NO: 283) (1) Obtain the purified mRNA by referring to step (1) in 2.2 of Example 2; (2) The integrity of mRNA was detected by capillary electrophoresis using the Qsep400 fully automated nucleic acid analyzer.

[0065] The test results are shown in Tables 5-1 and 5-2.

[0066] Table 5-1: Integrity of mRNA Products

[0067] Table 5-2: Integrity of mRNA Products

[0068] 3.3 saRNA template I (template SEQ ID NO: 284) (1) The stock solution of RNA polymerase variant K387Y was diluted with storage buffer (Vazyme, catalog number: DD4101) to an enzyme activity of 300 U / μL. In vitro transcription was performed according to the reaction system ratio in Table 2 to obtain purified mRNA; (2) The integrity of the saRNA product was detected by capillary electrophoresis using a Qsep400 fully automated nucleic acid analyzer.

[0069] The test results showed that the integrity of the transcription product was only 56.4% when using wild-type T7 RNA polymerase (WT) for in vitro transcription, while the integrity of the saRNA product could be increased to 73.1% when using the K387Y variant.

[0070] 3.4 saRNA template II (>10000 nt) (1) The stock solutions of RNA polymerase variants K387A and DEL172+K387Y were diluted with storage buffer (Vazyme, catalog number: DD4101) to an enzyme activity of 300 U / μL. In vitro transcription was performed according to the reaction system ratio in Table 6 to obtain purified mRNA; Table 6: Reaction System

[0071] (2) The integrity of the saRNA product was detected by capillary electrophoresis using a Qsep400 fully automated nucleic acid analyzer.

[0072] The test results showed that, compared with the saRNA product obtained by using the WT group polymerase, the variants in the K387A group and the DEL172+K387Y group could improve the integrity of the saRNA product by 3.1% and 4.9%, respectively.

[0073] Example 4: Preparation of Capped RNA by In Vitro Transcription (1) Dilute the stock solutions of K387Y and DEL172+K387S variant enzymes with storage buffer (Vazyme, catalog number: DD4101) to an enzyme activity of 300 U / μL. Prepare the MIX solution into EP tubes according to the reaction system (20 μL) in Table 7. Aliquot the MIX solution into octet, mix well, and centrifuge. Place the octet on a PCR instrument and react at 37℃ for 1 h. Add 36 μL of magnetic beads, mix well, and incubate at room temperature for 2-5 min. Place the mixture on a magnetic rack to purify the mRNA (Vazyme, catalog number: N412). After purification, transfer to an RNase-free centrifuge tube to obtain the purified mRNA.

[0074] Table 7: Reaction System

[0075] (2) Capping rate detection: ① The purified mRNA obtained in step (1) is bound to the probe. The reaction system is shown in Table 8 and the reaction conditions are shown in Table 9. Table 8: Reaction System

[0076] Table 9: Reaction Conditions

[0077] ②RNase H digestion: Prepare the digestion reaction system according to Table 10 (Thermo Scientific, catalog number: EN0201), vortex thoroughly to mix evenly, and place in a PCR instrument. React at 25°C for 20 min. Table 10: Reaction System

[0078] ③SA magnetic bead bonding: A. 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.

[0079] B. Reaction conditions: Remove the centrifuge tube from the magnetic rack, add the enzyme digestion product to the SA magnetic beads, 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.

[0080] ④ Rinsing and Washing: A. Place the product from step ③ on a magnetic rack for 2-3 minutes until the solution becomes clear, then use a pipette to remove the supernatant. B. Add 200 μL of rinsing solution to rinse, being careful not to blow away the magnetic beads, let stand for 0.5~1 min, and then use a pipette to remove the supernatant; C. Repeat step B; D. Remove the centrifuge tube from the magnetic rack, add 30 μL of elution buffer, and mix thoroughly by pipetting 10-20 times to ensure the magnetic beads are evenly dispersed and the elution is complete. E. Place in a PCR instrument and react at 85°C for 3 min. Immediately after reaction, place on a magnetic rack. After the solution clarifies (0.5~1 min), transfer the supernatant to a new centrifuge tube. The supernatant is the desired product. F. The capping rate of the product in step E is detected by capillary electrophoresis. The formula for calculating the capping rate is as follows: Cap1 capping rate % = [Cap1 peak area / (Uncap peak area + Cap1 peak area)] × 100%.

[0081] The results showed that in the co-transcriptional capping reaction, the capping rate of mRNA products in the WT (wild-type T7 RNA polymerase) group was only 87.2%, while the variants in the K387Y group could increase the capping rate of mRNA products to 91.3%, and the variants in the DEL172+K387S group could increase it to 100%.

Claims

1. A variant of an RNA polymerase having an amino acid sequence relative to SEQ ID NO: 1, comprising a mutation of the amino acid at position D130, said variant having an amino acid sequence as shown in any one of SEQ ID NO: 40, 44, 54-56, 58, 59, 61-64, 66, 67, 69, 70, 72-74, 76-79 or 140.

2. A nucleotide sequence encoding a variant as described in claim 1.

3. A method for preparing an RNA polymerase variant, comprising producing at least one RNA polymerase variant as described in claim 1 in a host cell; wherein, The host cell contains an expression vector carrying the nucleotide sequence as described in claim 2.

4. A method for reducing the generation of dsRNA impurities during in vitro transcription to prepare RNA, comprising contacting a DNA template with any of the variants shown in claim 1 and incubating it in an in vitro transcription system.

5. The use of at least one variant of claim 1 or the method of claim 4 in in vitro transcription to generate RNA.

6. A method for generating RNA by in vitro transcription, comprising contacting a DNA template with at least one variant as described in claim 1 and incubating it in an in vitro transcription system.

7. A method for generating capped RNA by in vitro transcription, comprising contacting a DNA template with at least one variant and cap analog as described in claim 1, and incubating in an in vitro transcription reaction system.

8. A composition or kit comprising at least one RNA polymerase variant as described in claim 1 and at least one buffer component.

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