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WO2024235228A1 - Mutants d'arn polymérase à performance améliorée - Google Patents

Mutants d'arn polymérase à performance améliorée Download PDF

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Publication number
WO2024235228A1
WO2024235228A1 PCT/CN2024/093147 CN2024093147W WO2024235228A1 WO 2024235228 A1 WO2024235228 A1 WO 2024235228A1 CN 2024093147 W CN2024093147 W CN 2024093147W WO 2024235228 A1 WO2024235228 A1 WO 2024235228A1
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amino acid
rna polymerase
mutant
mutated
seq
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Chinese (zh)
Inventor
杨广宇
秦伟彤
黑墨翰
罗漫杰
徐灿
聂挺
余佩
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Hzymes Biotechnology Co Ltd
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Hzymes Biotechnology Co Ltd
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Priority claimed from CN202310558557.6A external-priority patent/CN118956809A/zh
Priority claimed from CN202410499488.0A external-priority patent/CN120829885A/zh
Application filed by Hzymes Biotechnology Co Ltd filed Critical Hzymes Biotechnology Co Ltd
Publication of WO2024235228A1 publication Critical patent/WO2024235228A1/fr
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    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)

Definitions

  • the invention relates to an RNA polymerase mutant with improved performance, belonging to the technical field of genetic engineering and enzyme engineering.
  • T7 RNA polymerase is derived from Escherichia coli bacteriophage T7 and is a DNA-dependent single-subunit RNA polymerase that highly specifically recognizes the T7 promoter sequence.
  • the enzyme has a wide range of applications in in vitro RNA synthesis, including the synthesis of gRNA, siRNA, and isotope-labeled or non-isotope-labeled RNA probes, and the synthesis of capped mRNA with the participation of Cap analog for intracellular or in vivo expression and mRNA vaccine and drug research.
  • it can also be used for isothermal amplification, such as sequence-specific nucleic acid in vitro amplification (NASBA) and transcription-mediated amplification (TMA).
  • NASBA sequence-specific nucleic acid in vitro amplification
  • TMA transcription-mediated amplification
  • wild-type T7 RNAP has some shortcomings in practical applications, such as the generation of byproducts during RNA synthesis, including short fragments generated during transcription initiation, 3' end extension products, and dsRNA byproducts, which can affect the effects of mRNA vaccine and drug research.
  • the wild-type T7 RNA polymerase currently on the market cannot fully meet the growing research needs and needs to be optimized and improved to achieve better application value.
  • T7 RNAP some technologies have been used to improve T7 RNAP, such as mutants with improved enzyme activity, mutants with reduced dsRNA contaminants, and mutants with improved thermal stability.
  • US20150024435 A1 provides a mutant of T7 RNA polymerase with improved enzyme activity
  • CN 111212905 A provides a T7 RNAP mutant that reduces dsRNA contaminants and concatenated transcripts produced during in vitro transcription reactions
  • CN 112831484 B provides a method suitable for the synthesis of RNA containing internal termination signals and RNA with hairpin structures at the ends, which can synthesize Cas9 sgRNA with higher purity than T7 RNAP-WT
  • CN 108779446 A and US 9193959 B2 provide T7 RNAP mutants with improved thermal stability.
  • these mutants in the prior art often need to be improved in performance, such as insufficient performance improvement, balance between thermal stability and activity, or failure to effectively reduce byproducts.
  • the amino acid at position 217 is mutated to L, A or V, preferably, mutated to L;
  • the amino acid at position 121 is mutated to M, L or S, preferably, mutated to M;
  • the amino acid at position 223 is mutated to M, P, L, V or F, preferably, mutated to M;
  • the amino acid at position 632 is mutated to G or E, preferably, mutated to G;
  • amino acid at position 369 is mutated to T or E;
  • the RNA polymerase mutant also includes mutations at one or more of the following positions corresponding to the amino acid sequence of the parent RNA polymerase: position 430, position 633, position 786, position 744, position 724, position 43, and position 723.
  • the amino acid at position 430 is mutated to a non-polar hydrophobic amino acid; alternatively, it is mutated to P, L, V, M, F; preferably, it is mutated to P;
  • the amino acid at position 723 is mutated to E or S; preferably, mutated to E;
  • the amino acid at position 724 is mutated to N.
  • the RNA polymerase mutant comprises at least any one of the following mutations that is equivalent to or corresponds to the parent polymerase mutant: I217L, I217A, I217V, S397W, S397A, S397P, T121M, T121L, T121S, S223M, S223P, S223L, S223V, S223F, R632G, R632E, M369E, M369T, N579A, N579W, and N579D mutations.
  • the RNA polymerase mutant simultaneously has at least the following mutations equivalent to or corresponding to the parent polymerase mutant: I217L, S397W, S430P, S633P and Q786L, named I217L/S397W/S430P/S633P/Q786L (hereinafter referred to as M7).
  • the RNA polymerase mutant simultaneously has at least the following mutations equivalent to or corresponding to the parent polymerase mutant: I217L, S397W, S430P, S633P, Q786L, N579W, named I217L/S397W/S430P/S633P/Q786L/N579W.
  • the RNA polymerase mutant simultaneously has at least the following mutations equivalent to or corresponding to the parent polymerase mutant: I217L, S397W, S430P, S633P, Q786L, C723E, A724N, named I217L/S397W/S430P, S633P/Q786L/C723E/A724N.
  • the RNA polymerase mutant simultaneously has at least the following mutations equivalent to or corresponding to the parent polymerase mutant: I217L, S397W, S430P, S633P, Q786L, Q744R, S43Y, and is named I217L/S397W/S430P/S633P/Q786L/Q744R/S43Y (hereinafter referred to as M8).
  • the amino acid sequence of the RNA polymerase mutant is such as SEQ ID NO.2 (corresponding to I217L), SEQ ID NO.3 (corresponding to S397W), SEQ ID NO.4 (corresponding to T121M), SEQ ID NO.5 (corresponding to S223M), SEQ ID NO.6 (corresponding to R632G), SEQ ID NO.7 (corresponding to I217L/S397W/S430P/S633P/Q786L) or SEQ ID NO.
  • SEQ ID NO.8 (corresponding to I217L/S397W/S430P/S633P/Q786L/Q744R/S43Y), SEQ ID NO.79 (corresponding to M369T), SEQ ID NO.80 (corresponding to N579W), SEQ ID NO.81 (corresponding to I217L/S397W/S430P/S633P/Q786L/N579W), SEQ ID NO.82 (corresponding to I217L/S397W/S430P/S633P/Q786L/Q744R), and SEQ ID NO.83 (corresponding to I217L/S397W/S430P/S633P/Q786L/C723E/A724N).
