WO2023232075A1 - 一种rna聚合酶融合蛋白及其应用 - Google Patents

一种rna聚合酶融合蛋白及其应用 Download PDF

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WO2023232075A1
WO2023232075A1 PCT/CN2023/097436 CN2023097436W WO2023232075A1 WO 2023232075 A1 WO2023232075 A1 WO 2023232075A1 CN 2023097436 W CN2023097436 W CN 2023097436W WO 2023232075 A1 WO2023232075 A1 WO 2023232075A1
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rna polymerase
seq
amino acid
mutations
acid sequence
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French (fr)
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张平静
张颢译
张宁宁
肖何
钱其军
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上海吉量医药工程有限公司
浙江吉量科技有限公司
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Publication of WO2023232075A1 publication Critical patent/WO2023232075A1/zh

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    • 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)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1247—DNA-directed RNA polymerase (2.7.7.6)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
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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
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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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
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    • 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)
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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/07—Nucleotidyltransferases (2.7.7)
    • C12Y207/07006—DNA-directed RNA polymerase (2.7.7.6)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/85—Fusion polypeptide containing an RNA binding domain

Definitions

  • the invention belongs to the field of biotechnology, and specifically relates to a highly active RNA polymerase fusion protein and its application in RNA synthesis.
  • RNA polymerase also known as "DNA-dependent RNA polymerase”
  • DNA-dependent RNA polymerase has a unique molecular mechanism of single-enzyme driven transcription, which is able to transcribe DNA into RNA transcripts.
  • RNA polymerase has been widely expressed, isolated and purified, and widely used in in vitro production of RNA.
  • In vitro transcription is a procedure that allows DNA-directed synthesis of RNA molecules of any sequence, with RNA products ranging in size from short oligonucleotides to several thousand bases.
  • in vitro transcription involves the engineering of a template that includes a phage promoter sequence upstream of the sequence of interest (e.g., from T7 E. coli phage), followed by transcription using the corresponding RNA polymerase.
  • these RNA transcripts are subsequently modified (e.g., by capping, splicing, addition of poly-A tails, etc.). These transcripts are used in analytical techniques (e.g. hybridization analysis), structural studies (e.g. NMR and X-ray crystallography), biochemical and genetic studies (e.g. as antisense reagents), functional molecules (e.g. ribozymes and aptamers), and as therapeutic agents (e.g., RNA vaccines).
  • analytical techniques e.g. hybridization analysis
  • structural studies e.g. NMR and X-ray crystallography
  • biochemical and genetic studies e.g. as antisense reagents
  • functional molecules e.g. ribozymes and aptamers
  • therapeutic agents e.g., RNA vaccines.
  • Phage RNA polymerases such as SP6, T7, T3, syn5, KP34, and VSW-3 are highly specific for their respective 23-base promoters.
  • the development of cloning vectors containing promoters for these polymerases has made the in vitro synthesis of single-stranded RNA molecules a routine laboratory procedure, with T7 RNA polymerase being the most widely used.
  • T7 RNA polymerase is an RNA polymerase from T7 bacteriophage that catalyzes the formation of RNA from DNA in the 5' ⁇ 3' direction.
  • T7 polymerase has extremely strong promoter specificity and only transcribes DNA downstream of the T7 promoter. Its transcription yield per unit template has reached a relatively high level. Due to differences in application scope, T7RNA polymerase already has many mutants and fusion enzyme types. It is mainly reflected in the demand for diagnostic applications. For example, T7RNA high-temperature resistant mutants can improve the transcription of RNA with high GC content; improve the capping efficiency of co-transcribed cap analog RNA products; and improve long fragments.
  • the essence of the various mutations or fusions mentioned above is to improve the thermal stability of the T7 enzyme through mutation, which is manifested in maintaining high enzyme activity of T7 under high temperature conditions, such as 45-55°C.
  • the vast majority of mutants mainly play a role in delaying the decline of T7 enzyme activity at high temperatures.
  • the enzyme activity is significantly higher than that of the wild-type enzyme at high temperatures, it is not the same as the unit enzyme activity of the wild-type T7 enzyme at 37°C. Life has not improved significantly.
  • Phage RNA polymerase is a single-subunit DNA-dependent RNA polymerase, especially T7 enzyme, which is widely used in diagnostics, mRNA vaccines, and mRNA gene therapy. Except for a few special situations, most application scenarios are still Catalytically produce target RNA in a system at room temperature 25°C to mid-temperature 45°C. In view of the above reasons, how to improve the protein unit enzyme activity, salt tolerance, high sensitivity, and rapid reaction characteristics in a normal reaction system at a medium temperature of 25-45°C is a very large potential production demand.
  • RNA polymerase is also widely used in the production of mRNA vaccines and mRNA gene therapy drugs. Most of these productions also use RNA polymerase to perform transcription reactions under conditions between 37-45°C.
  • mRNA vaccines also known as “gene vaccines”—are derived from an innovative biotech approach that turns human cells into molecular factories to produce proteins that activate pathogen-specific immune responses. The production process of this innovative mRNA API begins with the synthesis of mRNA transcription via a biochemical reaction. The in vitro transcription reaction for mRNA vaccine production begins with the addition of all reaction components into the bioreactor.
  • T7 RNA polymerase connects the four nucleotides ATP, UTP, CTP, and GTP together according to the sequence of the linear template dsDNA (such as the spike protein of the SARS-CoV-2 virus) Form target RNA.
  • phage RNA polymerase is the core raw material in mRNA production, both in terms of its enzymatic activity and production.
  • the unit costs of the process all have a significant impact on the scale-up of mRNA industrial production.
  • the present invention carries out engineering transformation of this type of phage RNA polymerase, improves the unit enzyme activity, transcription speed, salt tolerance activity and other biological properties of T7 RNA polymerase, reduces the unit dosage in the production of mRNA process, and achieves increased production and reduced Comprehensive purpose of cost.
  • Patent CN1720324B discloses an SSO7-polymerase coupling protein that improves PCR performance.
  • the patent describes connecting the non-specific double-stranded nucleic acid binding domain Sso7d or Sac7d to the DNA polymerase catalytic domain to improve DNA polymerase activity, where The non-specific double-stranded nucleic acid binding domain is selected from Sso7d or Sac7d, and its application scenario is DNA polymerase chain reaction (PCR) under high temperature conditions.
  • PCR DNA polymerase chain reaction
  • US20030175738A1 discloses a high-temperature-resistant mutant RNA polymerase. This mutant polymerase can RNA transcription and synthesis dependent on DNA templates were achieved at the maximum tolerated temperature of 50°C, and the unit activity (units/ ⁇ g) of the combined mutant T7 RNA polymerase was only increased by about 36% at 37°C.
  • US9540670B2 discloses a combination of T7 RNA polymerase mutants based on the above-mentioned patent application. The combined mutation includes the high-temperature resistant mutation point in US20030175738A1 and a new mutation point. This combination of mutated T7 RNA polymerase operates at 37°C. The enzyme unit activity under the condition only increased by about 48% at most. There is no description of any fusion RNA polymerase protein in the above-mentioned patents or patent applications.
  • CN108779446A also discloses a heat-resistant mutant of T7 RNA polymerase.
  • This mutant only exhibits higher unit activity than wild-type T7 RNA polymerase at high temperatures above 42°C.
  • the patent discloses a fusion of a DNA binding domain. Examples of RNA polymerase with Sso7d domain, but are limited to applications above 42°C, and do not significantly improve the enzyme activity of the original mutant RNA polymerase.
  • the unit enzyme activity at 37°C is not involved. .
  • It is well known to those skilled in the art that most thermostable enzymes will experience a significant decrease in unit enzyme activity as the temperature rises.
  • the higher enzyme activity compared to the wild type described in the patent should actually be described as the enzyme activity changes with temperature. It decreases more slowly when increasing, thus showing a certain higher activity at high temperatures. This improvement in enzyme unit activity is more commonly used in high-temperature transcription or detection applications. There is no significant improvement in transcription activity at room temperature. Advantage.
  • JP2017178804A patent discloses a solution for AlbA fusion DNA polymerase, which uses the DNA binding domain of AlbA to improve the salt tolerance of DNA polymerase in PCR reactions.
  • This patent does not involve the characteristics of the RNA binding domain of Alba protein, nor does it involve When it comes to the application of RNA polymerase, we only focus on the high temperature resistance properties of these proteins and their PCR applications.
  • US patent US20130022980 discloses the application of a high-temperature-resistant non-specific RNA-binding domain fused to DNA polymerase with reverse transcription activity. It also discloses a chimeric type in which part of the DNA-binding domain polypeptide and part of the RNA-binding domain conserved polypeptide are connected in series. Polypeptides and proteins disclosed in the patent belong to the Csp protein family, but are limited to the application of high-temperature-resistant Csp proteins in PCR or reverse transcription of cDNA, and do not involve the application of in vitro transcribed RNA, because in vitro transcription cannot It involves high-temperature resistance characteristics, but also does not involve salt tolerance and resistance to transcription suspension.
  • RNA polymerase has low enzyme activity units and slow transcription speed, and more enzyme dosage brings There are disadvantages such as the risk of contamination.
  • the present invention provides an RNA polymerase fusion protein and its application. Specifically, the enzyme activity units of RNA polymerase are relatively low, resulting in high dosage of template and polymerase in production. Increasing the dosage of DNA template and enzyme leads to higher production costs, which is not conducive to the industrial production of mRNA. In addition, using more enzymes will bring more risks of contamination by RNase, host DNA, host proteins, etc., and also increase the complexity of the downstream purification and removal of enzyme proteins in RNA production, such as adding more organic solvents to high-concentration proteins. It can easily lead to protein precipitation, and more protein by-products can also lead to load-occupying effects on the purity chromatographic column.
  • RNA polymerase-mediated transcription synthesis system still has some shortcomings.
  • the current transcription speed of RNA polymerase is not fast enough, resulting in a relatively long transcription reaction time, which generally takes 4-6 hours or even longer. An overnight reaction is required.
  • RNA polymerase for example, in order to obtain a higher RNA yield per unit volume, it is necessary to conduct in vitro transcription reactions under higher template concentration or NTP concentration reaction conditions, but the above conditions often bring more
  • the high salt concentration effect inhibits the transcriptional activity of RNA polymerase, so it is necessary to engineer RNA polymerase to reduce the existence of this inhibitory effect; for example, in order to obtain less dsRNA non-specific transcription contaminants, in vitro experiments need to be performed at higher salt concentrations.
  • Transcription reaction requires increasing the salt tolerance concentration of wild-type RNA polymerase, etc.
  • RNA polymerase fusion protein which includes:
  • the RNA polymerase fusion protein has higher unit enzyme activity than the phage RNA polymerase.
  • the present invention fuses a DNA and/RNA binding domain.
  • This binding domain can not only bind the template dsDNA more firmly, but also better absorb the new RNA synthesis chain synthesized by transcription, so it can better It improves the speed and stability of transcription, which is expressed in the form of an increase in enzyme unit activity or an increase in transcription speed.
  • the higher unit enzyme activity in the present invention generally refers to an activity that is at least 1-fold higher than that of wild-type RNA polymerase.
  • the amino acid sequences of the phage RNA polymerase are as shown in SEQ ID NO: 1 to 6, which are respectively T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, Syn5 RNA polymerase, KP34 RNA polymerase and VSW3 RNA polymerase, or one or more mutants having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.
  • the DNA binding domain polypeptide and/or the RNA binding domain polypeptide is fused to the N-terminus of the phage RNA polymerase; and/or the DNA binding domain polypeptide and/or the RNA binding domain polypeptide It is connected to the bacteriophage RNA polymerase through a linker.
  • any flexible connector disclosed in the prior art can be used to replace the connectors mentioned in the present invention.
  • the linker used does not affect the functional improvement of the fusion protein.
  • the subunits are connected via a peptide linker, such as described in Chen et al. Adv Drug Deliv Rev 2013.
  • two subunits in a polypeptide are connected by a rigid linker.
  • the rigid linker consists of n EAAAAK (SEQ ID NO:7) motifs, represented as (EAAAAK) n .
  • n is a natural number from 0 to 10.
  • two subunits in the polypeptide are connected by a flexible linker.
  • the flexible linker consists of the motif (Gly)n. In some embodiments, the flexible linker consists of n GGGGS (SEQ ID NO:8) motifs, represented as (GGGGS) n . n is a natural number from 0 to 10. In some embodiments, a rigid or flexible linker consists of 1, 2, 3, 4, 5, 10, 15, or more amino acids in length to enable RNA transcription. In some embodiments, the linker consists of a combination of rigid and flexible linker motifs.
  • the mutant has one or more amino acid substitutions selected from the following on the amino acid sequences shown in SEQ ID NO: 1 to 6: S430P, S633P, F849I, F880Y, P266L and Q744R /L/P.
  • Q744R/L/P means Q is replaced by R, L or P at position 744.
