WO2024131998A2 - Rna聚合酶变体、其制备方法及其在rna合成中的应用 - Google Patents

Rna聚合酶变体、其制备方法及其在rna合成中的应用 Download PDF

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WO2024131998A2
WO2024131998A2 PCT/CN2024/079536 CN2024079536W WO2024131998A2 WO 2024131998 A2 WO2024131998 A2 WO 2024131998A2 CN 2024079536 W CN2024079536 W CN 2024079536W WO 2024131998 A2 WO2024131998 A2 WO 2024131998A2
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variant
mutation
mutation type
rna
amino acid
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PCT/CN2024/079536
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English (en)
French (fr)
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徐晓昱
金秋恒
何伟
王冲
胡荟雪
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南京诺唯赞生物科技股份有限公司
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Publication of WO2024131998A2 publication Critical patent/WO2024131998A2/zh

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  • the present application belongs to the field of biotechnology, and specifically relates to RNA polymerase variants, preparation methods thereof, and applications thereof in RNA synthesis.
  • RNA vaccines have the highest protective efficacy. mRNA vaccines have made great contributions to this war against the epidemic by preventing infection, reducing the rate of severe illness, and curbing the spread of the epidemic.
  • the research and development cycle of mRNA vaccines is relatively short, and new vaccine candidates can be developed quickly to respond to viral mutations.
  • the production process is simple, making it easy to efficiently develop and mass produce, allowing it to quickly achieve global supply for epidemics similar to the new coronavirus.
  • circRNA circular RNA
  • RNA has made great achievements in the research and development of vaccines and other drugs.
  • the removal of some impurities in the actual production process needs further study.
  • Double-stranded RNA (dsRNA) impurities can cause strong immunogenicity (Goubau et al., 2014; Kato et al., 2006; Mu et al., 2018). Therefore, it is urgent to develop methods to effectively reduce double-stranded RNA impurities.
  • the present application provides an RNA polymerase variant, a preparation method thereof and an application thereof in RNA synthesis.
  • the present application provides an RNA polymerase variant, whose amino acid sequence, relative to SEQ ID NO: 1, comprises at least one, at least two, at least three, at least four, at least five or at least six amino acid mutations at positions selected from D130, N171, K172, R173, Y178, R298, Y385, K387, D388 or F880, the type of mutation being 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.
  • the variant comprises one, two, three, four, five or six amino acid mutations at positions selected from D130, N171, K172, R173, Y178, R298, Y385, K387, D388 or F880; the type of mutation being selected from deletion or substitution.
  • the mutation type is deletion (indicated by DEL, such as DEL5, indicating the deletion of the amino acid at the 5th position).
  • the mutation type is substitution (such as K5A, indicating that the lysine at the 5th position is mutated to alanine).
  • the mutation type includes deletion and substitution, that is, deletion occurs at some positions and substitution occurs at some positions.
  • the RNA polymerase variant provided by the present application has an amino acid mutation relative to SEQ ID NO: 1, and the mutated amino acid position is selected from N171, K172, R173, Y178, R298, Y385, K387, D388 or F880, and the mutation type is substitution or deletion.
  • the amino acid of the variant The sequence has an amino acid mutation relative to SEQ ID NO: 1, wherein the mutation is selected from:
  • substituted amino acid can be selected from A, G, E, D, H, Y, S, W, P, I, M, V, F, T, C, N, L; or
  • substituted amino acid can be selected from A, C, G, E, D, H, Y, S, W, P, N, Q; or
  • substituted amino acid can be selected from A, Y, S, Q; or
  • substituted amino acid can be selected from A, G, and L;
  • the RNA polymerase variant provided herein whose amino acid sequence, relative to SEQ ID NO: 1, comprises mutations in 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 compared to SEQ ID NO: 1.
  • the variant comprises: (1) a mutation at the K172 position, and further comprises a mutation of one, two, three or four amino acids at a position selected from D130, R173, Y178, R298, Y385, K387, D388 or F880, wherein the mutation type is substitution or deletion; or (2) a mutation at the R173 position, and further comprises a mutation of one, two or three amino acids at a position selected from D130, Y178, R298, K387 or D388, wherein the mutation type is substitution or deletion; or (3) a mutation of two or three amino acids at a position of R298, Y385, K387 or D388, wherein the mutation type is substitution or deletion.
  • the amino acid sequence of the variant is relative to SEQ ID NO: 1, having any combination of mutation points selected from 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+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 ⁇ K13+K172+D388 ⁇ K
  • the mutation type of the variant at the K172 position can be selected from DEL172, K172A or K172G.
  • the mutation type of the variant at the R173 position can be selected from DEL173, R173A, R173G or R173C.
  • the mutation type of the variant at the Y178 position can be selected from Y178H or Y178P.
  • the mutation type of the variant at the R298 position is R298A.
  • the mutation type of the variant at the Y385 position is Y385A.
  • the mutation type of the variant at the K387 position can be selected from K387S, K387Y or K387G.
  • the mutation type of the variant at the D388 position can be selected from D388Y, D388A or D388G.
  • the mutation type of the variant at the F880 position can be selected from F880A or F880Y.
  • the variant comprises a mutation at position K172 and also comprises a mutation of one, two, three or four amino acids at positions selected from D130, R173, Y178, R298, Y385, 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 compared to SEQ ID NO: 1.
  • the variant comprises a mutation group selected from any one 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+
  • the variant has a mutation type of D130E at position D130.
  • the mutation type of the variant at the K172 position can be selected from DEL172, K172A or K172G.
  • the mutation type of the variant at the R173 position can be selected from DEL173, R173A, R173G or R173C. In some embodiments, further, the mutation type of the variant at the Y178 position is Y178H. Further, the mutation type of the variant at the R298 position is R298A. In some embodiments, further, the mutation type of the variant at the Y385 position is Y385A. In some embodiments, further, the mutation type of the variant at the K387 position can be selected from K387S, K387Y or K387G. In some embodiments, further, the mutation type of the variant at the D388 position can be selected from D388Y, D388A or D388G. In some embodiments, further, the mutation type of the variant at position F880 can be selected from F880A or F880Y.
  • the variant comprises a mutation at the R173 position, and further comprises a mutation of one, two or three amino acids at a position selected from D130, Y178, R298, K387, D388 or F880, 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 compared to SEQ ID NO: 1.
  • the variant comprises a mutation group selected from any of the following: D130+R173, R173+Y178, R173+D388, R173+R298, D130+R173+D388, D130+R173+Y178+K387.
  • the mutation type of the variant at the D130 position is D130E.
  • the mutation type of the variant at the R173 position can be selected from DEL173, R173A, R173G or R173C.
  • the mutation type of the variant at the Y178 position is Y178H or Y178P.
  • the mutation type of the variant at the R298 position is R298A.
  • the mutation type of the variant at the K387 position is K387Y.
  • the mutation type of the variant at the D388 position can be selected from D388Y or D388G.
  • the variant comprises a mutation of any two or three amino acids selected from the positions of R298, Y385, K387 or D388, 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.
  • 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.
  • the mutation type of the variant at the R298 position is R298A.
  • the substitution of the variant at the Y385 position is Y385A.
  • the variant may have a substitution at position K387 selected from K387S or K387Y.
  • the variant may have a substitution at position D388 selected from D388A or D388G.
  • the amino acid sequence of the variant described in the present 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 the present application is selected from SEQ ID NO: 2-141.
  • the present application provides a method for preparing an RNA polymerase variant, comprising producing at least one RNA polymerase variant described in the present application in a host cell.