  • the second object of the present invention is to provide a polynucleotide encoding the aforementioned RNA polymerase mutant.
  • the synthesis method comprises using an enzyme system comprising the RNA polymerase mutant to catalyze the synthesis of polynucleotides or using the whole cells of the above-mentioned cells to catalyze the synthesis of polynucleotides.
  • the synthesis method comprises in vitro transcription synthesis, isothermal amplification or transcription-mediated amplification.
  • the sixth object of the present invention is to provide an application of the above-mentioned RNA polymerase mutant or the above-mentioned polynucleotide synthesis method in at least one of mRNA vaccine preparation, nucleic acid drug preparation, gene editing and protein expression system construction, and the protein expression system construction includes but is not limited to in vivo protein expression system construction or cell-free protein expression in vitro translation system construction.
  • a seventh object of the present invention is to provide a kit for polynucleotide synthesis, which comprises the aforementioned RNA polymerase mutant.
  • the eighth object of the present invention is to provide a method for improving at least one performance of an RNA polymerase; the performance is selected from the group consisting of improved enzyme activity, improved thermal stability, improved 3' end consistency, higher in vitro transcription yield, fewer dsRNA byproducts and lower immunogenicity, the method comprising: based on the amino acid sequence corresponding to the parent RNA polymerase, one or more amino acid mutations are present at positions 121, 223, 632, 217, 397, 369 and 579, or one or more amino acid mutations occur at amino acid residues at equivalent positions in the parent RNA polymerase; the amino acid sequence of the parent RNA polymerase has at least 80% identity with the amino acids shown in SEQ ID NO.1 and has RNA polymerase activity.
  • the amino acid sequence of the parent RNA polymerase when the mutation site contains position 121, the amino acid sequence of the parent RNA polymerase has at least 90% identity with the amino acid shown in SEQ ID NO.1 and has RNA polymerase activity; when the mutation site contains position 369, the amino acid sequence of the parent RNA polymerase has at least 98.41% identity with the amino acid shown in SEQ ID NO.1 and has RNA polymerase activity.
  • the present invention provides a series of RNA polymerase mutants with improved performance, which have at least any of the following performance improvements: enzyme activity, thermal stability, 3' end consistency, dsRNA byproducts and immunogenicity, etc., and can meet various needs of practical applications and research.
  • the amino acid at position 223 was mutated to construct single-point mutants S223M, S223L, and S223V, which increased the enzyme activity by more than 18% compared with the wild-type WT.
  • S223M had the highest enzyme activity, which increased by 25% compared with the wild-type WT.
  • the amino acid at position 632 was mutated to construct single-point mutants R632G and R632E, which showed an enzyme activity increase of more than 20% compared with the wild-type WT.
  • R632G had the highest enzyme activity, which increased by 22% compared with the wild-type WT.
  • FIG4A shows the dsRNA content detection results of IVT products of T7 RNAP wild type and single mutants S430P, S633P, Q786L, I217L, S397W, combined mutants I217L/S397W/S430P/S633P/Q786L, I217L/S397W/S430P/S633P/Q786L/N579W, I217L/S397W/S430P/S633P/Q786L/Q744R, and I217L/S397W/S430P/S633P/Q786L/C723E/A724N;
  • Figure 5B shows the 3' end consistency test results of the IVT product of T7 RNAP mutant Q744R at 37°C;
  • Figure 5C shows the 3' end consistency test results of the IVT product of T7 RNAP mutant S43Y at 37°C;
  • Figure 5D shows the 3' end consistency test results of the IVT product of T7 RNAP mutant G47A+884G at 37°C;
  • Figure 5J shows the 3’ end consistency test results of the IVT product of T7 RNAP mutant S397W at 50°C;
  • Figure 7E is a sequence conservation analysis of T7 RNAP mutation sites 520 to 650 in the amino acid sequences of RNA polymerases from different sources.
  • the term "corresponding to” refers to a manner of determining a specific amino acid in a sequence where a specific amino acid sequence is referenced. For example, for the purposes of the present invention, when a specific amino acid position is referenced, a skilled person can compare another amino acid sequence with the amino acid sequence that has been referenced to determine which specific amino acid may be of interest in the other amino acid sequence. Alternative alignment methods may be used and are well known to those skilled in the art.
  • Wild-type enzyme when referring to an amino acid sequence or a nucleic acid sequence means that the amino acid sequence or nucleic acid sequence is a natural or naturally occurring sequence.
  • naturally occurring refers to any substance (e.g., a protein, amino acid, or nucleic acid sequence) found in nature.
  • non-naturally occurring refers to any substance not found in nature (e.g., a recombinant nucleic acid and protein sequence produced in a laboratory, or a modification of a wild-type sequence).
  • wild-type enzyme and “parent enzyme” are used interchangeably.
  • Sequence identity The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity”.
  • Recombinant When used in reference to a cell, nucleic acid, protein, or vector, the term “recombinant” means that it has been modified from its native state. Thus, for example, a recombinant cell expresses genes not found in the native (non-recombinant) form of the cell, or expresses native genes at different levels or under different conditions than found in nature.
  • a recombinant nucleic acid differs from a native sequence in one or more nucleotides and/or is operably linked to a heterologous sequence (e.g., a heterologous promoter in an expression vector).
  • a recombinant protein may differ from a native sequence in one or more amino acids and/or be fused to a heterologous sequence.
  • a vector comprising a nucleic acid encoding a polypeptide is a recombinant vector.
  • the term “recombinant” is synonymous with "genetically modified" and "transgenic.”
  • Droplet microfluidic cell sorting is based on a microfluidic ultra-high throughput fluorescence-activated droplet sorting platform (Fluorescence-Activated Droplet Sorting, FADS), and the FADS screening process includes three steps: Droplet encapsulation and incubation of single cells, incubation and reaction, and ultra-high throughput screening of single cell droplets after reaction.
  • FADS can encapsulate and culture single cells, use specific fluorescent substrates to perform fluorescence quantification of the production of secondary metabolites or specific enzymes of single cells, and use the screening platform to sort and collect droplets whose fluorescence intensity meets the screening threshold, and finally obtain a large number of candidate droplets.
  • This screening method has the advantages of high throughput and strong specificity.
  • the heat treatment condition is 40-50° C. for 5-10 min, preferably 45° C. for 7 min.
  • the target cells are the 0.4% to 0.45% of droplet-encapsulated cells that exhibit the highest viability.
  • the amplified polynucleotide encoding T7 RNA polymerase and the nucleotide sequence are as shown in SEQ ID NO.15, and the expressed amino acid sequence is the wild-type T7 RNA polymerase as shown in SEQ ID NO.1.