  • the mutants include S430P, S633P, F849I and F880Y mutations.
  • the mutant also includes Q744R and/or P266L mutations.
  • the mutants include S430P, S633P, Q744R, F849I and F880Y mutations, or P266L, S430P, S633P, Q744R, F849I and F880Y mutations.
  • the DNA binding domain polypeptide is selected from one or more of the Sul7d protein family derived from Archaea.
  • the cold shock domain is a protein domain containing approximately 70 amino acids that has been found in prokaryotic and eukaryotic DNA-binding proteins. Part of this domain is highly similar to the RNP-1 RNA binding motif. Sequence analysis revealed a short motif of eight amino acids corresponding to the RNP-1 motif found in canonical RNA-binding domains, which is common to two apparently unrelated protein families. Many RNA-binding proteins contain RNP-1 and RNP-2 motifs in the RNA-binding domain.
  • the cold shock domain (CSD) protein family which includes several transcription factors that have been shown to bind DNA, has been identified to contain a motif similar to RNP-1, and a search found that the CSD-containing protein family includes a motif similar to RNP-1 Almost the same motif.
  • the RNA binding domain polypeptide is selected from one or more cold shock domain CSD (PF00313) family proteins, preferably selected from the amino acid sequences such as SEQ ID NO: 15, 16, The cold shock domain CSD of Escherichia coli, Halobacteriales archaeon, Halobacterium salinarum, and Thermotoga maritima shown in 20-23, and has at least 80%, 90%, 95%, 96%, and 97% of the amino acid sequence , one or more of 98% or 99% identical mutants.
  • cold shock domain CSD PF00313 family proteins, preferably selected from the amino acid sequences such as SEQ ID NO: 15, 16, The cold shock domain CSD of Escherichia coli, Halobacteriales archaeon, Halobacterium salinarum, and Thermotoga maritima shown in 20-23, and has at least 80%, 90%, 95%, 96%, and 97% of the amino acid sequence , one or more of 98% or 99% identical mutants.
  • the DNA-binding domain polypeptide and/or RNA-binding domain polypeptide is a DNA/RNA-binding protein albA family protein (PFAM01918) derived from Archaea, preferably selected from but not limited to DNA/RNA binding domain Sac7D (SEQ ID NO:9) of Sulfolobus acidocaldarius, SEQ ID NO:10 of Sulfolobus genus, SEQ ID NO:11 of Sulfolobus Iceland, Sulfolobus sulphurifera SEQ ID NO:12 of Chrysococcum sp.
  • PFAM01918 DNA/RNA-binding protein albA family protein
  • SEQ ID NO:13 Acidophilus SEQ ID NO:14, CspA (SEQ ID NO:15) and CspE (SEQ ID NO:16) of Escherichia coli, or one or more mutants having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence.
  • the RNA polymerase fusion protein includes:
  • the amino acid sequence of the phage RNA polymerase is as shown in SEQ ID NO:1; or it has S430P, S633P, F849I and F880Y mutations on the amino acid sequence as shown in SEQ ID NO:1, S430P , S633P, Q744R, F849I and F880Y mutations, or P266L, S430P, S633P, Q744R, F849I and F880Y mutations.
  • the amino acid sequence of the phage RNA polymerase is as shown in SEQ ID NO:2; or it has S430P, S633P, F849I and F880Y mutations on the amino acid sequence as shown in SEQ ID NO:2, S430P, S633P, Q744R, F849I and F880Y mutation, or P266L, S430P, S633P, Q744R, F849I and F880Y mutations.
  • the amino acid sequence of the phage RNA polymerase is as shown in SEQ ID NO:3; or it has S430P, S633P, F849I and F880Y mutations on the amino acid sequence as shown in SEQ ID NO:3, S430P, S633P, Q744R, F849I and F880Y mutation, or P266L, S430P, S633P, Q744R, F849I and F880Y mutations.
  • the amino acid sequence of the phage RNA polymerase is as shown in SEQ ID NO: 4; or it has S430P, S633P, F849I and F880Y mutations on the amino acid sequence as shown in SEQ ID NO: 4, S430P, S633P, Q744R, F849I and F880Y mutation, or P266L, S430P, S633P, Q744R, F849I and F880Y mutations.
  • the amino acid sequence of the phage RNA polymerase is as shown in SEQ ID NO:5; or it has S430P, S633P, F849I and F880Y mutations on the amino acid sequence as shown in SEQ ID NO:5, S430P, S633P, Q744R, F849I and F880Y mutation, or P266L, S430P, S633P, Q744R, F849I and F880Y mutations.
  • the amino acid sequence of the phage RNA polymerase is as shown in SEQ ID NO: 6; or it has S430P, S633P, F849I and F880Y mutations on the amino acid sequence as shown in SEQ ID NO: 6, S430P, S633P, Q744R, F849I and F880Y mutation, or P266L, S430P, S633P, Q744R, F849I and F880Y mutations.
  • amino acid sequence of the DNA binding domain polypeptide is as shown in any one of SEQ ID NO: 9 to 14.
  • the RNA polymerase fusion protein includes:
  • the amino acid sequence of the phage RNA polymerase is as shown in SEQ ID NO:1; or it has S430P, S633P, F849I and F880Y mutations on the amino acid sequence as shown in SEQ ID NO:1, S430P , S633P, Q744R, F849I and F880Y mutations, or P266L, S430P, S633P, Q744R, F849I and F880Y mutations; in (ii), the amino acid sequence of the DNA binding domain polypeptide is as shown in SEQ ID NO: 9.
  • the phage RNA polymerase has S430P, S633P, Q744R, F849I and F880Y mutations on the amino acid sequence shown in SEQ ID NO:2; in (ii), the DNA binding domain polypeptide The amino acid sequence is shown in SEQ ID NO:10.
  • the phage RNA polymerase has S430P, S633P, Q744R, F849I and F880Y mutations on the amino acid sequence shown in SEQ ID NO:3; in (ii), the DNA binding domain polypeptide The amino acid sequence is shown in SEQ ID NO:11.
  • the phage RNA polymerase has S430P, S633P, Q744R, F849I and F880Y mutations on the amino acid sequence shown in SEQ ID NO:4; in (ii), the DNA binding domain polypeptide The amino acid sequence is shown in SEQ ID NO:12.
  • the phage RNA polymerase has S430P, S633P, Q744R, F849I and F880Y mutations on the amino acid sequence shown in SEQ ID NO:5; in (ii), the DNA binding domain polypeptide The amino acid sequence is shown in SEQ ID NO:13.
  • the phage RNA polymerase has S430P, S633P, Q744R, F849I and F880Y mutations on the amino acid sequence shown in SEQ ID NO: 6; in (ii), the DNA binding domain polypeptide The amino acid sequence is shown in SEQ ID NO:14.
  • a biological material selected from the following group:
  • the third aspect of the present invention provides a composition, which includes the RNA polymerase fusion protein as described in any one of the first aspect of the present invention.
  • buffers modified or unmodified ribonucleoside triphosphates, and cap analogs are also included.
  • the unmodified ribonucleoside triphosphates are ATP, CTP, UTP, and GTP; the modified ribonucleoside triphosphates are selected from pseudouridine triphosphate (), N1-methyl-pseudouridine triphosphate (m 1 ) , 2-amino-6-chloropurine ribonucleoside 5'triphosphate, 2-aminoadenosine 5'triphosphate, 2-thiocytidine 5'triphosphate, 2-thiouridine 5'triphosphate, 4-thiocytidine Uridine 3'triphosphate, 5-aminoallylcytidine 5'triphosphate, 5-aminoallyuridine 5'triphosphate, 5-bromocytidine 5'triphosphate, 5-bromouridine 5'triphosphate, 5-iodocytidine 5'triphosphate,
  • buffer refers to an agent that renders a solution resistant to changes in pH when an acid or base is added.
  • suitable non-naturally occurring buffers include, for example, one or more of Tris, HEPES, TAPS, MOPS, tricine, and MES.
  • cap refers to the cap structure found at the 5' end of an RNA molecule, usually consisting of a guanosine nucleotide linked by an unusual 5'-5' triphosphate Bonds to messenger RNA.
  • cap analogs refers to structures similar to caps that have the ability to stabilize RNA and/or enhance RNA translation, and can be linked to mRNA through capping enzymes or co-transcription.
  • the cap analogs include GAG, GAU, ARCA, GGG, etc. (http://www.syngenebio.com/product/37/).
  • the fourth aspect of the present invention provides a kit, which includes the RNA polymerase fusion protein as described in any one of the first aspect of the present invention.
  • buffers modified or unmodified ribonucleoside triphosphates, and cap analogs are also included.
  • the buffering agent includes: one or more of Tris, HEPES, TAPS, MOPS, tricine and MES.
  • the fifth aspect of the present invention provides a method for synthesizing RNA molecules, including incubating a mixture to transcribe template DNA molecules into RNA, the mixture comprising any one of the first aspects of the present invention.
  • RNA polymerase variant fusion protein or the composition of any one of the third aspects of the invention and a template DNA molecule are examples of RNA molecules.
  • the incubation is performed at a temperature of 25-45°C, such as 25°C, 37°C or 42°C.
  • the transcription is performed at a salt concentration of 0.05-0.2M
  • the salt is NaCl and the salt concentration is no higher than 0.1M.
  • the salt is selected from but not limited to Na ions, K ions, Li ions, Tris ions, etc., the unit enzyme activity of the fusion mutant RNA polymerase is significantly better than Non-fusion mutant RNA polymerase.
  • the mixture further contains modified or unmodified ribonucleoside triphosphates.
  • the mixture further contains a cap analog to achieve co-transcriptional capping.
  • concentration of the cap analog is ⁇ 8mM, such as 8mM, 6mM, 4mM, 2mM or 1mM.
  • the RNA polymerase fusion protein can maintain a high co-transcription yield under lower concentrations (for example, a minimum of 1 mM) of cap analogues, without significantly reducing capping efficiency, purity, or intracellular expression.
  • the sixth aspect of the present invention provides an RNA polymerase fusion protein as described in any one of the first aspects, a biological material as described in the second aspect, or a combination as described in the third aspect.
  • the seventh aspect of the present invention provides an application of an RNA polymerase fusion protein as described in any one of the first aspects in the preparation of an enzyme preparation with high activity under medium temperature conditions, such as 25-42°C. .
  • High activity in the present invention refers to having an activity that is at least doubled compared to wild-type RNA polymerase.
  • the eighth aspect of the present invention provides a nucleic acid diagnostic method, which includes the steps of nucleic acid transcription and nucleic acid determination; wherein, the nucleic acid amplification uses RNA as described in any one of the first aspects.
  • the polymerase variant fusion protein, the composition according to the third aspect or the kit according to any one of the fourth aspects transcribes or amplifies the nucleic acid template; the nucleic acid is determined using a sequencing method such as Sanger sequencing or NGS sequencing. conduct.
  • the nucleic acid diagnostic method is a method for non-disease diagnosis purposes, such as determining the specific nucleic acid used in a laboratory.
  • the reagents and raw materials used in the present invention are all commercially available.
  • the activity of the RNA polymerase fusion protein of the present invention is significantly increased; for example, the activity of the transcriptase unit of the 7kd DNA binding domain fusion RNA polymerase is significantly increased by 1-6 times.
  • the fusion mutant RNA polymerase maintains a high co-transcription yield under lower concentrations of cap analogues, and the capping efficiency, purity, and intracellular expression are not significantly reduced.
  • Figure 1 is a schematic diagram of the structure of the RNA polymerase fusion protein of the present invention.
  • Figure 2 is a schematic diagram of the conserved and highly conserved sequences of Sul7d.
  • Figure 3A shows the purified PAGE identification results of some RNA polymerases or mutants and their fusion proteins.
  • 1 Roche-T7; 2: WT-T7; 3: M4-T7; 4: HF-T7; 5: HF-T7-M4; 6: HF-T7-M5; 7: HF-T7-M6; 8: Sso7d-T7-M5; 9: Saz7d-T7-M5; 10: Sis7d-T7-M5; 11: RCPA-T7-M5; 12: RCPB-T7-M5; 13: RCP1-T7-M5; 14: RCP2-T7-M5.
  • Figure 3B shows the purified PAGE identification results of some RNA polymerases or mutants and their fusion proteins. Among them, 1: Roche-T7; 2: WT-T7; 3: HF-T7; 4: HF-T7-M4; 5: HF-T7-M5; 6: HF-T7-M3; 7: HF-T7- M2+M3-1;8:NEB007M6.
  • Figure 4A shows the fluorescence signal changes at each concentration point of the standard curve of the fluorescence real-time quantitative detection method of enzyme activity.
  • Figure 4B is a linear relationship diagram of the 60-minute standard curve of the fluorescence real-time quantitative detection method of enzyme activity.
  • Figure 5A shows the real-time quantitative fluorescence values of Sac7d fusion domain T7 enzyme and its control enzyme activity at 25°C.