  • the host cell contains an expression vector carrying a nucleotide sequence corresponding to the RNA polymerase variant described in the present application.
  • various modifications may be present in the coding region, as long as the variant amino acid sequence of the present application does not change with the degeneracy of the codon or does not change with the preferred codon in the organism expressing the variant.
  • the nucleotide sequence is selected from SEQ ID NO: 143-282.
  • the present application provides a method for reducing the generation of dsRNA impurities during the preparation of RNA by in vitro transcription, comprising contacting a DNA template with one or more RNA polymerase variants described in the present application and incubating them in an in vitro transcription system.
  • the present 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 the present application and incubating them in an in vitro transcription system.
  • the present application provides a method for generating RNA, comprising contacting a DNA template with one or more RNA polymerase variants described in the present application under conditions that result in the generation of RNA transcription products, and incubating in an in vitro transcription system.
  • the RNA prepared by the method described herein may be a coding RNA or a non-coding RNA, including but not limited to mRNA, siRNA, gRNA, saRNA, dsRNA, ssRNA, miRNA, piRNA, shRNA, etc.
  • the RNA product is mRNA. In some embodiments, the RNA product is saRNA.
  • the DNA template length can be selected from 1000-13000 bp. In some embodiments, the DNA template length can be selected from 8000-13000 bp. In some embodiments, the DNA template length can be selected from 10000-13000 bp.
  • the RNA produced by the methods described herein, after purification, has a residual amount of dsRNA impurities 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 more compared to a wild-type RNA polymerase (SEQ ID NO: 1).
  • the RNA produced by the methods described herein, after purification has a residual dsRNA impurity rate (dsRNA residual amount/total RNA amount) of 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%.
  • dsRNA residual amount/total RNA amount dsRNA impurity rate
  • the integrity of the mRNA product produced using the methods described in the present application is improved 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 using wild-type T7 RNA polymerase (SEQ ID NO: 1).
  • the integrity of the saRNA produced using the methods described herein, after purification is improved 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 using wild-type T7 RNA polymerase.
  • the present application provides a method for generating capped RNA by in vitro transcription, comprising incubating a DNA template with one or more RNA polymerase variants and cap analogs described in the present application in an in vitro transcription reaction system.
  • 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.
  • the trinucleotide cap is selected from m7GpppApA , m7GpppApC , m7GpppApG , m7GpppApU , m7GpppCpA , m7GpppCpC , m7GpppCpG, m7GpppCpU , m7GpppGpA, m7GpppGpC, m7GpppGpG , m7GpppGpU , m7GpppUpA , m7GpppUpC , m7GpppUpG , and m7GpppUpU .
  • the trinucleotide cap is selected from m7G 3′OMe pppApA, m7G 3′OMe pppApC, m7G 3′OMe pppApG , m7G 3′OMe pppApU, m7G 3′OMe pppCpA, m7G 3′OMe pppCpC , m7G 3′OMe pppCpG , m7G 3′OMe pppCpU, m7G 3′OMe pppGpA, m7G 3′OMe pppGpC, m7G 3′OMe pppGpG, m7G 3′OMe pppGpU , m7G 3′OMe pppUpA , m7G 3′OMe pppUpC , m7G 3′OMe pppUpC , m7G 3′OMe pppUpG
  • the trinucleotide cap is selected from m7G 3′OMe pppA 2′OMe pA , m7G 3′OMe pppA 2′OMe pC, m7G 3′OMe pppA 2′OMe pG , m7G 3′OMe pppA 2′OMe pU, m7G 3′OMe pppC 2′OMe pA, m7G 3′OMe pppC 2′OMe pC , m7G 3′OMe pppC 2′OMe pG, m7G 3′OMe pppC 2′OMe pU, m7G 3′OMe pppG 2′OMe pA, m7G 3′OMe pppG 2′OMe pC, m7G 3′OMe pppG 2′OMe pppG 2′OMe pG , m7G 3′OMe p
  • the trinucleotide cap is selected from m7GpppA 2′OMe pA, m7GpppA 2′OMe pC , m7GpppA 2′OMe pG, m7GpppA 2′OMe pU, m7GpppC 2′OMe pA , m7GpppC 2′OMe pC , m7GpppC 2′OMe pG, m7GpppC 2′OMe pU, m7GpppG 2′OMe pA , m7GpppG 2′OMe pC, m7GpppG 2′OMe pG, m7GpppG 2′OMe pU , m7GpppU 2′OMe pA , m7GpppG 2′OMe pG, m7GpppG 2′OMe pU , m7GpppU 2′OM
  • the in vitro transcription capping reaction using the polymerase variants described in the present application increases the capping rate of the mRNA product to 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% compared to the wild-type T7 RNA polymerase (SEQ ID NO: 1). In some embodiments, the capping rate of the mRNA product can be increased to 100%.
  • the present application provides a method for in vitro transcription, comprising contacting a DNA template with one or more RNA polymerase variants described in the present application under conditions that result in the generation of RNA transcription products, and incubating in an in vitro transcription system.
  • the method comprises the following steps: 1) providing a DNA template comprising a T7 promoter, wherein the T7 promoter is functionally linked to a target nucleotide sequence to be transcribed; 2) contacting the DNA template in step 1) with one or more RNA polymerase variants described in the present application; 3) incubating the DNA template and RNA polymerase variant in an in vitro transcription system.
  • 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.
  • the in vitro transcription system described in the present application comprises nucleoside triphosphates and a buffer component.
  • 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.
  • the nucleoside triphosphates can be selected from modified nucleoside triphosphates, including but not limited to m1A (N1-methyladenosine), m6A (N6-methyladenosine), m5C (5-methylcytidine), 5moU (5-methoxyuridine), ⁇ (pseudouridine), m1 ⁇ (N1-methyl-pseudouridine), nucleoside triphosphates with markers (the markers can be biotin, fluorescent substances, digoxin, radioactive elements, etc.).
  • the in vitro transcription system further comprises RNase inhibitor, inorganic pyrophosphatase, and magnesium ions. In some embodiments, the in vitro transcription system further comprises DEPC water.
  • the present application provides a composition or kit comprising one or more RNA polymerase variants described in the present application.
  • the composition or kit further comprises one or more buffer components.
  • the composition or kit further comprises one or more in vitro transcription system components, wherein the in vitro transcription components can be selected from nucleoside triphosphates, RNA enzyme inhibitors, inorganic pyrophosphatase, magnesium ions, etc.
  • the in vitro transcription system components can be selected from commercially available mRNA in vitro transcription reagents.
  • the composition or kit further comprises a cap analog.
  • the cap analog is a dinucleotide cap, a trinucleotide cap, or a tetranucleotide cap.
  • the cap analog is a trinucleotide cap.
  • the trinucleotide cap is selected from GAA, GAC, GAG, GAU, GCA, GCC, GCG, GCU, GGA, GGC, GGG, GGU, GUA, GUC, GUG, and GUU.
  • the trinucleotide cap is preferably m7GpppA2′OMepG.
  • the present application provides a composition comprising RNA and a pharmaceutically acceptable excipient, wherein the RNA is produced by the in vitro transcription method described in the present application.
  • the RNA product is not chemically modified.
  • the RNA product is chemically modified.
  • RNA polymerase variant whose amino acid sequence relative to SEQ ID NO: 1 contains one, two, three, four or five amino acid mutations at positions selected from D130, N171, K172, R173, Y178, R298, Y385, K387, D388 or F880, wherein the mutation type is substitution or deletion, and the amino acid sequence of the variant has a sequence identity of at least 97%, at least 98% or at least 99% with SEQ ID NO: 1.
  • a variant as described in item 1, wherein the amino acid sequence of the variant relative to SEQ ID NO: 1 comprises:
  • a mutation at position K172 and further comprising a mutation 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
  • variants as described in item 3, wherein the variant comprises a mutation group selected from any one 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, K172+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+R29
  • the mutation type at the K172 position is selected from DEL172, K172A, K172H, K172R, K172Y, K172S, K172E, K172D, K172W, K172F, K172I, K172M, K172V, K172P, K172T, K172C, K172N, K172Q, K172G or K172L;
  • the mutation type at the R173 position is selected from DEL173, R173A, R173H, R173R, R173Y, R173S, R173E, R173D, R173W, R173F, R173I, R173M, R173V, R173P, R173T, R173C, R173N, R173Q, R173G or R173L;
  • the mutation type at the Y385 position is selected from Y385A, Y385D or Y385E;
  • the mutation type at the K387 position is selected from K387Q, K387Y, K387S or K387A;
  • the mutation type at position D388 is selected from D388A, D388G or D388L;