  • the method further includes at least one step of: isolating a target fragment from a target cell sorted by a droplet microfluidic cell sorting method, transforming the fragment into another cell, and screening the cell again after culturing the fragment.
  • T7 RNA polymerase mutants expressed by positive cells are collected, the system containing the T7 RNA polymerase mutants is heat-treated, and then the enzyme activity is determined, and the positive clones whose enzyme activity is at least 1.5 times that of the wild type are sequenced.
  • T7 RNA polymerase mutants were screened using the following protocol:
  • the obtained mixed plasmid mutant library was electroporated into fresh E. coli BL21 (DE3) competent cells to obtain a mutant library containing about 107 clones.
  • E. coli was induced and expressed, and the single cell and reaction system were encapsulated using the FADS platform.
  • the single cell encapsulation rate was controlled at about 10% to improve the accuracy of screening.
  • the droplets were treated at a temperature of about 45°C for an appropriate time, and the activity was reduced to about one-third of the original to achieve the effect of high temperature treatment, and then incubated at 37°C for 3h.
  • the most suitable laser spot position, sorting speed and other conditions are determined by sorting FITC and ink. Then the incubated microdroplets are injected into the detection and sorting chip. The droplets of about 0.42% showing the highest activity are collected in a 1.5mL centrifuge tube, and the positive gene is recovered by PCR. The target fragment is treated with BamHI and HindIII double enzymes and reconstructed into the pQE-80L linearized vector. The ligated vector is transformed into the BL21 (DE3) competent state again, and the single clone grown on the plate is picked for rescreening.
  • the crude enzyme solution was transferred to a 384-well plate and the enzyme activity was measured using the STAR system (iSpinach aptamer-based monitoring of transcription activity in real-time, STAR, see application number: CN202211274914.8, the invention name is "A detection method for real-time monitoring of in vitro transcribed RNA synthesis", the introduction and use of the system are detailed in CN115896213A text). Then the mutants whose residual activity after heat treatment is at least 1.5 times that of the wild type are sequenced, and the distribution of mutation points is analyzed by sequencing results.
  • STAR system iSpinach aptamer-based monitoring of transcription activity in real-time, STAR, see application number: CN202211274914.8, the invention name is "A detection method for real-time monitoring of in vitro transcribed RNA synthesis", the introduction and use of the system are detailed in CN115896213A text.
  • a recombinant expression vector carrying a nucleotide sequence encoding a T7 RNAP mutant was constructed and transformed into BL21 (DE3) Escherichia coli competent cells to obtain recombinant strains of T7 RNP wild type and mutant, induced expression and prepared T7 RNAP mutants by protein purification. Then, a single-stranded RNA aptamer was synthesized by in vitro transcription reaction. The single-stranded RNA can bind to a specific chemical small molecule DFHBI to produce a significant fluorescence signal enhancement. The relative activity of different mutants and T7 RNAP-WT was determined by monitoring the change of real-time fluorescence during the reaction.
  • Tm values of different mutants were determined by differential scanning fluorimetry by combining the T7 RNAP mutant protein solution with Sypro Orange dye.
  • the application performance of T7 RNAP mutants is achieved by analyzing the products synthesized by in vitro transcription (IVT), including IVT yield, product purity, dsRNA content in the product and 3' end consistency index of the product.
  • IVT in vitro transcription
  • the present invention also provides a T7 RNA polymerase mutant screened out by the above screening process.
  • the T7 RNA polymerase mutant described herein is a polypeptide that has at least one amino acid residue difference compared to the wild-type T7 RNA polymerase.
  • the amino acid sequence of the wild-type T7 RNA polymerase is shown in SEQ ID NO.1.
  • the T7 RNA polymerase mutant provided by the present invention has at least one of the following amino acid residues: corresponding to the amino acid sequence shown in SEQ ID NO.1, position 217 is L (leucine), position 397 is A (alanine) or W (tryptophan), and position 579 is A (alanine) or W (tryptophan).
  • the T7 RNA polymerase mutant containing the aforementioned amino acid residues has good thermal stability.
  • the amino acid sequence corresponding to SEQ ID NO.1 as described herein refers to the amino acid residue determined by the position number of the position in the amino acid sequence shown in SEQ ID NO.1 when the T7 RNA polymerase mutant is aligned with the amino acid sequence shown in SEQ ID NO.1.
  • the position number of a certain amino acid residue in the amino acid sequence shown in SEQ ID NO.1 of the T7 RNA polymerase mutant corresponding to the amino acid sequence shown in SEQ ID NO.1 is different from the position number of the position in the T7 RNA polymerase mutant.
  • an amino acid residue is located at the i-th position in the amino acid sequence shown in SEQ ID NO.1 and is located at the i-N position in the amino acid sequence of the T7 RNA polymerase mutant.
  • the amino acid residue is defined by its position in the amino acid sequence corresponding to SEQ ID NO.1, rather than its position in the mutant.
  • the T7 RNA polymerase mutant provided by the present invention has at least 80% identity with SEQ ID NO.1, for example, it may be but is not limited to at least 80%, 85%, 90% or 95% identity with SEQ ID NO.1.
  • Identity or similar terms herein refer to the percentage of identical nucleotides or amino acids between the sequences to be compared under optimal alignment. The percentage is purely statistical, and the differences between the two sequences may be (but not necessarily) randomly distributed over the entire length of the sequences to be compared. The comparison of two sequences is typically performed by identifying local regions of corresponding sequences relative to fragments or "comparison windows" after optimal alignment.
  • a T7 RNA polymerase mutant having at least 80% identity with SEQ ID NO.1 contains at least one of the amino acid residues at position 217 being L, at position 397 being A or W, and at position 579 being A or W.
  • the T7 RNA polymerase mutant contains an amino acid residue corresponding to L at position 217 of the amino acid sequence shown in SEQ ID NO.1, and has at least 80%, 85%, 90% or 95% identity with SEQ ID NO.1.
  • the T7 RNA polymerase mutant contains an amino acid residue that is A or W at position 397 of the amino acid sequence shown in SEQ ID NO.1, and has at least 80%, 85%, 90% or 95% identity with SEQ ID NO.1, wherein preferably position 397 is W.
  • the T7 RNA polymerase mutant contains an amino acid residue that is A or W at position 579 of the amino acid sequence shown in SEQ ID NO.1, and has at least 80%, 85%, 90% or 95% identity with SEQ ID NO.1, wherein preferably position 579 is W.
  • the T7 RNA polymerase mutant removes at least one of the aforementioned amino acid residues and also includes at least one of the following: corresponding to the amino acid sequence shown in SEQ ID NO.1, at least one of the positions 369, 430, 633, 786, 744, 723 and 724 is different from SEQ ID NO.1.