  • Figure 5B shows the real-time quantitative fluorescence values of Sac7d fusion domain T7 enzyme and its control enzyme activity at 37°C.
  • Figure 5C shows the real-time quantitative fluorescence values of Sac7d fusion domain T7 enzyme and its control enzyme activity at 45°C.
  • Figure 6A shows the fluorescence signal values of different engineered T7 RNA polymerases and their reference enzymes in real-time transcription of RNA ligands combined with DHFI.
  • 1 Roche-T7
  • 2 WT-T7
  • 3 M4-T7
  • 4 HF-T7
  • 5 HF-T7-M4
  • 6 HF-T7-M5
  • 7 HF-T7-M6
  • 8 HF-M5-8aa
  • 10:NEB007M6 the fluorescence signal values of different engineered T7 RNA polymerases and their reference enzymes in real-time transcription of RNA ligands combined with DHFI.
  • 1 Roche-T7
  • 2 WT-T7
  • 3 M4-T7
  • 4 HF-T7
  • 5 HF-T7-M4
  • 6 HF-T7-M5
  • 7 HF-T7-M6
  • 8 HF-M5-8aa
  • Figure 6B is a comparison of the unit enzyme activities of different engineered T7 RNA polymerases and their reference enzymes. Note: 1: Roche-T7; 2: WT-T7; 3: M4-T7; 4: HF-T7; 5: HF-T7-M4; 6: HF-T7-M5; 7: HF-T7-M6; 8:HF-M5-8aa; 9:RCPA-T7-M5; 10:NEB007M6.
  • Figure 6C is an agarose gel electrophoresis diagram of different engineered T7 RNA polymerase proteins and their reference enzymes transcribing long-chain 4500nt RNA, reflecting the transcription speed, unit yield and product RNA purity of different enzymes.
  • 1 Roche-T7
  • 2 HF-T7
  • 3 HF-T7-M4
  • 4 HF-T7-M5
  • 5 HF-T7-M6
  • 6 WT-HF
  • Figure 7 shows the capillary electrophoresis diagram of different engineered T7 RNA polymerase proteins and their reference enzymes transcribing long-chain 4500nt RNA, reflecting the transcription speed, unit yield and product RNA purity of different enzymes.
  • Note 1 HF-T7-M5>HF-T7-M4>HF-T7-M6>M4-T7>HF-T7>Roche-T7>RCPA-T7;
  • Note 2 4 hours at 45°C due to the influence of high RNA concentration Capillary electrophoresis effect, so all samples were diluted 5 times before loading.
  • Figure 8A is a comparison of the real-time fluorescence values of different engineered T7 RNA polymerase proteins and their reference enzymes transcribing RNA ligands at 37°C.
  • Figure 8B is a comparison of the unit enzyme activity values of different engineered T7 RNA polymerase proteins and their reference substances at 37°C.
  • 1 WT-T7; 2: HF-T7; 3: HF-T7-M5; 4: Sso7d-T7-M5; 5: Saz7d-T7-M5; 6: Sis7d-T7-M5; 7: Met7d-T7-M5; 8: Asul7d-T7-M5; 9: RCPA-T7-M5; 10: RCPB-T7-M5; 11: RCP1-T7-M5; 12: RCP2-T7-M5; 13: NEB007M6.
  • Figure 8C is a comparison of agarose gel electrophoresis of different fusion mutant T7 RNA polymerase proteins and their reference enzyme-spot transcribed RNA. Note: 1: Roche-T7; 2: HF-T7-M5; 3: NEB007M6; 4: Hi-T7(NEB); 5: RCP2-T7; 6: HF-T7-M3; 7: HF-T7-M2 +M3-1.
  • Figure 8D shows the production of different engineered T7 RNA polymerase proteins and their control substances at 37°C.
  • Figure 8E shows the yields of different fusion mutant T7 RNA polymerase proteins and their reference enzyme fusion proteins at 45°C.
  • Figure 9 shows the quantitative fluorescence values of different engineered T7 RNA polymerases under the condition of additional addition of 0.1M NaCl.
  • Figures 10A to 10D show that the fusion mutant RNA polymerase significantly improves the sensitivity to RNA transcription templates.
  • Figure 11 is a comparison of the transcription yields of M5 and commercially available RNA polymerases.
  • Figure 12 shows the agarose gel electrophoresis diagram of M5 and commercially available RNA polymerase transcript products.
  • the samples from left to right are: 1.2ug-5:1, 1.2ug-10:1, 0.6ug-5:1, 0.6ug-10:1, 0.3ug-5:1, 0.3ug-10:1.
  • RNA polymerase achieves RNA synthesis under in vitro transcription conditions at room temperature 25-45°C, while the latter achieves RNA synthesis under PCR conditions at 50-100°C
  • fusion DNA-binding domain polypeptides are mainly derived from thermophilic archaea and mainly use their high-temperature resistance characteristics.
  • the main application point of the previous patent CN108779446A is also the DNA-binding domain fusion of a high-temperature-resistant T7 RNA polymerase mutant. Protein form, however, this fusion protein does not significantly improve the activity of the enzyme unit in a high-temperature environment, and the patent only shows a single example of a 7kd DNA binding domain SSO7D.
  • thermophilic archaea after being fused to RNA polymerase, can significantly improve the transcription activity of the enzyme unit under in vitro transcription conditions at room temperature of 25-42°C, especially if this DNA-binding domain protein is combined with a mutant After RNA polymerase is fused, its highest unit enzyme activity can actually be increased to 6 times, which is beyond expectation.
  • Sequence-nonspecific DNA-binding domain refers to a protein domain that binds to DNA without significant sequence preference.
  • the non-specific DNA binding domain is mainly derived from the 7kd DNA binding domain Sul7d protein family of Archaea (pfam02294).
  • the DNA binding domain binds double-stranded DNA.
  • Non-limiting exemplary DNA binding domains include Sso7d from Sulfolobus solfataricus, Sac7d from S.
  • the present invention also found that in addition to the 7kd DNA binding domain in archaea, there is also an albA protein family that not only has a DNA binding domain but also an RNA binding domain.
  • This binding domain can not only bind the template dsDNA more firmly, but also better It adsorbs the newly synthesized RNA chain for transcription and avoids features such as secondary structure transcription suspension, so it can better improve the speed and stability of transcription. Although its performance does not significantly reflect the increase in enzyme unit activity, it may have an impact on the RNA. Completeness helps.
  • thermophilic bacteria in most cases, their optimal survival temperatures also vary greatly, such as thermophiles and mesophiles. feature.
  • the suitable growth temperature of some bacteria in the genus Acidianus, Metallosphaera, Stygiolobus azoricus and Sulfolobus is between 60 and 80°C. These bacteria are called medium Thermophilic bacteria; there are also some bacteria whose suitable growth temperature exceeds 80°C. These bacteria are called extreme thermophiles, such as Thermococcus kodakarensis and Sulfolobus acidocaldarius.
  • Prior patents generally utilize the DNA-binding domains of hyperthermophiles among these archaea to adapt to the high-temperature environment of PCR.
  • Part of the DNA/RNA binding domain used in the present invention is derived from moderate thermophiles.
  • part of the AlbA protein thermoautotrophic Methanothermophilus has a temperature growth range of 30 to 70°C, and the optimal growth temperature is 65°C.
  • Methanococcus has a growth temperature of 20 to 40°C and an optimal growth temperature of 35 to 39°C.
  • these archaea originate from the ocean, such as Methanococcus marinus and Methanococcus thermoautotrophus, and their living conditions are in high-salt environments. Coupling this domain can be beneficial to the patented reaction system in high-salt environments (such as Under the same transcription conditions, an additional 0.1M NaCl) is added, which is of great benefit to the industrial RNA production of synthesizing high-yield and high-quality low dsRNA content or reducing redundant RNA transcript products.
  • Figure 1 is a schematic diagram of the structure of the RNA polymerase fusion protein of the present invention.
  • Figure 2 is a schematic diagram of the conserved and highly conserved sequences of Sul7d.
  • T7, SP6 RNA and other polymerase ORF regions were cloned into pQE-80L (Qiagen) to obtain recombinant plasmids pQE-80L-T7, pQE-80L-SP6, etc. Genes synthesize different DNA and/or RNA binding domain gene sequences.
  • YEASEN performs a 10 ⁇ L ligation reaction on the purified PCR product, and takes 5 ⁇ L of the ligation reaction product for transformation in TOP10 E. coli competent cells. For each mutant, 3 single clones were picked for culture, and half of each were sequenced, identified and preserved. bacteria, to identify mutants with correct sequences, use TIANprep Rapid Mini Plasmid Kit (TIANGEN) for plasmid extraction.
  • the cloning expression plasmids in the table are all PQE-80L, with His tag, and expressed in BL21(DE3).
  • the bacterial cells were resuspended in 10 times the volume of lysis buffer (50mM PB at pH 7.5, 500mM NaCl, 5mM imidazole, 1mM PMSF), and then subjected to high-pressure homogenization and lysis (800bar, 6 minutes). Centrifuge (13000rpm/min; 30min) Then take the supernatant and perform the first step of purification using immobilized metal affinity chromatography (IMAC). The supernatant was passed through a 5 ml Ni-NTA pre-packed column pre-equilibrated with binding buffer (50mM PB, pH 7.5, 500mM NaCl, 5mM imidazole).
  • IMAC immobilized metal affinity chromatography
  • This method utilizes the characteristics of fluorescent dyes that generate fluorescence after combining with RNA, and reflects the yield of RNA by monitoring changes in fluorescence signals, indirectly characterizing the rate of the enzymatic reaction, and using the enzymatic reaction rate to evaluate the enzyme activity of T7 RNA polymerase.
  • the corresponding iSpinach DNA template containing the T7 RNA polymerase promoter sequence was genetically synthesized, and then the iSpinach DNA sequence was cloned into the pUC57 plasmid vector, and the upstream and downstream primers of the pUC57 plasmid vector universal primer M13 were used to amplify iSpinach -D5 DNA transcription template, the amplified DNA product is used as the DNA substrate for the enzymatic reaction system evaluated by T7 RNA polymerase.
  • the activity detection transcription conditions were determined: 40mM Tris-HCl (pH8.0), 20mM MgCl2, 5mM DTT, 1mM Spermidine, 10mM rNTP, 16nmol/ml DFHBI, 200pmol/ml iSpinach-D5DNA template, RNase inhibitor 1KU/ml.
  • the enzyme activity of T7 RNA polymerase is defined as 1 U for each 1 pmol of iSpinach RNA produced after 60 minutes of reaction at 37°C under the conditions of a 100 ⁇ l enzyme activity detection system containing 20 pmol of iSpinach DNA template.
  • iSpinach RNA template molecular weight, M 39600.
  • the T7 enzyme continuously synthesizes iSpinach RNA.
  • the iSpinach RNA will combine with the DFHBI fluorescent dye to release a fluorescent signal.
  • concentration gradient dilution is used to establish a stable and accurate standard curve to quantitatively detect RNA production.
  • concentration gradient dilution is used to establish a stable and accurate standard curve to quantitatively detect RNA production.
  • concentration gradient dilution is used to establish a stable and accurate standard curve to quantitatively detect RNA production.
  • concentration gradient dilution is used to establish a stable and accurate standard curve to quantitatively detect RNA production.
  • the starting concentration of the standard curve is 60 ⁇ g/100 ⁇ l. After 2-fold gradient dilution, the lowest concentration of the standard curve is 3.75 ⁇ g/100 ⁇ l for real-time fluorescence detection.
  • RNA standard reference reaction system preparation reference is as follows. Take out the reagent and place it at room temperature. After melting, vortex and mix well and place it on ice for later use.
  • DFHBI is a 10 ⁇ l/tube storage solution, which needs to be diluted 50 times before use. After DFHBI is melted, add 490 ⁇ l Nuclease-free-Water, vortex and mix well before use. Prepare the enzymatic reaction system according to the table below, and prepare two duplicate wells for each sample.
  • the fluorescence signal at each concentration point of the standard curve gradually becomes stable over time, with a slight decreasing trend at high concentrations.
  • the upper limit of the standard curve is 60 ⁇ g/100 ⁇ l.
  • the fluorescence signal intensity reaches 340,000, which can cover the highest signal of the sample and meet the requirements for high-concentration enzymes. Quantitative detection of enzyme activity.
  • the above iSpinach RNA sequence reference is Alexis Autour, Eric Westhof. iSpinach: a fluorogenic RNA aptamer optimized for in vitro applications. Nucleic Acids Research, 2016, Vol. 44, No. 6 2491–2500.
  • the combined design of different T7RNA polymerase mutations and different DNA-binding proteins is compared, and the transcriptional activity, that is, the fluorescence value of all these new T7RNA polymerase variants under the detection system is compared, and is converted into unit mass. units of enzyme activity.