  • the mutation type at the F880 position is selected from F880A, F880G or F880W.
  • the mutation type at the K172 position can be selected from DEL172, K172A or K172G;
  • the mutation type at the R173 position can be selected from DEL173, R173A, R173G or R173C;
  • the mutation type at position Y178 can be selected from Y178H or Y178P;
  • the mutation type at position Y385 is Y385A;
  • the mutation type at the K387 position can be selected from K387S, K387Y or K387G;
  • the mutation type at position D388 can be selected from D388Y, D388A or D388G;
  • the mutation type at position F880 can be selected from F880A or F880Y.
  • a method for preparing an RNA polymerase variant comprising producing at least one RNA polymerase variant as described in items 1 to 7 in a host cell; wherein the host cell contains an expression vector carrying the nucleotide sequence as described in item 8.
  • a method for reducing the generation of dsRNA impurities during the preparation of RNA by in vitro transcription comprising contacting a DNA template with a variant as described in any one of items 1 to 7, and incubating the mixture in an in vitro transcription system.
  • a method for improving the integrity of in vitro transcribed RNA products comprising contacting a DNA template with a variant as described in any one of items 1 to 7, and incubating the mixture in an in vitro transcription system.
  • a method for generating RNA by in vitro transcription comprising contacting a DNA template with at least one variant as described in items 1 to 7, and incubating the mixture in an in vitro transcription system.
  • a method for generating capped RNA by in vitro transcription comprising contacting a DNA template with at least one variant and a cap analog as described in items 1 to 7, and incubating the mixture in an in vitro transcription reaction system.
  • composition or kit comprising at least one RNA polymerase variant as described in any one of items 1 to 7 and at least one buffer component.
  • the RNA polymerase variant of the present application has a high catalytic efficiency. When used in mRNA synthesis, it can reduce the production of double-stranded RNA contaminants. Compared with the existing reported mRNA preparation methods, the double-stranded RNA contaminants produced are the lowest, and it is a safe, green, and low double-stranded RNA contaminant mRNA preparation method.
  • the mRNA preparation method of the present application can greatly reduce the possibility of immunogenicity caused by double-stranded RNA impurities.
  • RNA polymerase variants provided in the present application can improve the integrity of the in vitro transcribed product RNA; the variant DEL172+K387S can also improve the utilization rate of cap analogs in co-transcription capping reactions, saving costs.
  • Figure 1 is a schematic diagram of the construction of the recombinant plasmid.
  • enzyme activity is defined as the amount of enzyme required to generate 1 ⁇ mol of product or convert 1 ⁇ mol of substrate within 1 minute under specific reaction conditions.
  • RNA polymerase and its variants shown in Table 1 were synthesized by DNA sequence (SEQ ID NO: 142-282) and then amplified by PCR.
  • the recombinant expression vector was then introduced into the BseRI and HindIII restriction sites of the expression vector pQE-80L to obtain the recombinant expression vector.
  • the constructed vector was introduced into E. coli BL21 (DE3) by chemical transformation technology, spread on an LB plate containing ampicillin resistance, and placed in a 37°C incubator overnight. The single colonies that grew were subjected to plasmid extraction and sequencing to finally obtain the recombinant engineered vector containing the target gene. Cheng Jun inoculated the successfully sequenced E.
  • coli recombinant strain into LB medium for overnight activation culture, and then inoculated 1% V/V into the fermentation liquid (LB medium) and cultured until the OD 600 value was 0.6-0.8.
  • IPTG IPTG with a final concentration of 0.5 mol/L and continuing to culture for 4-6 hours
  • the strain was collected by centrifugation at 12000 rpm and 5°C, and the collected strain was washed with PBS buffer with a pH value of 7.0 and 0.2 M to obtain the bacteria; after ultrasonic fragmentation, affinity chromatography purification was performed to obtain RNA polymerase stock solution.
  • WT is a wild-type T7 RNA polymerase variant, and its amino acid sequence is:
  • RNA polymerase variants and their mutation sites are shown in Table 1:
  • Example 1 The enzyme stock solution obtained in Example 1 was diluted with storage buffer (Vazyme, catalog number: DD4101) to an enzyme activity of 300U/ ⁇ L.
  • the reaction components (20 ⁇ L) in Table 2 were added to the eight-row strips, mixed, and centrifuged; the eight-row strips were placed on a PCR instrument and reacted at 37°C for 1 hour, and then 36 ⁇ L of magnetic beads were added and mixed, and then incubated at room temperature for 2-5 minutes; the mixed solution was placed on a magnetic rack to purify mRNA (Vazyme, catalog number: N412), and after purification, it was transferred to an RNase-free centrifuge tube to obtain purified mRNA;
  • dsRNA impurity content was tested using a dsRNA detection kit (Vazyme, catalog number: DD3509).
  • the dsRNA detection results are shown in Table 3. Compared with the WT group, most of the polymerase variants in Example 1 can effectively reduce the generation of dsRNA impurities during in vitro transcription, among which the variants in the DEL172-173+Y385A, DEL172-173+D388A, and DEL172-173+D388G groups can reduce the residual amount of dsRNA by 99%.
  • dsRNA impurity content was tested using a dsRNA detection kit (Vazyme, catalog number: DD3509).
  • the dsRNA detection results showed that the in vitro transcription reaction involving the enzyme variants DEL172-173+D388A and DEL172-173+D388G could reduce the residual dsRNA rate (dsRNA residual amount/total RNA amount) to 0.0003%; the in vitro transcription reaction involving the enzyme variant DEL172-173+Y385A could reduce the residual dsRNA rate to 0.0001%.
  • step (1) of 2.1 in Example 2 obtain purified mRNA
  • RNA polymerase variant K387Y stock solution Dilute the RNA polymerase variant K387Y stock solution with storage buffer (Vazyme, catalog number: DD4101) to an enzyme activity of 300 U/ ⁇ L. Perform in vitro transcription according to the reaction system ratio in Table 2 to obtain purified mRNA;
  • test results showed that in the in vitro transcription reaction using wild-type T7 RNA polymerase (WT), the integrity of the transcription product was only 56.4%, while the use of the K387Y variant could increase the integrity of the saRNA product to 73.1%.
  • WT wild-type T7 RNA polymerase
  • RNA polymerase variants K387A and DEL172+K387Y Dilute the enzyme stock solutions of RNA polymerase variants K387A and DEL172+K387Y with storage buffer (Vazyme, catalog number: DD4101) to an enzyme activity of 300 U/ ⁇ L. Perform in vitro transcription according to the reaction system ratio in Table 6 to obtain purified mRNA;
  • test results showed that compared with the saRNA products obtained using the WT group polymerase, the variants of the K387A group and the DEL172+K387Y group could increase the integrity of the saRNA products by 3.1% and 4.9%, respectively.
  • 2RNase H digestion Prepare the digestion reaction system (Thermo Scientific, Catalog No.: EN0201) according to Table 10, vortex thoroughly to mix well, place in a PCR instrument, and react at 25°C for 20 min.
  • Magnetic bead washing Take 9 ⁇ L SA magnetic beads into a centrifuge tube and place it on a magnetic rack. When the solution becomes clear, use a pipette to discard the supernatant. Remove the centrifuge tube from the magnetic rack, add 200 ⁇ L RNase-free H2O to rinse, place it on a magnetic rack, when the solution becomes clear, use a pipette to discard the supernatant, and then add 200 ⁇ L RNase-free H2O to repeat the rinse.
  • Reaction conditions Remove the centrifuge tube from the magnetic rack, add the enzyme cleavage product to the SA magnetic beads, pipette 20-30 times to mix thoroughly, place on a tumbling instrument and incubate at room temperature for 30 minutes to allow the magnetic beads and enzyme cleavage product to fully combine.
  • step 3 Place the product of step 3 on a magnetic rack for 2-3 minutes until the solution becomes clear and discard the supernatant with a pipette;
  • step E Use capillary electrophoresis to detect the capping rate of the product in step E.
  • the capping rate is calculated by the formula:
  • Cap1 capping rate % [Cap1 peak area/(Uncap peak area+Cap1 peak area)] ⁇ 100%.
  • the test results showed that in the co-transcriptional capping reaction, the capping rate of the mRNA product in the WT (wild-type T7 RNA polymerase) group was only 87.2%, while the variant in the K387Y group could increase the capping rate of the mRNA product to 91.3%, and the variant in the DEL172+K387S group could increase it to 100%.