  • the 633rd position corresponding to the amino acid sequence shown in SEQ ID NO.1 is a non-polar hydrophobic amino acid, further preferably P (proline), L (leucine), V (valine), M (methionine) or F (phenylalanine); further preferably P, and has at least 80%, 85%, 90% or 95% identity with SEQ ID NO.1.
  • the mutation at position 633 is beneficial to improve the stability of T7 RNA polymerase.
  • the 744th position corresponding to the amino acid sequence shown in SEQ ID NO.1 is R (arginine), P (proline) or L (leucine); more preferably R; and has at least 80%, 85%, 90% or 95% identity with SEQ ID NO.1.
  • the mutation at position 744 is beneficial to the improvement of T7 RNA polymerase activity.
  • the 786th position corresponding to the amino acid sequence shown in SEQ ID NO.1 is Y (tyrosine) or a non-polar hydrophobic amino acid; more preferably Y, L (leucine), M (methionine), W (tryptophan) or F (phenylalanine); more preferably L; and has at least 80%, 85%, 90% or 95% identity with SEQ ID NO.1.
  • the mutation at position 786 is beneficial to improve the stability of T7 RNA polymerase.
  • the 723rd position corresponding to the amino acid sequence shown in SEQ ID NO.1 is E (glutamic acid) or S (serine); more preferably E; and has at least 80%, 85%, 90% or 95% identity with SEQ ID NO.1.
  • the mutation at position 723 is beneficial to improve protein aggregation.
  • the 369th position corresponding to the amino acid sequence shown in SEQ ID NO.1 is T (threonine) or E (glutamate); and it has at least 80%, 85%, 90% or 95% identity with SEQ ID NO.1.
  • the 579th position corresponding to the amino acid sequence shown in SEQ ID NO.1 is W (tryptophan) or A (alanine); further preferably W; and it has at least 80%, 85%, 90% or 95% identity with SEQ ID NO.1.
  • the 430th position corresponding to the amino acid sequence shown in SEQ ID NO.1 is P (proline); and it has at least 80%, 85%, 90% or 95% identity with SEQ ID NO.1.
  • the 724th position corresponding to the amino acid sequence shown in SEQ ID NO.1 is N (asparagine); and it has at least 80%, 85%, 90% or 95% identity with SEQ ID NO.1.
  • the T7 RNA polymerase mutant is selected from any one of the following:
  • the amino acid sequence of the T7 RNA polymerase mutant is SEQ ID NO.2 (corresponding to I217L), SEQ ID NO.3 (corresponding to S397W), SEQ ID NO.4 (corresponding to T121M), SEQ ID NO.5 (corresponding to S223M), SEQ ID NO.6 (corresponding to R632G), SEQ ID NO.7 (corresponding to I217L/S397W/S430P/S633P/Q786L) or SEQ ID NO.8 (corresponding to I217L/S397W/S430P/S633P/Q786L /Q744R/S43Y), SEQ ID NO.79 (corresponding to M369T), SEQ ID NO.80 (corresponding to N579W), SEQ ID NO.81 (corresponding to I217L/S397W/S430P/S633P/Q786L/N579W), SEQ ID NO.82 (corresponding to I217L/S397W/S430P/
  • SEQ ID NO.2 is a T7 RNA polymerase mutant with a single point mutation I217L, whose Tm value is 50.5 ⁇ 0.21°C. The residual activity after treatment at 50°C for 5 minutes is 52%. Taking the enzyme activity of the wild type as 100% as a reference, the activity of this mutant is 134%.
  • SEQ ID NO.3 is a T7 RNA polymerase mutant with a single point mutation S397W, whose Tm value is 49.2 ⁇ 0.13°C. The residual activity after treatment at 50°C for 5 minutes is 47%. Taking the enzyme activity of the wild type as 100% as a reference, the activity of this mutant is 162%.
  • SEQ ID NO.80 is a T7 RNA polymerase mutant with a single point mutation N579W, whose Tm value is 48.0 ⁇ 0.23°C and the residual activity after treatment at 50°C for 5 minutes is 38%.
  • SEQ ID NO.7 is a T7 RNA polymerase mutant with combined mutations I217L, S397W, S430P, S633P and Q786L, with a Tm value of 54.52 ⁇ 0.20°C and a residual activity of 58% after heat treatment at 58°C for 15min.
  • SEQ ID NO.81 is a T7 RNA polymerase mutant with combined mutations I217L, S397W, S430P, S633P, Q786L and N579W, with a Tm value of 55.56 ⁇ 0.16°C and a residual activity of 65% after heat treatment at 58°C for 15 minutes.
  • SEQ ID NO.82 is a T7 RNA polymerase mutant with combined mutations I217L, S397W, S430P, S633P, Q786L and Q744R.
  • the Tm value is 54.50 ⁇ 0.18°C.
  • the residual activity after heat treatment at 58°C for 15min is 87%. Taking the enzyme activity of the wild type as 100% as a reference, the activity of this mutant is 195%.
  • SEQ ID NO.83 is a polymerase mutant with combined mutations I217L, S397W, S430P, S633P, Q786L, C723E and A724N.
  • the Tm value is 54.54 ⁇ 0.23°C.
  • the residual activity after heat treatment at 58°C for 15min is 81%. Taking the enzyme activity of the wild type as 100% as a reference, the activity of this mutant is 183%.
  • the present invention also provides a polynucleotide encoding the above-mentioned T7 RNA polymerase mutant.
  • polynucleotide refers to a polymeric form of nucleotides of any length, including ribonucleotides and/or deoxyribonucleotides.
  • examples of polynucleotides include, but are not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derived nucleotide bases.
  • the polynucleotide encodes the T7 RNA polymerase mutant described above, optionally encoding the sense strand or the antisense strand.
  • the polynucleotide may be naturally occurring, synthetic, recombinant, or any combination thereof.
  • the terms "polynucleotide” and “nucleic acid” are used interchangeably herein.
  • the present invention also provides a vector, which carries a polynucleotide encoding the above-mentioned T7 RNA polymerase mutant.
  • the vector is well known to those skilled in the art, including but not limited to: plasmid; phagemid; cosmid; artificial chromosome, such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC); bacteriophage such as ⁇ phage or M13 phage and animal virus, etc.
  • Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses.
  • the vector of the present invention contains regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRES) and other expression control elements (such as transcription termination signals, or polyadenylation signals and poly-U sequences, etc.).
  • the present invention further provides a cell, which carries the aforementioned polynucleotide or the aforementioned vector, or expresses the aforementioned T7 RNA polymerase mutant.