  • T7M4 S430P/S633P/F849I/F880Y;
  • T7M5 S430P/S633P/F849I/F880Y/Q744R;
  • T7M6 P266L/S430P/S633P/F849I/F880Y/Q744R;
  • HF Sac7d DNA-binding domain protein in the Sul7d family protein
  • 8aa T7M5 deletes 8 amino acids 167-174 (EEQLNKRV, SEQ ID NO:19).
  • the mutant T7RNA polymerase (M4-6) with Sac7d DNA-binding domain fusion all showed the transcriptional activity of the DNA-binding domain fusion alone or the mutant T7RNA polymerase alone, especially at 37-45 Under °C conditions, the fluorescence value increases significantly.
  • enzymatic activity in the present invention is as follows. In an enzymatic reaction evaluation system containing 20 pmol of DNA template in 100 uL, reacting at 37°C for 1 hour, the amount of 122nt RNA product produced per pmol of 1 unit is defined as 1 Unit of enzyme activity, measured by the fluorescence of standard RNA. The value can be used to calculate the enzyme activity units corresponding to each enzyme.
  • the present invention uses enzyme activity units and the fluorescence intensity of the transcript product of the same dose of enzyme as parameter diagrams ( Figure 6A and Figure 6B). The results show that the fusion RNA mutant polymerase designed by the inventor significantly increased the unit enzyme activity and/or increased the speed of enzyme transcription at various temperatures.
  • the transcription reaction system is as follows: 40mM Tris-HCl (pH8.0), 20mM MgCl 2 , 5mM DTT, 1mM Spermidine, 10mM rNTP, DNA template 0.1 ⁇ g/ ⁇ L, RNase inhibitor 1U/ ⁇ L, IPPase 1U/ ⁇ L, T7polymerase 0.01 ⁇ g/ ⁇ L.
  • the transcript products at different temperatures and different time points were subjected to agarose gel electrophoresis, and the results are shown in Figure 6C.
  • the results show that the mutant T7RNA polymerase (M4-6) fused with the Sac7d DNA binding domain shows the transcriptional activity of the DNA-binding domain fusion alone or the mutant T7RNA polymerase alone. This result is consistent with the above-mentioned fluorescence quantitative detection and enzyme activity calculation results. All highly consistent.
  • the increase in unit enzyme activity means that the amount of enzyme used when producing RNA is reduced, which can reduce production costs; the increase in transcription speed means that the time cost when producing RNA is reduced and labor costs are reduced; the decrease in enzyme amount also means that the enzyme protein content in the transcription system Reduction can reduce the difficulty of downstream purification and reduce the introduction of protein source impurities.
  • Example 5 Effect of fusion mutant T7 RNA polymerase enzyme on the yield and purity of long-chain RNA
  • a 4500nt Cas9mRNA transcribed DNA template was used to conduct a transcription experiment to compare the transcription performance of different engineered enzymes.
  • the transcription reaction conditions were the same as in Example 4, but inorganic pyrophosphatase was not added to the transcription reaction system in this example, and the reaction temperatures were set separately. Two conditions of 37°C and 45°C were used, and the same volume of transcripts was drawn at different time points of transcription for capillary electrophoresis to analyze the yield and purity of the RNA products.
  • the 4-hour transcript product at 45°C was too high in RNA concentration, so the present invention carried out 5 After dilution, perform capillary gel electrophoresis analysis.
  • the DNA transcription template and its transcribed RNA product sequence refer to the plasmid information of the Addgene database with the item number Plasmid#71310.
  • HF-T7-M4/M5/M6 is significantly better than the single fusion type HF-T7 enzyme and or non-fusion type in terms of transcription activity, RNA transcription elongation speed, and total yield.
  • the M4 mutant is better than the wild-type control group and/or the commercial control group.
  • the significant improvement in the unit enzyme activity and RNA yield of the fusion mutant T7 RNA polymerase is manifested in a significant increase in the speed of transcription and synthesis of RNA and/or a significant increase in the final yield.
  • the optimized fusion mutant RNA polymerase's transcript product yield in 2 hours under the same conditions far exceeds the wild-type polymerase's transcript product yield in 4 hours, greatly saving reaction time. between.
  • Example 6 Effects of different DNA binding domains and high temperature resistant mutant T7 RNA polymerase on unit enzyme activity
  • the present invention selected HF-M3 (Sac7D-V426L/A702V/V795I) and HF-M3-M2-1 (Sac7D-V426L/S633P/A702V/V795I/Q744R).
  • HF-M3 Sac7D-V426L/A702V/V795I
  • HF-M3-M2-1 Sac7D-V426L/S633P/A702V/V795I/Q744R
  • the present invention also selects the NEB007M6 fusion mutant T7 enzyme, namely SS07-T7M-6M (the fusion mutant T7 enzyme with the highest unit enzyme activity in the CN108779446A patent) I109L/H205S/D388E/L534V/V567P/G618Q, in which SS07 is derived from the Mycobacterium thermophilum. (007-T7) lacI-like protein.
  • the present invention also purchased the commercial high-temperature resistant T7 enzyme from NEB Company for comparison of transcription activities, Hi-T7NEB (product number: M0658S).
  • the present invention also selected the Alba DNA binding domain to be fused with the M5 mutation (RCP-2-HF-T7:ssh10b-S430P/S633P/F849I/F880Y/Q744R).
  • the present invention also selects other members of the Sul7d family (Sac7d, Sso7d, Saz7d, Sis7d, Met7d, Asul7d) for fusion expression, purification and enzyme activity testing of the mutant T7 enzyme.
  • the transcription conditions of the present invention are 40mM Tris-HCl (pH8.0), 20mM MgCl2, 5mM DTT, 1mM Spermidine, 10mM rNTP, 16nmol/ml DFHBI, 200pmol/ml iSpinach-D5 DNA template, RNase inhibitor 1KU/ml; the transcription temperature is 37°C.
  • Table 5 shows the effects of different DNA binding domains and high temperature resistant mutant T7 RNA polymerase on unit enzyme activity.
  • the fusion domains of the Sul7d family can significantly increase the enzyme activity, while the Alba-type fusion proteins have not significantly increased the enzyme activity.
  • This example also shows that not all mutations can significantly increase enzyme activity (as shown in Figures 8C, 8D and 8E).
  • NEB007M6 SS07-T7M-6M is a mutant fusion T7 enzyme, although it also shows a certain increase in unit enzyme activity.
  • the present invention also found that the commercial high-temperature resistant enzyme Hi-T7NEB and the high-temperature resistant M3 enzyme derived from CN102220294B cannot be used in the transcription condition system of the present invention. to produce sufficient transcripts. Therefore, we believe that the type of fusion protein and the type of mutation have a very important impact on improving the unit enzyme activity of T7 enzyme. Only by finding the best combination can we truly significantly improve the enzyme unit activity under normal temperature transcription conditions.
  • the only combination that can truly significantly improve T7 unit enzyme activity is a series of T7 variant proteins that are fused with M4 mutations or M4Plus mutations (ie, M5 and M6 mutations) of Sul7D family proteins. This shows that the highly thermostable mutant T7 enzyme does not all show a significant increase in unit enzyme activity after fusion with the DNA domain.
  • the fusion and mutation combination discovered in the present invention is a unique performance.
  • the transcription reactions of all T7 enzyme variants in the present invention are at the same enzyme protein concentration, that is, the final concentration of enzyme protein in the reaction system is 0.01 ⁇ g/ ⁇ L, and the transcription speeds shown at all temperatures of 25-45 degrees and transcription yield are the highest.
  • Example 7 DNA-binding domain RNA polymerase improves enzyme activity in high-salt environment
  • Example 8 Fusion of mutant RNA polymerase to improve DNA transcription template sensitivity and its diagnostic application
  • the present invention compares HF-T7-M4/M5/M6 with commercial enzymes, and dilutes the S gene DNA transcription DNA template of the new coronavirus COVID19 2 times for detection.
  • the fusion mutant RNA polymerase designed in the present invention significantly improves the sensitivity to RNA transcription templates.
  • the transcription-mediated detection signal of the engineered T7 RNA polymerase is 2-4 times that of the wild-type commercial T7 enzyme control, or the time required to generate the same signal value is significantly shortened. Therefore, we believe that the fusion mutant RNA polymerase of the present invention is of great significance in improving the sensitivity and efficiency of RNA transcription-mediated diagnostic methods.
  • Example 9 Effects of DNA-binding domain fusion with different RNA polymerases on unit enzyme activity
  • the inventors In addition to T7 RNA polymerase, the inventors also constructed and expressed fusion proteins of Sac7d DNA fusion domain and T3, sp6 and sny5 RNA polymerase, respectively detected their enzyme activities and compared them with wild-type enzyme activities.
  • the enzyme activity of each fusion RNA polymerase Both can reach 2-2.5 times that of wild-type RNA polymerase (Table 7).
  • the added amount of 10 ⁇ IVT Buffer is 1.5 ⁇ l
  • the added amounts of ATP, CTP, GTP, and Pseudo-UTP are all 3 ⁇ l
  • the added amount of cap analog GAG is 2.4 ⁇ l
  • the added amount of MgCl2 (1.5M) is 0.4 ⁇ l
  • the added amount of template is 1.5 ⁇ g
  • the added amount of RI (200U/ ⁇ l) is 0.15 ⁇ l
  • Ippase (0.6U/ ⁇ l ) was added in an amount of 0.03 ⁇ l
  • T7 RNA polymerase (1KU/ ⁇ l) was added in an amount of 1.5 ⁇ l
  • H 2 O was added to 30 ⁇ l.
  • the research variables in this experiment include the type of enzyme, NTP concentration, and NTP/GAG ratio.
  • the types of enzymes include: WT T7 enzyme, M4 and M5; NTP concentrations include 10mM, 7.5mM, 5mM; NTP/GAG ratios include (5:4), (5:3), (5:2), ( 5:1), (10:1).
  • WT T7 enzyme M4 and M5
  • NTP concentrations include 10mM, 7.5mM, 5mM
  • NTP/GAG ratios include (5:4), (5:3), (5:2), ( 5:1), (10:1).
  • RNA polymerase M5 sets the concentration of each NTP to 10mM, adjust the concentration of GAG and the amount of RNA polymerase added, and use commercially available RNA polymerase as a comparison to verify the transcription effect of M5.
  • the transcription yield is shown in Table 11 and Figure 11.
  • M5 is used for co-transcription.
  • the GTP/GAG ratio is 5:1 and 10:1
  • the transcription yield does not decrease significantly, reaching more than 7 mg/ml.
  • Roche's enzyme transcription yield is only is 1.1 mg/ml; the transcription yield corresponding to the nearshore enzyme is even lower, only 0.39 mg/ml.
  • RNA polymerase M5 still has a high transcription yield.
  • the agarose gel electrophoresis results of the transcript products are shown in Figure 12.
  • the electrophoresis graph shows that at the same loading volume, M5 has the highest transcription yield and no obvious non-specific bands.