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Abstract

本申请提供RNA聚合酶变体及其制备方法，将本申请的RNA聚合酶变体用于体外转录中，可获得低dsRNA污染、完整性较高的RNA产物，同时还可提高共转录加帽反应体系中帽类似物的利用效率，节约成本。此外，本申请还提供了一种体外转录生成RNA的方法。

Description

RNA聚合酶变体、其制备方法及其在RNA合成中的应用 技术领域
本申请属于生物技术领域，具体涉及RNA聚合酶变体、其制备方法及其在RNA合成中的应用。
背景技术
2019年末新型冠状病毒肺炎(Corona Virus Disease 2019，COVID-19)在全球爆发，造成全球几十亿人口感染，在Paxlovid(奈玛特韦片/利托那韦片组合包装)未上市前(2021年11月22日FDA紧急批准上市)，疫苗成了预防感染最有效的一道防线。在这次庞大的全球性战疫中，数十亿人接种了BioNTech公司与辉瑞研发的BNT162b2(mRNA疫苗)、美国Moderna公司研发的mRNA-1273(mRNA疫苗)或阿斯利康研发的AZD1222(腺病毒载体疫苗)等新冠疫苗，其中，RNA疫苗保护效力最高。mRNA疫苗通过预防感染降低重症率遏制疫情传播，为这场战疫作出了巨大贡献。
mRNA疫苗研发周期相对较短，能够快速开发新型候选疫苗应对病毒变异，通过体液免疫及T细胞免疫双重机制，其免疫原性强，效果显著，且生产工艺简单，易于高效研发和大规模生产，为新冠类似的战疫做到快速迅速做到全球供应。
根据最新消息，除mRNA外，circular RNA(circRNA)相关药物的研究，也有了突破性进展。Orna公司(Orna Therapeutics)利用circRNA开发了体内细胞治疗产品，在2022年美国基因和细胞治疗学会(ASGCT)年会上公布的研究报告中，已经证明了其在肿瘤治疗等其他领域也有着巨大的应用潜力。
RNA在疫苗等药物研发领域大放异彩，但在实际生产过程中的一些杂质的去除还有待进一步研究，其中双链RNA(dsRNA)杂质会引起强烈的免疫原性(Goubau et al.,2014；Kato et al.,2006；Mu et al.,2018)，因此亟急需开发有效降低双链RNA杂质的方法。
发明内容
本申请提供一种RNA聚合酶变体、其制备方法及其在RNA合成中的应用。申请第一方面，本申请提供一种RNA聚合酶变体，其氨基酸序列相对于SEQ ID NO：1，包含在选自D130、N171、K172、R173、Y178、R298、Y385、K387、D388或F880位置处至少一个、至少两个、至少三个、至少四个、至少五个或至少六个氨基酸的突变，所述突变的类型选自缺失或取代，且所述变体的氨基酸序列与SEQ ID NO：1具有至少95％、96％、97％、98％或99％的序列一致性。在一些实施方案中，所述变体包含在选自D130、N171、K172、R173、Y178、R298、Y385、K387、D388或F880位置处的一个、两个、三个、四个、五个或六个氨基酸的突变；所述的突变类型选自缺失或取代。在一些实施方案中，所述突变类型为缺失(用DEL表示，例如DEL5，表示在第5个位点处的氨基酸缺失)。在一些实施方案中，所述的突变类型为取代(例如K5A，表示在第5个位点处的赖氨酸突变为丙氨酸)。在一些实施方案中，所述突变类型包含缺失和取代，即一些位置发生缺失，一些位置发生取代。
在一些实施方案中，本申请提供的RNA聚合酶变体，其氨基酸序列相对于SEQ ID NO：1，具有一个氨基酸的突变，突变的氨基酸位点选自N171、K172、R173、Y178、R298、Y385、K387、D388或F880，所述突变类型为取代或缺失。在一些实施方案中，所述变体的氨基酸 序列相对于SEQ ID NO：1具有一个氨基酸突变，所述的突变选自：
(1)在N171位置处的取代，所述取代氨基酸为G；或
(2)在K172位置处的取代或缺失，所述取代氨基酸可选自A，G，E，D，H，Y，S，W，P，I，M，V，F，T，C，N，L；或
(3)在R173位置处的取代或缺失，所述取代氨基酸可选自A，C，G，E，D，H，Y，S，W，P，N，Q；或
(4)在Y178位置处的取代，所述取代氨基酸为H；或
(5)在R298位置处的取代，所述取代氨基酸为A；或
(6)在Y385位置处的取代，所述取代氨基酸可选自A，E，D；或
(7)在K387位置处的取代，所述取代氨基酸可选自A，Y，S，Q；或
(8)在D388位置处的取代，所述取代氨基酸可选自A，G，L；或
(9)在F880位置处的取代，所述取代氨基酸可选自A，G，W。
在一些实施方案中，本申请提供的RNA聚合酶变体，其氨基酸序列相对于SEQ ID NO：1，包含在选自D130、K172、R173、Y178、R298、Y385、K387、D388或F880位置处二个、三个、四个或五个氨基酸的突变，且所述变体的氨基酸序列与SEQ ID NO：1相比具有至少95％、96％、97％、98％、99％或99.5％的序列一致性。在一些实施方案中，所述变体包含：(1)在K172位置处的突变，并且还包含在选自D130、R173、Y178、R298、Y385、K387、D388或F880位置处一个、两个、三个或四个氨基酸的突变，所述突变类型为取代或缺失；或(2)在R173位置处的突变，并且还包含在选自D130、Y178、R298、K387或D388位置处一个、两个或三个氨基酸的突变，所述突变类型为取代或缺失；或(3)在R298、Y385、K387或D388位置处两个或三个氨基酸的突变，所述突变类型为取代或缺失。在一些实施方案中，所述变体的氨基酸序列相对于SEQ ID NO：1，具有选自下述任一的突变点组合：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+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+R173+D388+F880、K172+R173+Y178+D388、D130+K172+R173+D388、D130+K172+R173+Y178、D130+R173+Y178+K387、D130+K172+Y178+D388、D130+K172+R173+Y178+D388。在一些实施方案中，进一步的，所述变体在D130位置处的突变类型为D130E。在一些实施方案中，进一步的，所述变体在K172位置处的突变类型可选自DEL172、K172A或K172G。在一些实施方案中，进一步的，所述变体在R173位置处的突变类型可选自DEL173、R173A、R173G或R173C。在一些实施方案中，进一步的，所述变体在Y178位置处的突变类型可选自Y178H或Y178P。在一些实施方案中，进一步的，所述变体在R298位置处的突变类型为R298A。在一些实施方案中，进一步的，所述变体在Y385位置处的突变类型为Y385A。在一些实施方案中，进一步的，所述变体在K387位置处的突变类型可选自K387S、K387Y或K387G。在一些实施方案中，进一步的，所述变体在D388位置处的突变类型可选自D388Y、D388A或D388G。在一些实施方案中，进一步的，所述变体在F880位置处的突变类型可选自F880A或F880Y。
在一些实施方案中，所述变体包含在K172位置处的突变，并且还包含在选自D130、R173、Y178、R298、Y385、K387、D388或F880位置处一个、两个、三个或四个氨基酸的突变，所述突变类型为取代或缺失，且所述变体的氨基酸序列与SEQ ID NO：1相比具有至少95％、96％、97％、98％、99％或99.5％的序列一致性。在一些实施方案中，所述变体包含选自下述任一的突变组：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+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。在一些实施方案中，进一步的，所述变体在D130位置处的突变类型为D130E。在一些实施方案中，进一步的，所述变体在K172位置处的突变类型可选自DEL172、K172A或K172G。在一些实施方案中，进一步的，所述变体在R173位置处的突变类型可选自DEL173、R173A、R173G或R173C。在一些实施方案中，进一步的，所述变体在Y178位置处的突变类型为Y178H。进一步的，所述变体在R298位置处的突变类型为R298A。在一些实施方案中，进一步的，所述变体在Y385位置处的突变类型为Y385A。在一些实施方案中，进一步的，所述变体在K387位置处的突变类型可选自K387S、K387Y或K387G。在一些实施方案中，进一步的，所述变体在D388位置处的突变类型可选自D388Y、D388A或D388G。在一些实施方案中，进一步的，所述变体在F880位置处的突变类型可选自F880A或F880Y。