  • the cell may refer to a single cell, a cell line, or a cell culture.
  • the cell herein includes its progeny, which may not necessarily be completely identical to the primary cell due to natural, accidental or intentional mutations, and may differ from the primary cell in morphology and/or in genomic DNA.
  • the cell may be a natural cell or a transformant.
  • the present invention also provides a method for synthesizing a polynucleotide, wherein the method uses the RNA polymerase mutant of the present invention.
  • the method for synthesizing the polynucleotide comprises using an enzyme system comprising the aforementioned T7 RNA polymerase mutant to catalyze a synthesis reaction.
  • the synthesis method comprises in vitro transcription synthesis (In Vitro Transcription, IVT), isothermal amplification, such as sequence-specific nucleic acid in vitro amplification (Nucleic Acid Specific-Based Amplification, NASBA) or transcription-mediated amplification (Transcription mediated amplification, TMA)
  • the present invention also provides the use of the aforementioned T7 RNA polymerase mutant or the aforementioned polynucleotide synthesis method in any one of the following: at least one of mRNA vaccine preparation, nucleic acid drug preparation, gene editing and protein expression system construction, and the protein expression system construction includes but is not limited to in vivo protein expression system construction or cell-free protein expression in vitro translation system construction.
  • the present invention also provides a kit for polynucleotide synthesis, which comprises the aforementioned T7 RNA polymerase mutant.
  • kit for polynucleotide synthesis provided by the present invention may also comprise other detection reagents or consumables acceptable in the art, and those skilled in the art may select corresponding reagents and/or consumables based on the contents well known in the art and recorded in various general and more specific teaching materials, references, process manuals, product descriptions, standard documents and equipment manuals.
  • kits include, but are not limited to, one or more of buffers, salts, metal ions, dNTPs, enzymes, primers, probes, fluorescent dyes, luminescent substrates, reference substances, quality control substances and calibrators.
  • the wild-type T7 RNA polymerase gene nucleotide sequence (corresponding amino acid sequence such as SEQ ID NO.1) was optimized according to the Escherichia coli codon to obtain the nucleotide sequence SEQ ID NO.15, and then the optimized nucleotide sequence was inserted between the two restriction endonuclease sites of BamHI and HindIII of the vector pQE-80L (Qiagen, item number HG-VYQ0254), that is, the recombinant vector pQE-80L-T7 RNAP containing the wild-type T7 RNAP gene was obtained.
  • the plasmid structure is shown in Figure 1.
  • the recombinant vector pQE-80L-T7 RNAP was used as a template to amplify the T7 RNAP gene, and the degree of mutation was mainly achieved by changing the Mn 2+ concentration in the reaction system. 0.1mM, 0.2mM, 0.3mM, 0.4mM, 0.5mM, 0.6mM Mn 2+ concentration gradients were set for gene amplification, and the mutation rate was optimal with 1 to 3 mutation points per gene.
  • the amplification system was as follows: pQE-80L-T7 RNAP plasmid (0.2-1 ng/ ⁇ L), dATP (0.25 mM), dGTP (0.25 mM), dCTP (1.05 mM), dTTP (1.05 mM), DreamTaq Buffer (Takara), DreamTaq (Takara, 0.1 U/ ⁇ L), MnCl 2 (0.1 mM-0.6 mM), T7 RNAP-F (0.2 ⁇ M), T7 RNAP-R (0.2 ⁇ M), and the total PCR reaction system was 50 ⁇ L.
  • the target fragment size was analyzed by agarose gel electrophoresis, recovered by the kit, and then double-digested with BamHI and HindIII.
  • the digested product was then purified and recovered, and ligated with the linearized pQE-80L vector (double-digested with BamHI and HindIII) overnight under the action of T4 DNA ligase.
  • the recovered ligation product was electrotransformed into electrocompetent E. coli 10G, and the library capacity was calculated based on the clones grown, and the library capacity was more than 10 6.
  • the frequency of introduction under 0.2 mM Mn 2+ was 1.7 base mutations/gene, and the frequency of introduction under 0.3 mM Mn 2+ was 3.5 base mutations/gene. Therefore, in this example, the mutant plasmids obtained under 0.2 mM Mn 2+ and 0.3 mM Mn 2+ were mixed to obtain a mutant library in which an average of 2.6 mutant bases were introduced into each gene.
  • the obtained mixed plasmid mutant library was electroporated into fresh E. coli BL21 (DE3) competent cells to obtain a clone mutant library containing about 10 7.
  • the whole screening process is shown in Figure 2.
  • E. coli was induced and expressed, and the single cell and reaction system were encapsulated using the FADS platform (droplet microfluidic cell sorting based on microfluidic ultra-high throughput fluorescence-activated droplet sorting platform, Fluorescence-Activated Droplet Sorting, FADS).
  • the reaction system was published in patent CN 115896213 A, including single clones, 40-100nM DNA template ispinach, 100 ⁇ M DFHBI, 0.5-6mM NTPs, 30mM MgCl 2 , 5Mm NaCl, 20mM DTT, 0.002U/ ⁇ L inorganic pyrophosphatase, and 0.2U/ ⁇ L mouse ribonuclease inhibitor.
  • the single-cell encapsulation rate is controlled at about 10% to improve the accuracy of screening.
  • the droplets need to be heat-treated at 45°C for 7 minutes, and the activity will drop to about one-third of the original to achieve the effect of high-temperature treatment, and then incubated at 37°C for 3 hours.
  • the optimal laser spot position, sorting speed and other conditions are determined by sorting FITC and ink. The incubated microdroplets are then re-injected into the detection and sorting chip.
  • the PMT was set to 0.43V
  • the small and large thresholds of sorting were set to 0.2V and 0.6V respectively
  • the sorting voltage was 1.6V.
  • the droplets of about 0.42% showing the highest activity were collected in a 1.5mL centrifuge tube, and the positive genes were recovered by PCR.
  • the target fragments were double-digested with SacI and HindIII, reconstructed into the pQE-80L linearized vector, and the ligated vector was transformed into BL21 (DE3) competent cells again, and the single clones grown on the plate were picked for rescreening.
  • the enzyme activity was measured using the STAR system (iSpinach aptamer-based monitoring of transcription activity in real-time, STAR, see application number: CN202211274914.8, the invention name is "A detection method for real-time monitoring of in vitro transcribed synthetic RNA", the introduction and use of the system are detailed in CN115896213A text).
  • Mutants whose residual activity after heat treatment is at least 1.5 times that of the wild type were sequenced. Analysis of the sequencing results revealed that the frequencies of mutations at positions 217, 397, 121, 223, 632, 369 and 579 corresponding to the parental RNA polymerase (i.e., wild-type T7 RNAP, as shown in the amino acid sequence SEQ ID NO.1) were relatively high.