Abstract

本发明公开了一种RNA聚合酶融合蛋白及其应用。其中所述RNA聚合酶融合蛋白包括：(i)依赖于DNA模板的噬菌体RNA聚合酶；(ii)非特异性的DNA结合域多肽和/或RNA结合域多肽；其中，所述RNA聚合酶融合蛋白比所述噬菌体RNA聚合酶具有更高的单位酶活性。

Description

一种RNA聚合酶融合蛋白及其应用
本申请要求申请日为2022/6/2的中国专利申请2022106297834的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明属于生物技术领域，具体涉及一种高活性的RNA聚合酶融合蛋白及其在RNA合成中的应用。
背景技术
RNA聚合酶，也称为“DNA依赖性RNA聚合酶”，具有独特的单酶驱动转录的分子机制，其能够将DNA转录为RNA转录物。RNA聚合酶已被广泛表达、分离和纯化，大量应用于体外生产RNA。体外转录是一种允许DNA指导合成任何序列的RNA分子的程序，其RNA产物大小从短寡核苷酸到几千碱基不等。通常，体外转录涉及模板的工程化，该模板包括感兴趣序列上游的噬菌体启动子序列(例如，来自T7大肠杆菌噬菌体)，然后使用相应的RNA聚合酶进行转录。通常，这些RNA转录本随后会被修饰(例如，通过加帽、剪接、添加poly-A尾巴等)。这些转录本用于分析技术(例如杂交分析)、结构研究(例如NMR和X射线晶体学)、生化和遗传研究(例如，作为反义试剂)、功能分子(例如，核酶和适体)，并作为治疗剂(例如，RNA疫苗)。
SP6、T7、T3、syn5、KP34、VSW-3等噬菌体RNA聚合酶对其各自的23个碱基启动子具有高度特异性。含有这些聚合酶启动子的克隆载体的开发使单链RNA分子的体外合成成为常规的实验室程序，其中T7RNA聚合酶使用最为广泛。
T7RNA聚合酶是一种来自T7噬菌体的RNA聚合酶，可催化从DNA沿5'→3'方向形成RNA。T7聚合酶具有极强的启动子特异性，仅转录T7启动子下游的DNA，其单位模板转录产量已经达到比较高的水平。由于应用范围的差异，T7RNA聚合酶已经有了很多的突变体、融合酶类型。其主要体现在诊断学应用需求，例如，T7RNA耐高温突变体可以提高提升GC含量高的RNA的转录；提高共转录帽子类似物RNA产物的加帽效率；提高长片段。上述各种突变或融合的本质是通过突变提高T7酶的热稳定性，表现为在高温的条件下，例如45-55℃条件下使T7保持较高的酶活。实际情况下，绝大多数突变体在高温下主要起到延缓T7酶酶活下降的作用，在高温下虽可见明显比野生型酶活要高，但与野生型T7酶37℃下的单位酶活并未见显著提升。
噬菌体RNA聚合酶作为单一亚基的DNA依赖的RNA聚合酶，尤其是T7酶在诊断学、mRNA疫苗、mRNA基因治疗中都有广泛的应用，除了个别特殊情景下，绝大多数应用场景仍然是在室温25℃至中温45℃的体系下催化生产目标RNA。鉴于上述原因，如何提高25-45℃中温正常反应体系下的蛋白单位酶活、耐盐、高灵敏度、快速反应特征是一种非常大的潜在生产需求。
一方面，很多等温扩增相关的检测发生在37℃-42℃的条件下，如TMA(转录介导的扩增技术)、NASBA(核酸依赖性扩增检测技术)、SHERLOCK(转录介导的CRISPR检测技术)等基于转录和反转录的横等温扩增，增加RNA聚合酶酶单位活性、速度、耐盐等特征无疑可以大大提高反应的灵敏度，降低检测限，还可以大幅减少酶的使用量从而降低成本。
另一方面，RNA聚合酶还广泛应用于mRNA疫苗、mRNA基因治疗药物的生产，这些生产绝大多数也是RNA聚合酶在介于于37-45℃的条件下进行转录反应。mRNA疫苗——也称为“基因疫苗”——源于一种创新的生物技术方法，该方法将人体细胞转变为分子工厂，以产生激活病原体特异性免疫反应的蛋白质。这种创新的mRNA原料药的生产过程始于进行生化反应的mRNA转录合成。mRNA疫苗生产的体外转录反应始于将所有反应组分添加到生物反应器中，其主要成分包括：核苷酸、线性模板、T7RNA聚合酶、核糖核酸酶抑制剂、转录反应缓冲液等。在这个约2-6小时完成的反应中，T7RNA聚合酶按照线性模板dsDNA(例如SARS-CoV-2病毒的刺突蛋白)的序列将ATP、UTP、CTP、GTP四种核苷酸连接在一起形成目标RNA。
鉴于国内外mRNA医学及RNA诊断产业化崛起，对mRNA生产的工业化产能放大需求日趋迫切，其中噬菌体RNA聚合酶尤其T7RNA聚合酶是mRNA生产中的最核心原料，无论是其酶活性能，还是生产过程中的单位成本都对mRNA工业生产放大产生重大影响。本发明基于此目的对此类噬菌体RNA聚合酶进行工程化改造，提高T7RNA聚合酶的单位酶活，转录速度、耐盐活性等生物学性状，减少mRNA工艺生产中的单位使用剂量，达到增产降成本的综合目的。
专利CN1720324B公开了一种改善PCR性能的SSO7-聚合酶偶联蛋白质，该专利描述了将非特异性双链核酸结合域Sso7d或Sac7d与DNA聚合酶催化结构域相连接从而改进DNA聚合酶活性，其中非特异双链核酸结合域选自Sso7d或Sac7d，其应用场景为高温条件下的DNA聚合酶链反应即PCR。在该专利中未见任何涉及依赖DNA模板的RNA聚合酶融合蛋白描述。
US20030175738A1公开了一种耐高温的突变型RNA聚合酶，这种突变聚合酶能够 达到最高耐受温度50℃条件下实现依赖于DNA模板的RNA转录合成，在37℃条件下组合突变T7RNA聚合酶的单位活性(units/μg)仅提高了36％左右。US9540670B2在上述专利申请的基础上公开了一种T7RNA聚合酶突变体组合，该组合突变包含了US20030175738A1中的耐高温突变点和一个新增突变点，这种组合突变的T7RNA聚合酶在37℃条件下的酶单位活性最多也仅提高了48％左右。上述专利或专利申请中没有任何融合型RNA聚合酶蛋白的描述。
CN108779446A也公开了一种T7RNA聚合酶的耐热突变体，这种突变体只在高温42℃以上表现出比野生型T7RNA聚合酶更高的单位活性，同时该专利中公开了融合了DNA结合域Sso7d结构域的RNA聚合酶的实施例，但是只局限于42℃以上的应用，且对原有突变RNA聚合酶的酶活无显著提升，在37℃条件下的单位酶活情况则无任何涉及。本领域内技术人员周知，绝大多数耐热酶随温度上升都会出现明显的单位酶活下降，专利中所描述的相对于野生型更高的酶活性，实际上应该描述为其酶活随温度提高时降低幅度更慢，从而在高温下表现出一定的更高活性，这种酶单位活性的改善更多的也是应用于高温转录或检测应用场景，在常温下的转录活性并无非常显著的优势。
JP2017178804A专利公开了一种AlbA融合DNA聚合酶的方案，利用AlbA的DNA结合域提高DNA聚合酶在PCR反应中的盐耐受性，该专利不涉及到Alba蛋白的RNA结合域特征，也没有涉及到RNA聚合酶的应用，只关注于这些蛋白的耐高温性质和PCR应用。
美国专利US20130022980公开了一种耐高温的非特异RNA结合域融合具有反转录活性的DNA聚合酶应用，同时还公开了将DNA结合域部分多肽和RNA结合域部分保守性多肽串联的嵌合型多肽，专利中所公开的此类蛋白属于Csp蛋白家族，但仅局限于于耐高温特征的Csp蛋白在PCR或反转录cDNA中的应用，不涉及到体外转录RNA的应用，因为体外转录不涉及到耐高温特征，也不涉及到耐盐和抗转录中止特征。
上述关于DNA/RNA结合蛋白的专利表明本技术领域内，对Sul7d家族、Alba等家族蛋白融合聚合酶的设计都集中应用在高温的应用场景中，包括需要高温介导的DNA链扩增反应即PCR、涉及高温转录和反转录的核酸聚合酶。上述专利还表明目前用于融合聚合酶的Sul7d家族蛋白主要为耐90度高温的Sso7d等DNA结合域蛋白，几乎没有对其它嗜中温的古细菌Sul7d家族蛋白进行利用和设计。
发明内容
为解决现有技术存在RNA聚合酶的酶活单位偏低、转录速度慢，而更多的酶用量带 来了污染风险等缺点，本发明提供了一种RNA聚合酶融合蛋白及其应用。具体地，RNA聚合酶的酶活单位偏低，导致生产上模板和聚合酶的用量偏高，提升了DNA模板和酶用量导致生产成本提高不利于工业化生产mRNA。此外，酶用量更多的情况下会带来更多的RNase、宿主DNA、宿主蛋白等污染风险，同时增加RNA生产下游纯化去除酶蛋白步骤的复杂度，例如在高浓度蛋白下加入有机溶剂更容易导致蛋白沉淀，更多的蛋白副产物还导致纯度色谱柱的载量占位效应等。
而且，现有技术RNA聚合酶介导的转录合成体系还存在一些缺点，例如目前RNA聚合酶的转录速度还不够快，导致转录反应时间比较长，一般都要4-6小时，甚至更长时间需要过夜反应。例如长链RNA转录过程中存在二级结构导致中断；例如为了得到更高的单位体积RNA产量需要在更高的模板浓度或NTP浓度反应条件下进行体外转录反应，但上述条件经常会带来更高的盐浓度效应抑制RNA聚合酶的转录活性，因此需要工程化RNA聚合酶减少这种抑制效应的存在；例如为了得到更少的dsRNA非特异性转录污染物，需要在更高的盐浓度进行体外转录反应，需要提升野生型RNA聚合酶的盐耐受浓度等。
为解决上述技术问题，本发明的第一方面提供了一种RNA聚合酶融合蛋白，其包括：
(i)依赖于DNA模板的噬菌体RNA聚合酶；
(ii)非特异性的DNA结合域多肽和/或RNA结合域多肽；
其中，所述RNA聚合酶融合蛋白比所述噬菌体RNA聚合酶具有更高的单位酶活性。
本发明在RNA聚合酶的基础上，融合一段DNA和/RNA结合域，这段结合域不仅可以更牢固的结合模板dsDNA，还可以更好的吸附转录合成的新RNA合成链，因此可以更好的提升转录的速度和稳定性，其表现形式为酶单位活性的提高或转录速度的提高。所述更高的单位酶活性，在本发明中通常指与野生型RNA聚合酶相比具有至少提高1倍的活性。
在一些优选的实施例中，所述噬菌体RNA聚合酶的氨基酸序列如SEQ ID NO:1～6所示，分别为T7RNA聚合酶、T3RNA聚合酶、SP6RNA聚合酶、Syn5RNA聚合酶、KP34RNA聚合酶和VSW3RNA聚合酶，或与其具有至少80％、90％、95％、96％、97％、98％或99％序列同一性的突变体中的一种或多种。
在一些优选的实施例中，所述DNA结合域多肽和/或RNA结合域多肽融合于所述噬菌体RNA聚合酶的N端；和/或，所述DNA结合域多肽和/或RNA结合域多肽与所述噬菌体RNA聚合酶之间通过连接子连接。
本领域技术人员应知，采用现有技术已公开的任何柔性连接子可以替代本发明中所 使用的连接子，且不影响融合蛋白的功能提升。在一些实施方案中，亚基通过肽接头连接子，例如Chen等人Adv Drug Deliv Rev 2013中所述。在一些实施方案中，多肽中的两个亚基由刚性连接器连接。在一些实施方案中，刚性接头由n个EAAAAK(SEQ ID NO:7)基序组成，即表示为(EAAAAK)n。n为0～10的自然数。在其他实施方案中，多肽中的两个亚基通过柔性接头连接。在一些实施方案中，柔性接头由基序(Gly)n组成。在一些实施方案中，柔性接头由n个GGGGS(SEQ ID NO:8)基序组成，即表示为(GGGGS)n。n为0～10的自然数。在一些实施方案中，刚性或柔性接头由长度为1、2、3、4、5、10、15或更多个氨基酸组成以实现RNA转录。在一些实施方案中，接头由刚性和柔性接头基序的组合组成。
在一些优选的实施例中，所述突变体在如SEQ ID NO:1～6所示的氨基酸序列上具有选自以下的一个或多个氨基酸取代：S430P、S633P、F849I、F880Y、P266L和Q744R/L/P。Q744R/L/P表示在744位上由R、L或P取代了Q。
优选地，所述突变体包括S430P、S633P、F849I和F880Y突变。
更优选地，所述突变体还包括Q744R和/或P266L突变。
进一步更优选地，所述突变体包括S430P、S633P、Q744R、F849I和F880Y突变，或P266L、S430P、S633P、Q744R、F849I和F880Y突变。
本领域内技术人员难以预测到Sul7d作为古细菌来源的dsDNA结合蛋白，在常温下也有dsDNA结合并显著促进RNA聚合酶的转录效果。因此，在一些优选的实施例中，所述DNA结合域多肽选自源自古细菌(Archaea)的Sul7d蛋白家族中的一种或多种。
分子生物学中，冷休克结构域(CSD)是一个包含约70个氨基酸的蛋白质结构域，已在原核和真核DNA结合蛋白中发现。该结构域的一部分与RNP-1RNA结合基序高度相似。序列分析表明，有一个由8个氨基酸组成的短基序，对应于典型RNA结合结构域中发现的RNP-1基序，这对两个明显不相关的蛋白质家族来说很常见。许多RNA结合蛋白在RNA结合域中包含RNP-1和RNP-2基序。冷休克域(CSD)蛋白质家族，包括已显示与DNA结合的几种转录因子，现已被鉴定包含类似于RNP-1的基序，搜索发现含有CSD的蛋白质家族包括一个与RNP-1基序几乎相同的基序。