在一些实施方案中，所述变体包含在R173位置处的突变，并且还包含在选自D130、Y178、R298、K387、D388或F880位置处一个、两个或三个氨基酸的突变，所述突变类型为取代或缺失，且所述变体的氨基酸序列与SEQ ID NO：1相比具有至少95％、96％、97％、98％、99％或99.5％的序列一致性。在一些实施方案中，所述变体包含选自下述任一的突变组：D130+R173、R173+Y178、R173+D388、R173+R298、D130+R173+D388、D130+R173+Y178+K387。在一些实施方案中，进一步的，所述变体在D130位置处的突变类型为D130E。在一些实施方案中，进一步的，所述变体在R173位置处的突变类型可选自DEL173、R173A、R173G或R173C。在一些实施方案中，进一步的，所述变体在Y178位置处的突变类型为Y178H或Y178P。在一些实施方案中，进一步的，所述变体在R298位置处的突变类型为R298A。在一些实施方案中，进一步的，所述变体在K387位置处的突变类型为K387Y。在一些实施方案中，进一步的，所述变体在D388位置处的突变类型可选自D388Y或D388G。
在一些实施方案中，所述变体包含在选自R298、Y385、K387或D388位置处中任意两个或三个氨基酸的突变，所述突变类型为取代或缺失，且所述变体的氨基酸序列与SEQ ID NO：1具有至少95％、96％、97％、98％、99％或99.5％的序列一致性。在一些实施方案中，所述变体包含选自下述任一的突变组：K387+R298、Y385+R298、D388+R298、Y385+K387、Y385+D388、K387+D388、Y385+K387+D388。在一些实施方案中，进一步的，所述变体在R298位置处的突变类型为R298A。在一些实施方案中，进一步的，所述变体在Y385位置处的取代为Y385A。在一些实施方案中，进一步的，所述变体在K387位置处的取代可选自K387S或K387Y。在一些实施方案中，进一步的，所述变体在D388位置处的取代可选自D388A或D388G。
在一些实施方案中，本申请所述的变体的氨基酸序列与任一选自SEQ ID NO：2-141所示的氨基酸序列具有至少99.0％、至少99.1％、至少99.2％、至少99.3％、至少99.4％、至少99.5％、至少99.6％、至少99.7％、至少99.8％、至少99.9％或更高的序列一致性。在一些实施方案中，本申请所述的变体的氨基酸序列选自SEQ ID NO：2-141。
第二方面，本申请提供一种制备RNA聚合酶变体的方法，包括在宿主细胞中生产至少一种本申请所述的RNA聚合酶变体。在一些实施方案中，所述宿主细胞中含有携带本申请所述RNA聚合酶变体对应的核苷酸序列的表达载体。在一些实施方案中，所述多核苷酸序列中，在编码区中可以存在各种修饰，只要本申请的变体氨基酸序列不随密码子的简并性变化或不随表达该变体的生物体中优选密码子变化即可。在一些实施方案中，所述的核苷酸序列选自SEQ ID NO：143-282。
第三方面，本申请提供一种降低体外转录制备RNA过程中dsRNA杂质生成的方法，包括使DNA模板与本申请所述的一种或多种RNA聚合酶变体接触，在体外转录体系中孵育。
第四方面，本申请提供一种提高体外转录RNA产物完整性的方法，包括使DNA模板与本申请所述的一种或多种RNA聚合酶变体接触，在体外转录体系中孵育。
第五方面，本申请提供一种生成RNA的方法，包括在导致RNA转录产物生成的条件下，使DNA模板与本申请所述的一种或多种RNA聚合酶变体接触，在体外转录体系中孵育。
在一些实施方案中，利用本申请所述方法制备的RNA可为编码RNA或非编码RNA，包括但不限于mRNA、siRNA、gRNA、saRNA、dsRNA、ssRNA、miRNA、piRNA、shRNA等。在一些实施方案中，所述RNA产物为mRNA。在一些实施方案中，所述RNA产物为saRNA。
在一些实施方案中，所述的DNA模板长度可选自1000-13000bp。在一些实施方案中，所述DNA模板长度可选自8000-13000bp。在一些实施方案中，所述DNA模板长度可选自10000-13000bp。
在一些实施方案中，与利用野生型RNA聚合酶(SEQ ID NO：1)相比，采用本申请所述方法生产的RNA，经纯化后，dsRNA杂质的残留量降低至少50％、至少55％、至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少95％或更高。在一些实施方案中，利用本申请所述的方法产生的RNA，经纯化后dsRNA杂质残留率(dsRNA残留量/总RNA的量)低于0.04％、低于0.03％、低于0.02％、低于0.01％、低于0.005％、低于0.004％、低于0.003％、低于0.002％、低于0.001％、低于0.0005％、低于0.0003％或低于0.0001％。
在一些实施方案中，利用本申请所述的方法产生的mRNA，经纯化后，同利用野生型的T7 RNA聚合酶(SEQ ID NO：1)相比，mRNA产物的完整性提高至少约1％、约2％、约3％、约4％、约5％、约6％、约7％、约8％、约9％、约10％、约11％、约12％、约13％、约14％或约15％。
在一些实施方案中，利用本申请所述的方法产生的saRNA，经纯化后，同利用野生型的T7 RNA聚合酶相比，saRNA产物的完整性提高至少约3％、约5％、约10％、约12％、约14％、约15％、约16％、约17％、约18％、约19％、约20％、约21％、约22％、约23％、约24％、约25％、约26％、约27％、约28％、约29％或约30％。
第六方面，本申请提供一种体外转录生成加帽RNA的方法，包括使DNA模板与本申请所述的一种或多种RNA聚合酶变体及帽类似物在体外转录反应体系中孵育。
在一些实施方案中，所述帽类似物是二核苷酸帽、三核苷酸帽或四核苷酸帽。在一些实施方案中，所述帽类似物是三核苷酸帽。在一些实施方案中，所述三核苷酸帽选自GAA、GAC、GAG、GAU、GCA、GCC、GCG、GCU、GGA、GGC、GGG、GGU、GUA、GUC、GUG和GUU。在一些实施方案中，所述三核苷酸帽选自m7GpppApA、m7GpppApC、m7GpppApG、m7GpppApU、m7GpppCpA、m7GpppCpC、m7GpppCpG、m7GpppCpU、m7GpppGpA、m7GpppGpC、m7GpppGpG、m7GpppGpU、m7GpppUpA、m7GpppUpC、m7GpppUpG，和m7GpppUpU。在一些实施方案中，所述三核苷酸帽选自m7G3′OMepppApA、m7G3′OMepppApC、m7G3′OMepppApG、m7G3′OMepppApU、m7G3′OMepppCpA、m7G3′OMepppCpC、m7G3′OMepppCpG、m7G3′OMepppCpU、m7G3′OMepppGpA、m7G3′OMepppGpC、m7G3′OMepppGpG、m7G3′OMepppGpU、m7G3′OMepppUpA、m7G3′OMepppUpC、m7G3′OMepppUpG，以及m7G3′OMepppUpU。在一些实施方案中，所述三核苷酸帽选自m7G3′OMepppA2′OMepA、m7G3′OMepppA2′OMepC、m7G3′OMepppA2′OMepG、m7G3′OMepppA2′OMepU、m7G3′OMepppC2′OMepA、m7G3′OMepppC2′OMepC、m7G3′OMepppC2′OMepG、m7G3′OMepppC2′OMepU、m7G3′OMepppG2′OMepA、m7G3′OMepppG2′OMepC、m7G3′OMepppG2′OMepG、m7G3′OMepppG2′OMepU、m7G3′OMepppU2′OMepA、m7G3′OMepppU2′OMepC、m7G3′OMepppU2′OMepG，和m7G3′OMepppU2′OMepU。在一些实施方案中，所述三核苷酸帽选自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，和m7GpppU2′OMepU。在一些实施方式中，所述三核苷酸帽优选为m7GpppA2′OMepG。
在一些实施方案中，利用本申请所述的聚合酶变体进行体外转录加帽反应，同利用野生型T7 RNA聚合酶(SEQ ID NO：1)相比，mRNA产物的加帽率提高至90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％。在一些实施方案中，mRNA产物的加帽率可提高至100％。
第七方面，本申请提供了一种进行体外转录的方法，包括在导致RNA转录产物生成的条件下，使DNA模板与本申请所述的一种或多种RNA聚合酶变体接触，在体外转录体系中孵育。在一些实施方案中，所述方法包括下述步骤：1)提供包含T7启动子DNA模板，所述T7启动子功能性连接到待转录的靶核苷酸序列上；2)使用本申请所述的一种或多种RNA聚合酶变体与步骤1)中的DNA模板相接触；3)在体外转录体系中，孵育所述的DNA模板和RNA聚合酶变体。