  • the wild-type T7 RNAP shown in the amino acid sequence SEQ ID NO.1 was determined as the parent, and the 217th amino acid, the 397th amino acid, the 121st amino acid, the 223rd amino acid and the 632nd amino acid of the parent amino acid sequence were used as key sites to design mutants and combination mutants with amino acid sequences as shown in SEQ ID NO.2 to 8; mutants S430P, S633P, Q786L, Q744R, S43Y and mutant combinations reported in the prior art were designed as controls, and the amino acid sequences were as shown in SEQ ID NO.9 to 14, namely the following mutants:
  • S430P (US7507567B2), S633P (US7507567B2) and Q786L (CN102177236B) are mutation points reported in the prior art to improve the thermal stability of T7 RNAP
  • Q744R (US20150024435A1) is a mutation point reported in the prior art to improve the activity of T7 RNAP
  • S43Y (CN112831484B) is a mutation point reported in the prior art to reduce the RdRp activity of T7 RNAP
  • G47A+884G refers to the addition of an additional amino acid G at the C-terminus of the mutant amino acid sequence on the basis of G47A (total length 883), so that the total length of the mutant is 884.
  • This mutation method (CN111212905A) can reduce dsRNA contaminants and concatenated transcripts generated during in vitro transcription reactions.
  • the specific steps for constructing the mutant vector are as follows: reverse transcribe the above amino acid sequences (SEQ ID NO.1-14, SEQ ID NO.79-83) and optimize them according to the Escherichia coli codon to obtain nucleotide sequences (SEQ ID NO.15-28, SEQ ID NO.84-88); then insert the nucleotide sequences between the two restriction endonuclease sites of BamHI and HindIII of the vector pQE-80L to form the recombinant expression vectors of wild-type T7 RNAP and mutant T7 RNAP.
  • T7 RNAP-WT for expressing wild type T7 RNAP
  • T7 RNAP-I217L for expressing single point mutant I217L
  • T7 RNAP-S397W for expressing single point mutant S397W
  • 7 RNAP-T121M for expressing mutant T121M
  • T7 RNAP-S223M for expressing mutant S223M
  • T7 RNAP-R632G for expressing mutant R632G
  • T7 RNAP-I217L/S397W/S430P/S633P/Q786L for expressing combined mutant M7: I217L/S397W/S430P/S633P/Q786L
  • the wild-type and mutant recombinant strains prepared in the previous step were placed in LB medium containing ampicillin resistance and cultured in a shaking incubator at 37°C until the OD600 value was close to 1.2. Then, isopropyl- ⁇ -D-thiogalactopyranoside (IPTG) with a final concentration of 1 mM was added to induce expression in a shaking incubator at 25°C overnight (16 hours). The bacterial precipitate was then collected by centrifugation at 4°C and 5000 rpm for 20 min.
  • IPTG isopropyl- ⁇ -D-thiogalactopyranoside
  • the bacteria were fully resuspended in a lysis buffer containing 300 mM NaCl, 20 mM Tris HCl (pH 7.5), 0.5 mg/mL lysozyme, and 0.5 mM DTT, and the protein lysate was centrifuged in a high-speed refrigerated centrifuge at 4°C and 18000 rpm for 1 h. The supernatant was then separated and filtered to remove impurities. The filtered supernatant can be temporarily stored on ice for subsequent nickel column purification.
  • the nickel column Before the protein passes through the nickel column, it is necessary to equilibrate the nickel column with 10 times the volume of elution buffer (20mM Tris HCl (PH7.5), 300mM NaCl, 0.5mM DTT), and then add the previously filtered protein solution to the nickel column. After all the protein solution has passed through the nickel column filler, first use a certain concentration of Triton X-ray to wash the impurities, and then use different gradients of imidazole solution (50mM 100mM 150mM-200mM-500mM) to elute, and use several test tubes to number them in order of outflow to collect the outflow.
  • elution buffer 20mM Tris HCl (PH7.5), 300mM NaCl, 0.5mM DTT
  • the enzyme storage buffer consisted of 50mM Tris-HCl, 100mM NaCl, 20mM 2-Mercaptoethanol, 1mM EDTA, 50% Glycerol, 0.1% Triton X-100, pH 7.9.
  • the wild-type T7 RNA and the mutant T7 RNA prepared in Example 2 were taken and their enzyme activities were tested.
  • the reaction system for real-time fluorescence detection of the relative enzyme activity of T7 RNAP mutants is shown in Table 3.
  • Table 3 For specific experimental methods, please refer to the Chinese patent application with application number: CN202211274914.8, and the invention name is "A detection method for real-time monitoring of RNA synthesized by in vitro transcription", in which the added protein concentration and volume of the T7 RNAP mutant are consistent with the wild type with known enzyme activity and protein concentration.
  • the reaction is carried out in a 96-well plate, and the fluorescence value generated by the binding of the aptamer produced by the reaction and DFHBI is measured in real time by an enzyme reader.
  • the reaction system is determined according to the experimental needs.
  • the reaction conditions are 37°C.
  • the enzyme reader When the enzyme reader is used for real-time measurement, it takes 1 hour to measure a value every 1 minute. After the reaction is completed, the Origin software is used to draw a curve graph, and the standard curve of the protein concentration and fluorescence intensity of the wild type is calculated at the time with the best linear relationship, as shown in Figure 3, so as to calculate the relative enzyme activity of the mutant. All reagents in the reaction system need to be prepared with Nuclease-free H 2 O, and the consumables used are all RNase-free grade to prevent the generated RNA from being degraded by RNase.
  • the relative enzyme activity detection of mutants is shown in Table 4-1, Table 4-2, and Table 4-3.
  • the enzyme activities of single point mutant I217L, single point mutant N579W, single point mutant S397W, single point mutant T121M, single point mutant S223M, and single point mutant R632G at 37°C are significantly improved compared with T7 RNAP-WT, and are significantly better than the single point mutants S430P, G47A+884G, single point mutant S633P, and single point mutant Q786L reported in the prior art.
  • the enzyme activity of the combined mutant M8 is significantly higher than that of WT and the single point mutants S430P, S633P, Q744R, Q786L, S43Y, and G47A+884G mutants reported in the prior art.
  • the relative activities of the combined mutants M7 and I217L/S397W/S430P/S633P/Q786L/N579W at 37°C were not different from those of T7 RNAP-WT.
  • the residual activity of the combined mutants I217L/S397W/S430P/S633P/Q786L/Q744R and I217L/S397W/S430P/S633P/Q786L/C723E/A724N was significantly higher than that of T7 RNAP-WT, and was significantly higher than the residual activity of T7-RNAP-WT, single point mutant S430P, single point mutant S633P and single point mutant Q786L after treatment at 50°C for 5 min.