因此，在一些优选的实施例中，所述RNA结合域多肽选自冷休克结构域CSD(PF00313)家族蛋白的一种或多种，优选选自氨基酸序列分别如SEQ ID NO:15、16、20-23所示的大肠杆菌、嗜盐古菌Halobacteriales archaeon、Halobacterium salinarum、Thermotoga maritima的冷休克结构域CSD，及与所述氨基酸序列具有至少80％、90％、95％、96％、97％、98％或99％同一性的突变体中的一种或多种。
在一些优选的实施例中，所述的DNA结合域多肽和/或RNA结合域多肽为源自古细菌(Archaea)的DNA/RNA结合蛋白albA家族蛋白(PFAM01918)，优选选自但不局限于嗜酸热硫化叶菌Sulfolobus acidocaldarius的DNA/RNA结合域Sac7D(SEQ ID NO:9)、憎叶菌属的SEQ ID NO:10、冰岛硫化叶菌的SEQ ID NO:11、硫磺矿硫化叶菌的SEQ ID NO:12、生金球形菌属SEQ ID NO:13、嗜酸两面菌的SEQ ID NO:14、大肠杆菌的CspA(SEQ ID NO:15)和CspE(SEQ ID NO:16)，或与所述氨基酸序列具有至少80％、90％、95％、96％、97％、98％或99％同一性的突变体中的一种或多种。
在一些更优选的实施例中，所述RNA聚合酶融合蛋白包括：
(i)依赖于DNA模板的噬菌体RNA聚合酶；
(ii)非特异性的DNA结合域多肽和/或RNA结合域多肽；
其中，(i)中，所述噬菌体RNA聚合酶的氨基酸序列如SEQ ID NO:1所示；或在如SEQ ID NO:1所示的氨基酸序列上具有S430P、S633P、F849I和F880Y突变，S430P、S633P、Q744R、F849I和F880Y突变，或P266L、S430P、S633P、Q744R、F849I和F880Y突变。
所述噬菌体RNA聚合酶的氨基酸序列如SEQ ID NO:2所示；或在如SEQ ID NO:2所示的氨基酸序列上具有S430P、S633P、F849I和F880Y突变，S430P、S633P、Q744R、F849I和F880Y突变，或P266L、S430P、S633P、Q744R、F849I和F880Y突变。
所述噬菌体RNA聚合酶的氨基酸序列如SEQ ID NO:3所示；或在如SEQ ID NO:3所示的氨基酸序列上具有S430P、S633P、F849I和F880Y突变，S430P、S633P、Q744R、F849I和F880Y突变，或P266L、S430P、S633P、Q744R、F849I和F880Y突变。
所述噬菌体RNA聚合酶的氨基酸序列如SEQ ID NO:4所示；或在如SEQ ID NO:4所示的氨基酸序列上具有S430P、S633P、F849I和F880Y突变，S430P、S633P、Q744R、F849I和F880Y突变，或P266L、S430P、S633P、Q744R、F849I和F880Y突变。
所述噬菌体RNA聚合酶的氨基酸序列如SEQ ID NO:5所示；或在如SEQ ID NO:5所示的氨基酸序列上具有S430P、S633P、F849I和F880Y突变，S430P、S633P、Q744R、F849I和F880Y突变，或P266L、S430P、S633P、Q744R、F849I和F880Y突变。
所述噬菌体RNA聚合酶的氨基酸序列如SEQ ID NO:6所示；或在如SEQ ID NO:6所示的氨基酸序列上具有S430P、S633P、F849I和F880Y突变，S430P、S633P、Q744R、F849I和F880Y突变，或P266L、S430P、S633P、Q744R、F849I和F880Y突变。
(ii)中，所述DNA结合域多肽的氨基酸序列如SEQ ID NO:9～14任一所示。
在一些最优选的实施例中，所述RNA聚合酶融合蛋白包括：
(i)依赖于DNA模板的噬菌体RNA聚合酶；
(ii)非特异性的DNA结合域多肽和/或RNA结合域多肽；
其中，(i)中，所述噬菌体RNA聚合酶的氨基酸序列如SEQ ID NO:1所示；或在如SEQ ID NO:1所示的氨基酸序列上具有S430P、S633P、F849I和F880Y突变，S430P、S633P、Q744R、F849I和F880Y突变，或P266L、S430P、S633P、Q744R、F849I和F880Y突变；(ii)中，所述DNA结合域多肽的氨基酸序列如SEQ ID NO:9所示。
或，(i)中，所述噬菌体RNA聚合酶在如SEQ ID NO:2所示的氨基酸序列上具有S430P、S633P、Q744R、F849I和F880Y突变；(ii)中，所述DNA结合域多肽的氨基酸序列如SEQ ID NO:10所示。
或，(i)中，所述噬菌体RNA聚合酶在如SEQ ID NO:3所示的氨基酸序列上具有S430P、S633P、Q744R、F849I和F880Y突变；(ii)中，所述DNA结合域多肽的氨基酸序列如SEQ ID NO:11所示。
或，(i)中，所述噬菌体RNA聚合酶在如SEQ ID NO:4所示的氨基酸序列上具有S430P、S633P、Q744R、F849I和F880Y突变；(ii)中，所述DNA结合域多肽的氨基酸序列如SEQ ID NO:12所示。
或，(i)中，所述噬菌体RNA聚合酶在如SEQ ID NO:5所示的氨基酸序列上具有S430P、S633P、Q744R、F849I和F880Y突变；(ii)中，所述DNA结合域多肽的氨基酸序列如SEQ ID NO:13所示。
或，(i)中，所述噬菌体RNA聚合酶在如SEQ ID NO:6所示的氨基酸序列上具有S430P、S633P、Q744R、F849I和F880Y突变；(ii)中，所述DNA结合域多肽的氨基酸序列如SEQ ID NO:14所示。
为解决上述技术问题，本发明的第二方面提供了一种生物材料，其选自以下组：
(1)分离的核酸，其编码如本发明的第一方面任一项所述的RNA聚合酶融合蛋白；
(2)重组表达载体，其包含如(1)中的分离的核酸；
(3)转化体，其包含如(2)中的重组表达载体。
为解决上述技术问题，本发明的第三方面提供了一种组合物，其包括如本发明第一方面任一项中所述的RNA聚合酶融合蛋白。
优选地，还包括缓冲剂、修饰或无修饰的核糖核苷三磷酸、和帽子类似物的一种或多种。所述无修饰的核糖核苷三磷酸为ATP、CTP、UTP、GTP；修饰的核糖核苷三磷酸选自假尿苷三磷酸()、N1-甲基-假尿苷三磷酸(m1)、2-氨基-6-氯嘌呤核糖核苷5'三磷酸、2-氨基腺苷5'三磷酸、2-硫胞苷5'三磷酸、2-硫尿苷5'三磷酸、4-硫尿苷3'三磷酸， 5-氨基烯丙基胞苷5'三磷酸、5-氨基烯丙尿苷5'三磷酸、5-溴胞苷5'三磷酸，5-溴尿苷5'三磷酸、5-碘胞苷5'三磷酸、5-碘尿苷5'三磷酸、5-甲基胞苷5'三磷酸、5-甲基尿苷5'三磷酸、6-氮杂胞苷5'三磷酸、6-氮杂尿嘧啶5'三磷酸、6-氯尿苷5'三磷酸、7-脱氮腺苷5'三磷酸、7-去氮鸟苷5'三磷酸、8-氮杂腺苷5'三磷酸，8-叠氮腺苷5'三磷酸、苯并咪唑核苷5'三磷酸、N1甲基腺苷5'三磷酸、N1甲基鸟苷5'三磷酸、N6甲基腺苷5'三磷酸、O6甲基鸟苷3'三磷酸、嘌呤霉素腺苷5'三磷酸和黄氨酸腺苷5'三磷酸。
用于本申请的术语“缓冲剂”是指使得溶液在被加入酸或碱时能够抵抗pH变化的试剂。可用于本发明的组合物、试剂盒和方法的合适的非天然存在的缓冲剂的实例包括例如：Tris、HEPES、TAPS、MOPS、tricine和MES中的一种或多种。
用于本申请的术语“帽子(cap)”指在RNA分子5'端发现的帽子结构，通常由一个鸟苷核苷酸组成，该核苷酸通过一个不寻常的5'-5'三磷酸键与信使核糖核酸连接。术语“帽子类似物(cap analogs)”是指与帽子结构类似，具有稳定RNA和/或增强RNA翻译的能力，可以通过加帽酶或共转录的方式连接到mRNA上。所述帽子类似物包括GAG、GAU、ARCA、GGG等(http://www.syngenebio.com/product/37/)。
为解决上述技术问题，本发明的第四方面提供了一种试剂盒，其包括如本发明第一方面任一项中所述的RNA聚合酶融合蛋白。
优选地，还包括缓冲剂、修饰或无修饰的核糖核苷三磷酸、和帽子类似物的一种或多种。
所述缓冲剂包括：Tris、HEPES、TAPS、MOPS、tricine和MES中的一种或多种。
为解决上述技术问题，本发明的第五方面提供了一种合成RNA分子的方法，包括温育混合物以将模板DNA分子转录成RNA，所述混合物包含本发明第一方面任一项所述的RNA聚合酶变体融合蛋白或本发明第三方面任一项所述的组合物以及模板DNA分子。
在一些优选的实施例中，所述温育在25-45℃，例如25℃、37℃或42℃的温度下进行。
在一些优选的实施例中，所述转录在0.05～0.2M的盐浓度下进行；
更优选地，所述盐为NaCl，所述盐浓度不高于0.1M。在同等转录条件下，额外增加不高于0.1M的离子浓度，所述盐选自但不局限于Na离子、K离子、Li离子、Tris离子等，融合突变RNA聚合酶单位酶活显著优于非融合突变RNA聚合酶。
在一些优选的实施例中，所述混合物还包含修饰或无修饰的核糖核苷三磷酸。
在一些优选的实施例中，所述混合物还包含帽子类似物，实现共转录加帽。较佳地，所述帽子类似物的浓度≤8mM，例如8mM、6mM、4mM、2mM或1mM。本发明的 RNA聚合酶融合蛋白可以在较低浓度(例如最低1mM)的帽子类似物条件下，保持较高的共转录产量，且加帽效率、纯度、胞内表达量也没有显著降低。
为解决上述技术问题，本发明的第六方面提供了一种如第一方面任一项所述的RNA聚合酶融合蛋白、如第二方面所述的生物材料或如第三方面所述的组合物在制备扩增RNA的试剂中或RNA转录介导的诊断和/或体外翻译试剂盒的应用。
为解决上述技术问题，本发明的第七方面提供了一种如第一方面任一项所述的RNA聚合酶融合蛋白在制备中温例如25-42℃条件下具有高活性的酶制剂中的应用。本发明的高活性指与野生型RNA聚合酶相比，具有至少提高1倍的活性。
为解决上述技术问题，本发明的第八方面提供了一种核酸诊断方法，其包括核酸转录和核酸确定的步骤；其中，所述核酸扩增使用如第一方面任一项中所述的RNA聚合酶变体融合蛋白、第三方面所述的组合物或第四方面任一项所述的试剂盒对核酸模板进行转录或扩增；所述核酸确定使用测序的方法例如Sanger测序或者NGS测序进行。
优选地，所述核酸诊断方法为非疾病诊断目的的方法，例如在实验室中确定具体使用的核酸。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于：
(1)本发明的RNA聚合酶融合蛋白的活性明显增加；例如7kd DNA结合域融合RNA聚合酶的转录酶单位活性显著提高1-6倍。
(2)在同等转录体系下，具有更高的盐耐受性，在额外添加不大于0.1M NaCl情况下，融合突变型RNA聚合酶单位酶活提高至非突变融合酶的1-6倍。
(3)融合突变型RNA聚合酶在更低的DNA模板条件下表现出更好的灵敏度。
(4)同等转录条件下，融合突变型RNA聚合酶转录速度和效率提高至1-6倍。
(5)同等转录条件下，融合突变型RNA聚合酶转录RNA产物产量更高。
(6)融合突变型RNA聚合酶在较低浓度的帽子类似物条件下，保持较高的共转录产量，且加帽效率、纯度、胞内表达量也没有显著降低。
附图说明
图1为本发明RNA聚合酶融合蛋白结构示意。
图2为Sul7d保守序列和高度保守序列示意图。
图3A为部分RNA聚合酶或突变体及其融合蛋白纯化PAGE鉴定结果。注：1:Roche-T7；2:WT-T7；3:M4-T7；4:HF-T7；5:HF-T7-M4；6:HF-T7-M5；7:HF-T7-M6；8:Sso7d-T7-M5；9:Saz7d-T7-M5；10:Sis7d-T7-M5；11:RCPA-T7-M5；12:RCPB-T7-M5；13:RCP1-T7-M5；14:RCP2-T7-M5。
图3B为部分RNA聚合酶或突变体及其融合蛋白纯化PAGE鉴定结果。其中，1:Roche-T7；2:WT-T7；3:HF-T7；4:HF-T7-M4；5:HF-T7-M5；6:HF-T7-M3；7:HF-T7-M2+M3-1；8:NEB007M6。
图4A为荧光实时定量检测酶活方法的标准曲线各浓度点的荧光信号变化。
图4B为荧光实时定量检测酶活方法的60min标准曲线线性关系图。
图5A为Sac7d融合域T7酶及其对照酶活实时定量荧光值25℃。
图5B为Sac7d融合域T7酶及其对照酶活实时定量荧光值37℃。
图5C为Sac7d融合域T7酶及其对照酶活实时定量荧光值45℃。