在一些实施方案中，步骤3)所述的孵育温度为30～50℃，优选为37℃。在一些实施方案中，步骤3)的孵育时间为20～240min，优选为60min。
本申请所述的体外转录体系中包含三磷酸核苷和缓冲组分。
在一些实施方案中，所述的三磷酸核苷可选自修饰或未修饰的三磷酸核苷(包括其类似物)。在一些实施方案中，所述三磷酸核苷可选自未修饰的ATP、GTP、CTP、UTP。在一些实施方案中，所述三磷酸核苷可选自选自修饰三磷酸核苷，包括但不限于m1A(N1-甲基腺苷)、m6A(N6-甲基腺苷)、m5C(5-甲基胞苷)、5moU(5-甲氧基尿苷)、ψ(假尿苷)、m1ψ(N1-甲基-假尿苷)、带有标记物的三磷酸核苷(标记物可为生物素、荧光物质、地高辛、放射性元素等)。
在一些实施方案中，体外转录体系中还包含RNA酶抑制剂、无机焦磷酸酯酶、镁离子。在一些实施方案中，体外转录体系中进一步包含DEPC水。
第八方面，本申请提供一种组合物或试剂盒，包含本申请所述的一种或多种RNA聚合酶变体。
在一个实施方式中，所述组合物或试剂盒还包含一种或多种缓冲组分。在一个实施方案中，所述组合物或试剂盒还包含一种或多种体外转录体系组分，其中所述体外转录组分可选自三磷酸核苷、RNA酶抑制剂、无机焦磷酸酯酶、镁离子等。在一个实施方案中，所述体外转录体系组分可选自市售的mRNA体外转录试剂。
在一些实施方案中，所述组合物或试剂盒中还包含帽类似物。在一些实施方案中，所述帽类似物是二核苷酸帽、三核苷酸帽或四核苷酸帽。在一些实施方案中，所述帽类似物是三核苷酸帽。在一些实施方案中，所述三核苷酸帽选自GAA、GAC、GAG、GAU、GCA、GCC、GCG、GCU、GGA、GGC、GGG、GGU、GUA、GUC、GUG和GUU。在一些实施方式中，所述三核苷酸帽优选为m7GpppA2′OMepG。
第九方面，本申请提供了一种组合物，所述组合物包含RNA和药学上可接受的赋形剂，其中所述RNA为本申请所述体外转录方法所产生。在一些实施方案中，所述RNA产物未经化学修饰。在一些实施方案中，所述RNA产物经过化学修饰。
其它实施方案：
1、一种RNA聚合酶变体，其氨基酸序列相对于SEQ ID NO：1包含在选自D130、N171、K172、R173、Y178、R298、Y385、K387、D388或F880位置处的一个、两个、三个、四个或五个氨基酸的突变，所述突变类型为取代或缺失，且所述变体的氨基酸序列与SEQ ID NO：1具有至少97％、至少98％或至少99％的序列一致性。
2、如第1项中所述的变体，所述变体的氨基酸序列相对于SEQ ID NO：1具有一个氨基酸的突变，所述突变氨基酸的位置选自N171、K172、R173、Y178、R298、Y385、K387、D388或F880。
3、如第1项中所述的变体，所述变体的氨基酸序列相对于SEQ ID NO：1，包含：
(1)在K172位置处的突变，并且还包含在选自D130、R173、Y178、R298、Y385、K387、D388或F880位置处任意一个、两个、三个或四个氨基酸的突变，所述突变类型为取代或缺失；或
(2)在K173位点处的突变，并且还包含在选自D130、Y178、R298、K387或D388位置处任意一个、两个或三个氨基酸的突变，所述突变类型为取代或缺失；或
(3)在R298、Y385、K387或D388位置处任意两个或三个氨基酸的突变，所述突变类型为取代。
4、如第3项中所述的变体，所述变体包含选自下述任一的突变组：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+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+R173+D388+F880、K172+R173+Y178+D388、D130+K172+R173+D388、D130+K172+R173+Y178、D130+R173+Y178+K387、D130+K172+Y178+D388、或D130+K172+R173+Y178+D388。
5、如第2项中所述的变体，其中，
1)N171位置处的突变类型为N171G；
2)K172位置处的突变类型选自DEL172、K172A、K172H、K172R、K172Y、K172S、K172E、K172D、K172W、K172F、K172I、K172M、K172V、K172P、K172T、K172C、K172N、K172Q、K172G或K172L；
3)R173位置处的突变类型选自DEL173、R173A、R173H、R173R、R173Y、R173S、R173E、R173D、R173W、R173F、R173I、R173M、R173V、R173P、R173T、R173C、R173N、R173Q、R173G或R173L；
4)Y178位置处的突变类型为Y178H；
5)R298位置处的突变类型为R298A；
6)Y385位置处的突变类型选自Y385A、Y385D或Y385E；
7)K387位置处的突变类型选自K387Q、K387Y、K387S或K387A；
8)D388位置处的突变类型选自D388A、D388G或D388L；以及
9)F880位置处的突变类型选自F880A、F880G或F880W。
6、如第3项或第4项中所述的变体，其中，
1)D130位置处的突变类型为D130E；
2)K172位置处的突变类型可选自DEL172、K172A或K172G；
3)R173位置处的突变类型可选自DEL173、R173A、R173G或R173C；
4)Y178位置处的突变类型可选自Y178H或Y178P；
5)R298位置处的突变类型为R298A；
6)Y385位置处的突变类型为Y385A；
7)K387位置处的突变类型可选自K387S、K387Y或K387G；
8)D388位置处的突变类型可选自D388Y、D388A或D388G；以及
9)F880位置处的突变类型可选自F880A或F880Y。
7、如第1项中所述的变体，所述变体的氨基酸序列如SEQ ID NO：2-141中任一所示。
8、核苷酸序列，其编码如第1-7项中任一所述的变体。
9、RNA聚合酶变体的制备方法，其包括在宿主细胞中生产至少一种如第1-7项中所述的RNA聚合酶变体；其中，所述宿主细胞中含有携带如第8项中所述的核苷酸序列的表达载体。
10、一种降低体外转录制备RNA过程中dsRNA杂质生成的方法，其包括使DNA模板与如第1-7项中任一所示的变体接触，在体外转录体系中孵育。
11、一种提高体外转录RNA产物完整性的方法，其包括DNA模板与如第1-7项中任一所述的变体接触，在体外转录体系中孵育。
12、如第1-7项中至少一种变体或如第10-11项所述的方法在体外转录生成RNA中的应用。
13、一种体外转录生成RNA的方法，其包括DNA模板与如第1-7项中所述的至少一种变体接触，在体外转录体系中孵育。
14、一种体外转录生成加帽RNA的方法，其包括DNA模板与如第1-7项中所述的至少一种变体及帽类似物接触，在体外转录反应体系中孵育。
15、一种组合物或试剂盒，其包含至少一种如第1-7项中任一所述的RNA聚合酶变体和至少一种缓冲组分。
16、如第15项中所述的组合物或试剂盒，还包含帽类似物。
本申请具有以下有益效果：
本申请的RNA聚合酶变体催化效率较高，将其用于mRNA合成中，可减少双链RNA污染物的产生，与现有报道的mRNA制备方法相比，所产生双链RNA污染物最低，是一种安全、绿色、低双链RNA污染物的mRNA制备方法。利用本申请的mRNA制备方法可极大幅度降低由双链RNA杂质引起免疫原性的可能。此外，本申请提供的部分RNA聚合酶变体能够提高体外转录产物RNA的完整性；变体DEL172+K387S还可提高共转录加帽反应中帽类似物的利用率，节约了成本。
附图说明
图1为重组质粒的构建示意图。
具体实施方式
本申请实施例中，酶活的定义为：在特定反应条件下1分钟内生成1μmol产物或者转化1μmol底物所需要的酶量。
实施例1 RNA聚合酶变体的制备
将表1中所示的RNA聚合酶及其变体经过DNA序列合成后(SEQ ID NO：142-282)进行PCR扩增，然后导入表达载体pQE-80L的BseRI和HindIII酶切位点获得重组表达载体，将构建好的载体通过化转技术导入E.coli BL21(DE3)中，涂布在含氨苄抗性的LB平板中，放入37℃培养箱中过夜，将长出来的单菌落进行质粒提取和测序，最终获得含目的基因的重组工程菌将测序成功的E.coli重组菌株接种到LB培养基中进行过夜活化培养后1～5％V/V接种至发酵液(LB培养基)，培养至OD 600值为0.6～0.8，加入终浓度为0.5mol/L的IPTG继续培养4-6h后，12000rmp，5℃下离心收集菌株，采用pH值为7.0，0.2M的PBS缓冲液洗涤收集后菌株，获得菌体；通过超声波破碎后，进行亲和层析纯化，获得RNA聚合酶原液。
WT为野生型T7 RNA聚合酶变体，其氨基酸序列为：
RNA聚合酶变体及其突变位点如表1所示：
表1：RNA聚合酶变体突变位点及对应的氨基酸序列编号