  • the residual relative activity after heat treatment indicated that the combined mutants I217L/S397W/S430P/S633P/Q786L/Q744R and the combined mutants I217L/S397W/S430P/S633P/Q786L/C723E/A724N had higher thermal stability than the existing technology.
  • T7 RNAP protein solution 0.2 mg/mL
  • 2 ⁇ L Sypro Orange dye 100 ⁇
  • PBS 0.1 mg/mL
  • the Tm of a protein is an indicator of thermal stability. Generally speaking, the higher the Tm, the more stable the protein.
  • the in vitro transcription (IVT) reaction system is shown in Table 6.
  • the reaction system can be scaled up in equal proportions. After the system is configured, it is placed at 37°C and 50°C for 2 hours, respectively. Then, 10U DNaseI is added to each reaction according to 1 ⁇ g template and reacted at 37°C for 30 minutes to digest the DNA template. The IVT product is then recovered by LiCl precipitation, the RNA concentration is measured, and the mRNA yield ( ⁇ g) corresponding to 1 ⁇ g template DNA is calculated.
  • the mRNA yield ( ⁇ g) corresponding to 1 ⁇ g template DNA is shown in Table 7-1.
  • the IVT yield of the combined mutant M8 of the present invention is significantly improved compared with the wild-type WT and the single-point mutants S430P, S430P, Q786L, and G47A+884G reported in the prior art, and is comparable to the IVT yields of the mutants Q744R and S43Y reported in the prior art, and is also somewhat improved compared to the combined mutant M7 IVT yield; at the same time, under 50°C reaction conditions, the WT and Q744R reported in the prior art are significantly improved compared to the combined mutant M7.
  • the mutants 744R, S43Y, and G74A+884G produced almost no IVT products, indicating that WT, Q744R, S43Y, and G47A+884G could not tolerate the reaction at 50°C; the IVT yield of the combined mutant I217L/S397W/S430P/S633P/Q786L/N579W at 50°C increased by 60% compared with the IVT yield of WT at 37°C; the combined mutant I217L/S397W/S430P/S633P/Q786L/N579W at 50°C increased by 60% compared with the IVT yield of WT at 37°C.
  • the IVT yield of L/Q744R at 37°C or 50°C was significantly increased compared with WT and single-point mutants I217L, S397W, S430P, S633P, and Q786L.
  • the IVT yield of the combined mutant I217L/S397W/S430P/S633P/Q786L/C723E/A724N at 37°C was increased by about 20% compared with WT and single-point mutants I217L, S430P, S633P, and Q786L.
  • the IVT yield at 50°C was increased by about 120% compared with the IVT yield of WT and single point mutants I217L, S430P, S633P, and Q786L at 37°C; the IVT yield of the combined mutant M8 at 50°C was significantly higher than that of WT and mutants reported in the prior art, indicating that the combined mutants M8 and M7 have better IVT application performance, can produce higher yields of in vitro transcription products, can tolerate 50°C reactions, and have good thermal stability.
  • the product purities of the single mutants S430P, S633P, Q786L, I217L, S397W, the combined mutants M7, I217L/S397W/S430P/S633P/Q786L/N579W, I217L/S397W/S430P/S633P/Q786L/Q744R, and I217L/S397W/S430P/S633P/Q786L/C723E/A724N are shown in Table 7-2.
  • the purity of the IVT product of N579W at 37°C was significantly improved compared with WT and single point mutants I217L, S397W, S430P, S633P, and Q786L, and the product purity improvement rate was all above 10%; the purity of the product of the combination mutants I217L/S397W/S430P/S633P/Q786L/Q744R and the combination mutants I217L/S397W/S430P/S633P/Q786L/C723E/A724N at 37°C was significantly improved compared with WT and single point mutants I217L, S397W, S430P, S633P, and Q786L, and the product purity improvement rate was all above 5%.
  • the purity of the IVT product of P/Q786L/C723E/A724N at 50°C is within 5% relative to the purity of the IVT product of WT and single point mutants I217L, S397W, S430P, S633P, Q786L at 37°C and single point mutants I217L, S397W, S430P, S633P, Q786L at 50°C, and it is considered that there is no significant difference. This shows that the purity of the IVT product of the T7 RNAP
  • the wild type WT, single mutants Q744R, S43Y, G47+884G, T121M, S223M, R632G, S430P, S633P, Q786L, I217L, S397W, combined mutants M7 and M8 were tested using our company's self-developed dsRNA content ELISA quantitative detection kit.
  • the purified IVT product RNA samples obtained in 3 were tested for dsRNA content.
  • the specific operation method is as follows: (1) The dsRNA standard was diluted with STE Buffer to 8 concentrations of 2pg/ ⁇ L, 1pg/ ⁇ L, 0.5pg/ ⁇ L, 0.25pg/ ⁇ L, 0.125pg/ ⁇ L, 0.0625pg/ ⁇ L, 0.0312pg/ ⁇ L, and 0pg/ ⁇ L. The purified RNA samples to be tested were diluted to 3 different concentrations respectively.
  • FIG4A The experimental results are shown in FIG4A , wherein the dsRNA content of the combined mutant M8 of the present invention in the unit mass IVT product of the conventional 37° C. reaction of the wild-type WT, Q744R, S43Y and G47A+884G is significantly lower than that of the wild-type WT and the single point mutants Q744R, S43Y, S430P, S633P, Q786L and M7 combined mutants reported in the prior art; in the unit mass IVT product of the in vitro transcription reaction at 50° C., the combined mutant M The dsRNA content of 8 is significantly lower than that produced by the reaction at 37°C.
  • the dsRNA content of the IVT product per unit mass under the reaction conditions of 50°C is significantly reduced compared to the single point mutants S430P, S633P, and Q786L reported in the prior art, and the dsRNA content of its 50°C in vitro transcription product can reach the same level as the G47A+884G mutant reported in CN111212905A that can reduce dsRNA contaminants and concatenated transcripts in the in vitro transcription product.
  • the dsRNA content level of the combined mutant M8 of the present invention per unit mass of the IVT product is lower than that of the combined mutant M7.
  • Wild type WT single mutants S430P, S633P, Q786L, I217L, S397W, combined mutants I217L/S397W/S430P/S633P/Q786L, I217L/S397W/S430P/S633P/Q786L/N579W, I217L/S397W/S430P/S633P/Q786L/Q744R, I217L/S397W/S430P/S633P/Q786L
  • the dsRNA content in the IVT product of /C723E/A724N was detected by dot blot.