图6A为不同工程化T7RNA聚合酶及其对照品酶实时转录RNA配体结合DHFI荧光信号值。注：1:Roche-T7；2:WT-T7；3:M4-T7；4:HF-T7；5:HF-T7-M4；6:HF-T7-M5；7:HF-T7-M6；8:HF-M5-8aa；9:RCPA-T7-M5；10:NEB007M6。
图6B为不同工程化T7RNA聚合酶及其对照品酶单位酶活对比。注：1:Roche-T7；2:WT-T7；3:M4-T7；4:HF-T7；5:HF-T7-M4；6:HF-T7-M5；7:HF-T7-M6；8:HF-M5-8aa；9:RCPA-T7-M5；10:NEB007M6。
图6C为不同工程化T7RNA聚合酶蛋白及其对照品酶转录长链4500nt RNA的琼脂糖凝胶电泳图，反映出不同酶的转录速度、单位产量和产物RNA纯度。注：1:Roche-T7；2:HF-T7；3:HF-T7-M4；4:HF-T7-M5；5:HF-T7-M6；6:WT-HF；7:M4-T7；8:RCPA-T7-M5。
图7为不同工程化T7RNA聚合酶蛋白及其对照品酶转录长链4500nt RNA的毛细管电泳图，反映出不同酶的转录速度、单位产量和产物RNA纯度。注1：HF-T7-M5＞HF-T7-M4＞HF-T7-M6＞M4-T7＞HF-T7＞Roche-T7＞RCPA-T7；注2：45℃4小时由于RNA浓度过高影响毛细管电泳效果，故将所有样品进行5倍稀释后再进行上样。
图8A为不同工程化T7RNA聚合酶蛋白及其对照品酶37℃转录RNA配体实时荧光值对比。
图8B为不同工程化T7RNA聚合酶蛋白及其对照品37℃单位酶活值对比。注：1:WT-T7；2:HF-T7；3:HF-T7-M5；4:Sso7d-T7-M5；5:Saz7d-T7-M5；6:Sis7d-T7-M5；7: Met7d-T7-M5；8:Asul7d-T7-M5；9:RCPA-T7-M5；10:RCPB-T7-M5；11:RCP1-T7-M5；12:RCP2-T7-M5；13:NEB007M6。
图8C为不同融合突变型T7RNA聚合酶蛋白及其对照品酶点转录RNA琼脂糖凝胶电泳对比。注：1:Roche-T7；2:HF-T7-M5；3:NEB007M6；4:Hi-T7(NEB)；5:RCP2-T7；6:HF-T7-M3；7:HF-T7-M2+M3-1。
图8D为不同工程化T7RNA聚合酶蛋白及其对照品37℃时的产量。
图8E为不同融合突变型T7RNA聚合酶蛋白及其对照品酶融合蛋白45℃时的产量。
图9为不同工程化T7RNA聚合酶在额外添加0.1M NaCl条件下的实施定量荧光值。
图10A～10D为融合突变型RNA聚合酶显著提高了对RNA转录模板的灵敏度。
图11为M5和市售RNA聚合酶转录产量的比较。
图12为M5和市售RNA聚合酶转录产物的琼脂糖凝胶电泳图，从左至右的样品依次为：1.2ug-5:1、1.2ug-10:1、0.6ug-5:1、0.6ug-10:1、0.3ug-5:1、0.3ug-10:1。
具体实施方式
1)已有大量专利公开源自古细菌的7kd DNA结合域应用于DNA聚合酶融合表达，这种融合表达的蛋白能显著提升这些原有野生型DNA聚合酶的扩增速度和扩增长度，其表现形式既为酶单位活性的提高，但目前没有任何专利公开过7kd DNA结合域融合RNA聚合酶及在常温转录条件下的应用以及其对聚合酶活性的显著提升。已公开专利的不足之处：a)RNA聚合酶与DNA聚合酶的重大区别在于前者是在常温25-45℃体外转录条件下实现的RNA合成，而后者是在50-100℃PCR条件下的聚合酶链式反应DNA合成，行业内技术人员并没有能够预测到在低温和/或中温环境下，这种DNA结合蛋白也能在特定突变酶基础上起到促进RNA转录的作用。b)另一方面，融合DNA结合域多肽主要源自嗜热古细菌，主要也是应用其耐高温的特征，例如在先专利CN108779446A的主要应用点也是耐高温T7RNA聚合酶突变体的DNA结合域融合蛋白形式，然而在高温环境下这种融合蛋白对酶单位活性的提升并不明显，且该专利中仅展示了一种7kd DNA结合域SSO7D的单独实施例。本发明发现这些嗜热古细菌来源的DNA结合域融合RNA聚合酶后，在常温25-42℃体外转录条件下反而可以显著提升酶单位转录活性，尤其是如果这种DNA结合域蛋白与突变型RNA聚合酶融合后，其最高单位酶活性居然可以提高至 6倍，这是超乎预料的。
“序列非特异性DNA结合域”、“非特异性的DNA结合域”或“DNA结合域”是指与DNA结合而没有显着序列偏好的蛋白质域。非特异性的DNA结合域主要源自古细菌Archaea的7kd DNA结合域Sul7d蛋白家族(pfam02294)。在一些实施方案中，DNA结合结构域结合双链DNA。非限制性示例性DNA结合结构域包括来自Sulfolobus solfataricus的Sso7d、来自S.acidocaldarius的Sac7d、以及憎叶菌Stygiolobus azoricus、冰岛硫化叶菌Sulfolobus islandicus、硫磺矿硫化叶菌Sulfolobus solfataricus、生金球形菌Metallosphaera、嗜酸两面菌Acidianus sulfidivorans的DNA结合域蛋白(SEQ ID NO:9-14)。
2)本发明还发现古细菌中除了7kd DNA结合域，还存在albA蛋白家族不仅具有DNA结合域还兼具有RNA结合域，这段结合域不仅可以更牢固的结合模板dsDNA，还可以更好的吸附转录的新合成RNA链，避免二级结构转录中止等特征，因此可以更好的提升转录的速度和稳定性，其表现形式虽然没有显著体现出酶单位活性的提高，但可能对RNA的完整性有所帮助。
3)本发明还发现含有7kd DNA结合域、AlbA DNA/RNA结合域的古细菌虽然大多数情况下为嗜热细菌，但是其最适生存温度也有较大的变化，例如有嗜高温和嗜中温特征。酸菌属(Acidianus)、金属球菌属(Metallosphaera)、憎叶菌属(Stygiolobus azoricus)和硫化叶菌属(Sulfolobus)中的部分细菌适宜的生长温度在60～80℃，这部分细菌称为中等嗜热菌；还有部分细菌的适宜生长温度超过80℃，这部分细菌称为极度嗜热菌，极端嗜热古菌Thermococcus kodakarensis、Sulfolobus acidocaldarius。在先专利一般利用这些古细菌中的极度嗜热菌的DNA结合域从而适应PCR的高温环境。本发明中所使用的DNA/RNA结合域则有一部分来源于中等嗜热菌，例如部分AlbA蛋白热自养甲烷嗜热球菌温度生长范围是30～70℃，最适生长温度是65℃，海沼甲烷球菌、生长温度20～40℃，最适生长温度：35～39℃，这些蛋白并不符合在先专利的耐高温DNA结合域范畴。另一方面，这些些古细菌源自海洋例如海沼甲烷球菌、热自养甲烷嗜热球菌，其生存条件在高盐环境中，偶联该结构域可以有利于专利反应体系在高盐环境(例如同等转录条件下，额外添加0.1M NaCl)进行，这对合成高产和高质量的低dsRNA含量或减少冗余RNA转录产物的工业RNA生产有很大的裨益。
1)Sac7D嗜酸热硫化叶菌DNA-binding protein[Sulfolobus acidocaldarius]NCBI Reference Sequence:WP_011276993.1

2)Saz7d憎叶菌属DNA-binding protein Stygiolobus azoricus WP_156007647.1；
3)冰岛硫化叶菌DNA-binding protein[Sulfolobus islandicus]Sis7d WP_012953380.1
4)硫磺矿硫化叶菌Sso7d DNA-binding protein[Saccharolobus solfataricus]WP_009990119.1
5)生金球形菌属Metallosphaera Met7d WP_009071314.1
6)嗜酸两面菌DNA-binding protein Acidianus sulfidivorans Asul7d WP_110380358.1
7)大肠杆菌CspA(RCP)
8)大肠杆菌CspE(RCP2)
9)Ssh Alba：
10)MTH Alba：
11)Aenirpl:cold-shock protein[Halobacteriales archaeon]GenBank:MCA1820174.1
12)NNJ93774.1Halobacteria archaeon
13)TPA_inf:cold shock protein[Halobacterium salinarum NRC-1]GenBank:DAC77414.1
14)TmCspB(RCP3):cold-shock protein[Thermotoga maritima].WP_004082199
图1为本发明RNA聚合酶融合蛋白结构示意。
图2为Sul7d保守序列和高度保守序列示意图。
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
实施例1:7kd DNA结合域RNA聚合酶的克隆表达和纯化
将T7、SP6RNA等聚合酶ORF区域分别克隆到pQE-80L(Qiagen)中，获得重组质粒pQE-80L-T7、pQE-80L-SP6等。基因合成不同的DNA和/或RNA结合域基因序列。使用High-Fidelity DNA Polymerase(New England BioLabs)分别扩增出带有同源臂的重组质粒骨架结构与DNA和/或RNA结合域序列，用琼脂糖凝胶DNA回收试剂盒(天漠生物)纯化PCR产物，然后使用HieffPlus Multi One Step Cloning Kit(YEASEN)对纯化的PCR产物进行10μL连接反应，将DNA和/或RNA结合域构建到RNA聚合酶的N端。取5μL连接反应产物在TOP10大肠杆菌感受态细胞中进行转化。对于每个重组载体挑取3个单克隆进行培养，分别各取一半进行测序鉴定和保菌，对于鉴定序列正确的重组载体使用TIAN prep Rapid Mini Plasmid Kit(TIANGEN)进行质粒提取。对于突变体的构建，High-Fidelity DNA Polymerase(New England BioLabs)用于所有定点诱变,使用多个PCR反应诱变后，用琼脂糖凝胶DNA回收试剂盒(天漠生物)纯化PCR产物，使用HieffPlus Multi One Step Cloning Kit(YEASEN)对纯化的PCR产物进行10μL连接反应，取5μL连接反应产物在TOP10大肠杆菌感受态细胞中进行转化。对于每个突变体挑取3个单克隆进行培养，分别各取一半进行测序鉴定和保 菌，对于鉴定序列正确的突变体使用TIANprep Rapid Mini Plasmid Kit(TIANGEN)进行质粒提取。
实施例2：不同DNA/RNA结合域RNA聚合酶的克隆表达和纯化
质粒被转化到BL21(DE3)感受态细胞中。细胞在37℃下的2xYT培养基中过夜培养，传代培养物在37℃的新鲜2xYT培养基中，生长至OD600＝0.6-0.8时，加入1mM/L的IPTG进行蛋白诱导，4小时后结束诱导并离心收集菌体纯化。
表1.DNA/RNA结合域融合的T7RNA聚合酶信息表
注：表中克隆表达质粒均为PQE-80L，带有His标签，在BL21(DE3)中表达。
菌体在10倍体积的裂解缓冲液(PH 7.5的50mM PB,500mM NaCl,5mM咪唑,1mM PMSF)重悬后，进行高压均质破碎裂解(800bar，6分钟)。离心(13000rpm/min；30min) 后取上清液，采用固定化金属亲和色谱(IMAC)进行第一步纯化。上清液通过用结合缓冲液(PH 7.5的50mM PB,500mM NaCl,5mM咪唑)预平衡过的5ml Ni-NTA预装柱。10倍柱体积的缓冲液进行线性梯度洗脱(洗脱范围：PH 7.5的50mM PB,500mM NaCl,5mM咪唑的缓冲液—PH 7.5的50mM PB,500mM NaCl,300mM咪唑的缓冲液)，SDS-PAGE电泳鉴定后，合并可用的洗脱液进行离子交换层析进一步纯化。IMAC纯化洗脱液通过用结合缓冲液(PH8.0的50mM Tris-HCl,100mM NaCl)预平衡过的5ml SP Purose 6FF预装柱。10倍柱体积的缓冲液进行线性梯度洗脱(洗脱范围：PH8.0的50mM Tris-HCl,100mM NaCl的缓冲液—PH8.0的50mM Tris-HCl,500mM NaCl的缓冲液)，SDS-PAGE电泳鉴定后，合并可用洗脱液，得到最终蛋白。结果请参见图3A和图3B。
实施例3：T7RNA酶活检测方法的建立
本方法利用荧光染料与RNA结合后产生荧光的特性，通过监测荧光信号的变化反应RNA的产量，间接表征酶促反应的速率，并利用酶促反应速率对T7RNA聚合酶的酶活力进行评估。