实施例2体外转录中dsRNA杂质生成情况
2.1未修饰NTP
(1)将实施例1中获得的酶原液用储存buffer(Vazyme，货号：DD4101)稀释成酶活为300U/μL。按表2中的反应组分(20μL)装至八联排中混匀，离心；将八联排，置于PCR仪上37℃反应1h后加入36μL的磁珠混匀后室温孵育2～5min；将混合液置于磁力架上以纯化mRNA(Vazyme，货号：N412)，纯化后转移至RNase-free的离心管中，获得纯化后的mRNA；
(2)通过dsRNA检测试剂盒(Vazyme，货号：DD3509)测试dsRNA杂质含量。
表2：反应体系配比

dsRNA检测结果见表3，与WT组相比，实施例1中大多数聚合酶变体均能有效降低体外转录过程中dsRNA杂质的生成，其中DEL172-173+Y385A、DEL172-173+D388A、DEL172-173+D388G组的变体能将dsRNA的残留量降低99％。
表3：dsRNA残留情况



2.2修饰的NTP(m1ψ)
(1)将RNA聚合酶变体DEL172-173+Y385A、DEL172-173+D388A及DEL172-173+D388G的酶原液用储存buffer(Vazyme，货号：DD4101)稀释成酶活为300U/μL。按表4中的反应组分(20μL)装至八联排中混匀，离心；将八联排，置于PCR仪上37℃反应1h后加入36μL的磁珠混匀，室温孵育2～5min；将混合液置于磁力架上来纯化mRNA(Vazyme，货号：N412)，纯化后转移至RNase-free的离心管中，获得纯化后mRNA；
(2)通过dsRNA检测试剂盒(Vazyme，货号：DD3509)测试dsRNA杂质含量。
表4：反应体系配比

dsRNA检测结果显示，酶变体DEL172-173+D388A、DEL172-173+D388G参与的体外转录反应可将dsRNA的残留率(dsRNA残留量/总RNA的量)降低至0.0003％；酶变体DEL172-173+Y385A参与的体外转录反应可将dsRNA的残留率降低至0.0001％。
实施例3体外转录RNA产物完整性的比对
3.1未修饰NTP(模板SEQ ID NO：283)
(1)参考实施例2中2.1的步骤(1)获取纯化后的mRNA；
(2)经Qsep400全自动核酸分析仪进行毛细管电泳，检测mRNA的完整性(完整的RNA产物/总RNA)。
检测结果见表5-1和表5-2。
3.2修饰的NTP(m1ψ，模板SEQ ID NO：283)
(1)参考实施例2中2.2的步骤(1)获取纯化后的mRNA；
(2)经Qsep400全自动核酸分析仪进行毛细管电泳，检测mRNA的完整性。
检测结果见表5-1和表5-2。
表5-1：mRNA产物的完整性
表5-2：mRNA产物的完整性


3.3 saRNA模板I(模板SEQ ID NO：284)
(1)将RNA聚合酶变体K387Y的酶原液用储存buffer(Vazyme，货号：DD4101)稀释成酶活为300U/μL。按照表2中的反应体系配比进行体外转录，获得纯化后的mRNA；
(2)经Qsep400全自动核酸分析仪进行毛细管电泳，检测saRNA产物的完整性。
检测结果显示，利用野生型的T7 RNA聚合酶(WT)进行体外转录反应中，转录产物的完整性仅为56.4％，而利用K387Y变体可将saRNA产物的完整性提升至73.1％。
3.4 saRNA模板II(＞10000nt)
(1)将RNA聚合酶变体K387A和DEL172+K387Y的酶原液用储存buffer(Vazyme，货号：DD4101)稀释成酶活为300U/μL。按照表6中的反应体系配比进行体外转录，获得纯化后的mRNA；
表6：反应体系