  • the IVT RNA sample recovered by LiCl precipitation obtained in 3 was added dropwise to the positively charged Hybond N membrane (GE Amersham), and then the membrane was blocked in 5% (w/v) skim milk powder TBS-T buffer (20mM Tris, pH 7.4, 150mM NaCl, 0.1% v/v Tween-20). Then J2 anti-dsRNA antibody was diluted 1:5000; incubated overnight at 4°C. The blot was detected with HRP-conjugated secondary antibody (Cell Signaling Technologies). A dsRNA standard with a known concentration was used as a positive control.
  • the 3' end consistency detection of IVT products is achieved through RNase T1 digestion experiment.
  • RNase T1 can specifically degrade single-stranded RNA at the G residue, which forms the corresponding intermediate 2', 3'-cyclic phosphate (-mP) salt, cleaving the phosphodiester bond between the 3'-guanosine residue and the 5'-OH residue of the adjacent nucleotide; the reaction products are 3'-GMP and oligonucleotides containing terminal 3'-GMP. Then, the molecular weight of the 3' end product is analyzed by LC-MS and compared with the theoretical molecular weight.
  • -mP 2', 3'-cyclic phosphate
  • the 3' end of the normal mRNA product is a G base ending with -OH
  • the 3' end G base of the heterogeneous product ends with -mP
  • the molecular weight of the heterogeneous product is 80 more than that of the normal product.
  • the 3' end consistency (%) is calculated by analyzing the proportion of the theoretical molecular weight product.
  • Figures 5A to 5J The experimental results are shown in Figures 5A to 5J.
  • the molecular weight of the pure product with 3' end consistency is 9663.1, and the molecular weight of the 3' end heterogeneous conjugation product is 9743.1.
  • Figures 5A to 5J are the results of LC-MS analysis of 3' end products of IVT products of wild type WT (37°C), Q774R (37°C), S43Y (37°C), G47A+884G (37°C), M7 (37°C), M7 (50°C), M8 (37°C), M8 (50°C), I217L (50°C), and S397W (50°C).
  • Table 8 The detailed data analysis is shown in Table 8.
  • the immune response of cells to dsRNA byproducts may lead to adverse consequences.
  • the purified IVT product RNA sample was used for cell response detection.
  • the specific operation method is as follows: (1) Thaw the cryotube containing the Raw cell suspension and mix it evenly, and add the cell suspension to a culture flask or culture dish containing culture medium for overnight culture. When the RAW cell density reaches 80%-90%, subculture is performed. (2) Inoculate cells: Prepare IVT product-lipid complexes and transfect cells with cationic liposome transfection reagent Lipofectamine 3000.
  • IFN- ⁇ and IL-6 are cytokines produced by the body's immune cells. They are immune response factors produced by immune cells through immune response when the body is infected. The levels of IFN- ⁇ and IL-6 detected in this study reflect the immunogenicity of the T7 RNA polymerase IVT products.
  • T7 RNAP mutants were prepared, and their activity changes were detected (the activity changes were detected using the relative activity detection method of Example 3, and the Tm values of each mutant were determined using the differential scanning fluorimetry of Example 3.
  • the Tm value reflects the stability of each mutant protein. The higher the Tm value, the more stable the protein).
  • T7 RNAP mutants with mutation sites Q786M, Q786F, I217A, I217V, S397P, N579A, and M369E have higher thermal stability than T7 RNAP-WT.
  • I121L, I121S, S223L, S223V, and R632E mutants have no obvious advantage in thermal stability over wild-type T7 RNAP, they have higher enzyme activity. These mutants will have broad application prospects in in vitro transcription, mRNA vaccine and drug research and production, gene editing, isothermal amplification, in vivo protein expression, or cell-free protein expression in vitro translation systems.
  • RNA polymerases from different sources with an amino acid identity of more than 80% were compared, and the results are shown in Figures 7A to 7E. It was found that some sequences in the sequence are highly conserved, including positions 217, 223, 397, 579, and 632. When the amino acid substitutions at positions 217, 223, 397, 579, and 632 described in the present application are transferred to the corresponding positions in other closely related phage RNA polymerases (SEQ ID NO. 29 to SEQ ID NO. 62), they are expected to have the same effect.
  • the closely related bacteriophage RNA polymerase amino acid sequences SEQ ID NO. 29 to SEQ ID NO.
  • the amino acid substitutions at positions 121 and 369 are expected to have the same effect when transferred to the corresponding positions in other closely related phage RNA polymerases.
  • the amino acid sequences of the closely related phage RNA polymerases at position 121 (SEQ ID NO.29 to SEQ ID NO.39, SEQ ID NO.41 to SEQ ID NO.42, SEQ ID NO.44 to SEQ ID NO.45, SEQ ID NO.47, SEQ ID NO.49 to SEQ ID NO.53) have 97.96%, 98.07%, 98.64%, 98.07%, 98.66%, 98.67%, 98.68%, 98.69%, 98.70%, 98.71%, 98.72%, 98.73%, 98.74%, 98.76%, 98.77%, 98.78%, 98.80%, 98.81%, 98.82%, 98.83%, 98.84%, 98.86
  • the closely related bacteriophage RNA polymerase amino acid sequence SEQ ID NO.44 at position 369 has a sequence identity of 98.41% with the wild-type amino acid sequence of T7 bacteriophage RNA polymerase shown in SEQ ID NO.1 in Examples 1-4.
  • amino acid sequence of the wild-type parent T7 RNA polymerase of the present invention (SEQ ID NO.1):

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Abstract

La présente invention concerne une série de mutants de l'ARN polymérase à performance améliorée. Les mutants, par comparaison avec une séquence d'acides aminés d'une ARN polymérase parentale, ont une ou plusieurs mutations d'acides aminés aux positions 121 223 632, 217, 397, 369 et 579 ou ont une ou plusieurs mutations d'acides aminés qui se produisent au niveau de résidus d'acides aminés à des positions équivalentes dans l'ARN polymérase parent. Les mutants présentent au moins une amélioration quant à l'un des aspects suivants : activité enzymatique, stabilité thermique, cohérence de la terminaison 3', rendement de la transcription in vitro, sous-produits d'ARNdb, immunogénicité et autres, et peuvent répondre à diverses exigences d'application pratique et de recherche.
PCT/CN2024/093147 2023-05-15 2024-05-14 Mutants d'arn polymérase à performance améliorée Pending WO2024235228A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CN202310558557.6A CN118956809A (zh) 2023-05-15 2023-05-15 热稳定性提高的t7 rna聚合酶突变体及其应用
CN202310558557.6 2023-05-15
CN202410499488.0 2024-04-24
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