(2)经Qsep400全自动核酸分析仪进行毛细管电泳，检测saRNA产物的完整性。
检测结果显示，相对于利用WT组聚合酶获得的saRNA产物，K387A组和DEL172+K387Y组的变体能将saRNA产物的完整性分别提高3.1％和4.9％。
实施例4体外转录制备加帽RNA
(1)将K387Y及DEL172+K387S变体酶的原液用储存buffer(Vazyme，货号：DD4101)稀释成酶活为300U/μL。按表7中的反应体系(20μL)配制MIX溶液至EP管中，将MIX溶液分装至八联排中，混匀，离心；将八联排置于PCR仪上37℃反应1h后加入36μL的磁珠混匀后室温孵育2～5min；将混合液置于磁力架上以纯化mRNA(Vazyme，货号：N412)，纯化后转移至RNase-free的离心管中，获得纯化后mRNA。
表7：反应体系
(2)加帽率检测：
①将步骤(1)中获得的纯化的mRNA与探针结合，反应体系见表8，反应条件见表9；
表8：反应体系
表9：反应条件
②RNase H酶切：按表10配制反应酶切反应体系(Thermo Scientific，货号：EN0201)，充分涡旋震荡混合均匀后放置于PCR仪中，25℃反应20min；
表10：反应体系
③SA磁珠结合：
A.磁珠清洗：取9μL SA磁珠到离心管中，放置于磁力架上，待溶液变澄清后，用移液器吸弃上清液；将离心管从磁力架上取下，加入200μL RNase-free H2O润洗，放置于磁力架上，待溶液变澄清后用移液器吸弃上清液，再加入200μL RNase-free H2O重复润洗一次。
B.反应条件：将离心管从磁力架上取下，将酶切产物加入到SA磁珠中，用移液器吹打20-30次，充分混合均匀，置于翻滚仪上室温翻滚孵育30min，使磁珠与酶切产物充分结合。
④漂洗与洗脱：
A.将步骤③的产物置于磁力架上2～3min，至溶液澄清后用移液器吸弃上清液；
B.加入200μL漂洗液漂洗，注意避免吹散磁珠，静置0.5～1min，用移液器吸弃上清液；
C.重复步骤B；
D.将离心管从磁力架上取下，加入30uL洗脱液，用移液器吹打10-20次混合均匀，使磁珠均匀分散，充分洗脱；
E.放置于PCR仪中，85℃反应3min后立即放置于磁力架上，溶液澄清后(0.5～1min)将上清吸取至新的离心管中，上清即所需产物；
F.采用毛细管电泳检测步骤E产物的加帽率，加帽率计算公式：
Cap1加帽率％＝[Cap1峰面积/(Uncap峰面积+Cap1峰面积)]×100％。
检测结果显示，在共转录加帽反应中，WT(野生型T7 RNA聚合酶)组的mRNA产物加帽率仅为87.2％，而K387Y组的变体可将mRNA产物的加帽率提高至91.3％，DEL172+K387S组的变体可提高至100％。

Claims (14)

  1. 一种RNA聚合酶的变体，其氨基酸序列相对于SEQ ID NO：1，包含在选自D130、N171、K172、R173、Y178、R298、Y385、K387、D388或F880位置处的一个、两个、三个、四个或五个氨基酸的突变，其中，所述突变类型为取代或缺失，且所述变体的氨基酸序列与SEQ ID NO：1具有至少97％、至少98％或至少99％的序列一致性。
  2. 如权利要求1所述的变体，其中，所述变体的氨基酸序列相对于SEQ ID NO：1具有一个氨基酸的突变，所述突变的氨基酸的位置选自N171、K172、R173、Y178、R298、Y385、K387、D388或F880。
  3. 如权利要求1所述的变体，其中，所述变体的氨基酸序列相对于SEQ ID NO：1，包含：
    (1)在K172位置处的突变，并且还包含在选自D130、R173、Y178、R298、Y385、K387、D388或F880位置处任意一个、两个、三个或四个氨基酸的突变，所述突变类型为取代或缺失；或
    (2)在K173位点处的突变，并且还包含在选自D130、Y178、R298、K387或D388位置处任意一个、两个或三个氨基酸的突变，所述突变类型为取代或缺失；或
    (3)在R298、Y385、K387或D388位置处任意两个或三个氨基酸的突变，所述突变类型为取代。
  4. 如权利要求3中所述的变体，其中，所述变体包含选自下述任一的突变组：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+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+R173+D388+F880、K172+R173+Y178+D388、D130+K172+R173+D388、D130+K172+R173+Y178、D130+R173+Y178+K387、D130+K172+Y178+D388、或D130+K172+R173+Y178+D388。
  5. 如权利要求2中所述的变体，其中，
    1)N171位置处的突变类型为N171G；
    2)K172位置处的突变类型选自DEL172、K172A、K172G、K172E、K172D、K172H、K172Y、K172S、K172W、K172P、K172I、K172M、K172V、K172F、K172T、K172C、K172N或K172L；
    3)R173位置处的突变类型选自DEL173、R173A、R173C、R173G、R173E、R173D、R173H、R173Y、R173S、R173W、R173P、R173N或R173Q；
    4)Y178位置处的突变类型为Y178H；
    5)R298位置处的突变类型为R298A；
    6)Y385位置处的突变类型选自Y385A、Y385D或Y385E；
    7)K387位置处的突变类型选自K387Q、K387Y、K387S或K387A；
    8)D388位置处的突变类型选自D388A、D388G或D388L；以及
    9)F880位置处的突变类型选自F880A、F880G或F880W。
  6. 如权利要求3或4中所述的变体，其中，
    1)D130位置处的突变类型为D130E；
    2)K172位置处的突变类型可选自DEL172、K172A或K172G；
    3)R173位置处的突变类型可选自DEL173、R173A、R173G或R173C；
    4)Y178位置处的突变类型可选自Y178H或Y178P；
    5)R298位置处的突变类型为R298A；
    6)Y385位置处的突变类型为Y385A；
    7)K387位置处的突变类型可选自K387S、K387Y或K387G；
    8)D388位置处的突变类型可选自D388Y、D388A或D388G；以及
    9)F880位置处的突变类型可选自F880A或F880Y。
  7. 如权利要求1所述的变体，其中，所述变体的氨基酸序列如SEQ ID NO：2-141中任一所示。
  8. 一种核苷酸序列，其编码如权利要求1-7中任一所述的变体。
  9. 一种RNA聚合酶变体的制备方法，其包括在宿主细胞中生产至少一种如权利要求1-7中所述的RNA聚合酶变体；其中，所述宿主细胞中含有携带如权利要求8中所述的核苷酸序列的表达载体。
  10. 一种降低体外转录制备RNA过程中dsRNA杂质生成的方法，其包括使DNA模板与如权利要求1-7中任一所示的变体接触，在体外转录体系中孵育。
  11. 如权利要求1-7中至少一种变体或权利要求10中所述的方法在体外转录生成RNA中的应用。
  12. 一种体外转录生成RNA的方法，其包括DNA模板与如权利要求1-7中所述的至少一种变体接触，在体外转录体系中孵育。
  13. 一种体外转录生成加帽RNA的方法，其包括DNA模板与如权利要求1-7中所述至少一种变体及帽类似物接触，在体外转录反应体系中孵育。
  14. 一种组合物或试剂盒，其包含至少一种如权利要求1-7中任一所述的RNA聚合酶变体和至少一种缓冲组分。
PCT/CN2024/079536 2023-03-01 2024-03-01 Rna聚合酶变体、其制备方法及其在rna合成中的应用 WO2024131998A2 (zh)

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