根据参考文献中Spinach RNA序列，基因合成相应的含有T7RNA聚合酶启动子序列的iSpinach DNA模板，然后将iSpinach DNA序列克隆到pUC57质粒载体中，使用pUC57质粒载体通用引物M13的上下游引物扩增iSpinach-D5DNA转录模板，扩增DNA产物作为T7RNA聚合酶评价的酶促反应体系的DNA底物。
参考文献中的酶促反应体系，结合实际的评价体系筛选的条件，确定了活性检测转录条件：40mM Tris-HCl(pH8.0)，20mM MgCl2，5mM DTT，1mM Spermidine，10mM rNTP，16nmol/ml DFHBI，200pmol/ml iSpinach-D5DNA模板，RNase inhibitor 1KU/ml。
表2.T7酶酶活性评价体系
模板DNA使用量计算：以通常DNA浓度检测结果单位为ng/μl为例进行计算，体积(μl)＝20/{(C*1000)/M}＝(20*M)/(C*1000)，M为DNA模板分子量，M＝99980.7，C＝DNA模板检测浓度。
T7RNA聚合酶的酶活力定义，在含有20pmol的iSpinach DNA模板的100μl酶活性检测体系条件下，37℃反应60min后，每产生1pmol的iSpinach RNA定义为1U。iSpinach RNA模板分子量，M＝39600。
在本方法转录体系中T7酶不断地合成iSpinach RNA，iSpinach RNA会与DFHBI荧光染料结合，释放荧光信号。本研究可以使用已知浓度的iSpinach RNA标准参考品，来定量反应体系中RNA的产量，从而定量评估酶活力。本实施例中通过浓度梯度稀释来建立稳定准确的标准曲线来定量检测RNA产量。使用转录纯化的iSpinach RNA标准参考品，标准曲线起始浓度为60μg/100μl，进行2倍梯度稀释后标准曲线最低浓度为3.75μg/100μl，进行实时荧光检测。
RNA标准参考品反应体系配制参考如下，取出试剂置于室温，融化后涡旋混匀放于冰上备用。DFHBI为10μl/支的保存液，需进行50倍稀释后使用，DFHBI融化后加入490μl Nuclease-free-Water，涡旋混匀后备用。按下表配制酶促反应体系，按照每个样本配制两个复孔进行配制。
表3.iSpinach RNA标准曲线反应体系
配制完成后，每孔50μl加到96孔板中，上机前将RNA参考品加入到检测孔中。打开ELISA仪器，创建新程序，选择激发波长为490nm，发射波长为516nm，检测时间为60s/次，60个循环，保存备用，试验结果如图4A所示。
标准曲线各浓度点的荧光信号随时间的延长逐渐趋于稳定，高浓度有轻微的降低趋势，分析标准曲线60min时间点RNA浓度和荧光信号之间的关系，从图4B中能够看出，标准曲线线性关系较好，R2＝0.998，达到标准曲线的建立要求。标准曲线上限60μg/100μl在60min后荧光信号强度达到340000，能够覆盖样本最高的信号，满足对于高浓度酶的 酶活力定量检测。以上iSpinach RNA序列参考文献为Alexis Autour,Eric Westhof.iSpinach:a fluorogenic RNA aptamer optimized for in vitro applications.Nucleic Acids Research,2016,Vol.44,No.6 2491–2500。
实施例4：7kd DNA结合域Sul7DRNA聚合酶酶活检测
本实施例中比较了不同T7RNA聚合酶突变与不同DNA结合蛋白的组合设计、并比较了所有这些新的T7RNA聚合酶变体的在检测体系下的转录活性即荧光值，同时换算为单位质量下的酶活力单位。
DNA结合结构域和T7突变氨基酸说明如下：
T7M4：S430P/S633P/F849I/F880Y；
T7M5：S430P/S633P/F849I/F880Y/Q744R；
T7M6：P266L/S430P/S633P/F849I/F880Y/Q744R；
HF＝Sul7d家族蛋白中的Sac7d DNA结合域蛋白；
8aa＝T7M5删除167-174(EEQLNKRV,SEQ ID NO:19)8个氨基酸。
如图5A-5C所示，Sac7d DNA结合域融合的突变型T7RNA聚合酶(M4-6)，均表现出单独DNA结合域融合或单独突变型T7RNA聚合酶的转录活性，尤其是在37-45℃条件下，荧光值提高幅度非常显著。
表4.在不同温度下不同策略的突变RNA聚合酶融合蛋白及其对照品的RNA转录配体产物荧光值情况。
本发明中酶活的定义如下，在100uL含20pmol的DNA模板的酶促反应评价体系中，37℃反应1小时，每产生1pmol的122nt RNA产物量，定义为1Unit酶活力，通过标准RNA的荧光值可以计算出每种酶所对应的酶活单位。本发明分别用酶活单位和相同剂量酶的转录产物荧光强度来作为参数作图(图6A和图6B)体现。结果表明，发明人设计的融合型RNA突变聚合酶在各种温度下，都显著提升了单位酶活和/或提高了酶转录的速度。
以2000nt的PB mRNA DNA转录模板为试验对象，转录反应体系如下所示：40mM Tris-HCl(pH8.0)，20mM MgCl2，5mM DTT，1mM Spermidine，10mM rNTP，DNA模板0.1μg/μL，RNase inhibitor 1U/μL，IPPase 1U/μL,T7polymerase0.01μg/μL。
将不同温度和不同时间点的转录产物进行琼脂糖凝胶电泳，结果如图6C所示。结果表明Sac7d DNA结合域融合的突变型T7RNA聚合酶(M4-6)，均表现出单独DNA结合域融合或单独突变型T7RNA聚合酶的转录活性，该结果与上述荧光定量检测和酶活计算结果均高度吻合。
上述两种论证方法都能够说明我们开发的融合突变型T7RNA聚合酶在单位酶活和转录速度性能上都比野生型T7RNA聚合酶有非常显著的提升。单位酶活的提升意味着生产RNA时的酶用量减少可以降低生产成本；转录速度的提升意味着生产RNA时的时间成本降低减少了人工成本；酶用量的减少还意味转录体系中的酶蛋白含量减少可以降低下游纯化的难度和减少蛋白源杂质的带入。
实施例5：融合突变型T7RNA聚合酶酶对长链RNA的产量和纯度影响
以4500nt的Cas9mRNA转录DNA模板进行转录试验比较不同工程化酶的转录性能，转录反应条件与实施例4中相同，但本实施例中的转录反应体系中不添加无机焦磷酸酶，反应温度分别设置了37℃和45℃两种条件，在转录不同时间点吸取相同体积的转录产物进行毛细管电泳分析RNA产物的产量和纯度，其中45℃4小时转录产物因为RNA浓度太高，本发明进行了5倍的稀释后再进行毛细管凝胶电泳分析。DNA转录模板及其转录RNA产物序列参考Addgene数据库货号为Plasmid#71310的质粒信息。
如图7所示，结果表明，HF-T7-M4/M5/M6，无论是转录活性，RNA转录延伸速度上，总产量上都要显著优于单独融合型HF-T7酶和或非融合型M4突变体，更优于野生型对照组和/或商品对照组。
融合突变型T7RNA聚合酶单位酶活和RNA产量的显著提升表现为转录合成RNA的速度明显提高和/或终产量明显提高。优化后的融合突变RNA聚合酶在同等条件下2小时的转录产物产量远远超过野生型聚合酶4小时的转录产物产量，大大节约了反应时 间。
实施例6：不同DNA结合域和耐高温突变T7RNA聚合酶对单位酶活的影响
已有专利描述了若干不同的耐高温突变，这些突变体表明可以一定程度上提高T7酶的耐热稳定性，虽然这些突变体一定程度上延缓了因温度升高导致的活性下降的幅度，但基本上没有在25度-42度的相关酶活显著提高数据，有一些提到了在中高温情况下提高了稳定性或酶活的数据，但提高幅度很有限。例如NEB专利中只有42度以上的略微提高。鉴于此，本发明将这些已知的不同融合蛋白和不同突变进行组合形成了不同融合突变T7RNA聚合酶，进行了一系列表达、纯化和酶活对比。
本发明选取了HF-M3(Sac7D-V426L/A702V/V795I)和HF-M3-M2-1(Sac7D-V426L/S633P/A702V/V795I/Q744R)，上述两个融合突变T7酶组合中的氨基酸突变源自专利CN102220294B及其衍生突变体，如专利中所述这些突变可以提高其在50度温度下的热稳定性。
本发明还选取了NEB007M6融合突变T7酶即SS07-T7M-6M(CN108779446A专利中单位酶活最高的融合突变T7酶)I109L/H205S/D388E/L534V/V567P/G618Q，其中SS07源自高温神袍菌(007-T7)的lacI样蛋白。本发明还购买了NEB公司的商品化耐高温T7酶进行转录活性比较，Hi-T7NEB(货号为：M0658S)。
本发明还选取了Alba DNA结合域融合了M5突变(RCP-2-HF-T7:ssh10b-S430P/S633P/F849I/F880Y/Q744R)。
本发明还选取了Sul7d家族(Sac7d,Sso7d,Saz7d,Sis7d,Met7d,Asul7d)的其它成员进行突变T7酶的融合表达、纯化和酶活测试。
本发明的转录条件为40mM Tris-HCl(pH8.0)，20mM MgCl2，5mM DTT，1mM Spermidine，10mM rNTP，16nmol/ml DFHBI，200pmol/ml iSpinach-D5DNA模板，RNase inhibitor 1KU/ml；转录温度为37℃。
下表5显示了不同DNA结合域和耐高温突变T7RNA聚合酶对单位酶活的影响。
表5融合蛋白的单位酶活

如图8A和8B所示，Sul7d家族(Sac7d,Sso7d,Saz7d,Sis7d,Met7d,Asul7d)融合域均能显著提高酶活性，而Alba类融合蛋白则没有见到明显提高酶活性。本实施例还表明不是所有突变都可以显著提高酶活性(如图8C、8D和8E)，其中NEB007M6(SS07-T7M-6M)为突变型融合T7酶，虽然也表现出一定的单位酶活提升，但其提升幅度远远不如本发明的融合突变型T7酶方案；与此同时本发明还发现商品化耐高温酶Hi-T7NEB和源自CN102220294B的耐高温M3酶不能在本发明的转录条件体系下产生足够的转录产物。因此我们认为融合蛋白的类型和突变的类型对T7酶的单位酶活提高都具有非常重要的影响，只有找到最佳的组合才可以真正显著提高常温转录条件下的酶单位活力。
在本发明中，真正显著性提高T7单位酶活的组合只有Sul7D家族蛋白融合M4突变或M4Plus突变(即M5、M6突变)的系列T7变体蛋白。这表明热稳定高的突变体T7酶融合DNA结构域后，并不都表现出单位酶活的显著提高，本发明所发现的融合和突变组合是独特的表现。
本发明中的所有T7酶变体的转录反应都在相同的酶蛋白浓度下，即反应体系下0.01μg/μL终浓度酶蛋白，其在所有的25-45度温度下所表现出的转录速度和转录产量都是最高的。
实施例7：DNA结合域RNA聚合酶提高在高盐环境下的酶活
本实施例验证了设计的多种工程化融合突变型RNA聚合酶在高盐环境下即0.1M NaCl下对RNA转录酶单位酶活的影响(图9)，结果表明本发明中的HF-T7-M4/M5/M6 显著高于单独突变酶或单独融合野生型酶。
实施例8：融合突变RNA聚合酶提高DNA转录模板灵敏度及其诊断学应用
Sherlock诊断是一种基于核酸恒温扩增和cas13a介导的CRISPR RNA(crRNA)切割作用的体外核酸检测平台，该方法与体外转录过程结合，将DNA转为RNA，并通过Cas13a切割靶DNA产生非特异性RNase活性激活荧光探针信号，从而实现在高灵敏度的核酸检测，其中转录的关键酶为T7RNA聚合酶。本发明基于上述诊断学方法，通过RNase Alert检测试剂盒监测转录介导的Cas13产生的非特异性RNase信号，进而反映出不同工程化RNA聚合酶对转录模板的灵敏度影响。(本实验所需序列和具体实验步骤参考文献Gootenberg JS,Abudayyeh OO,Lee JW.Nucleic acid detection with CRISPR-Cas13a/C2c2.Science.2017Apr 28；356(6336):438-442.doi:10.1126/science.aam9321.Epub2017Apr 13.PMID:28408723；PMCID:PMC5526198.)。
本发明将HF-T7-M4/M5/M6与商品酶进行对比，分别将新冠病毒COVID19的S基因DNA转录DNA模板2倍倍比稀释用于检测。
反应体系如下表6所示：
表6反应体系中各组分及用量
如图10A、图10B、图10C和图10D所示，本发明设计的融合突变型RNA聚合酶显著提高了对RNA转录模板的灵敏度。工程化T7RNA聚合酶在相同DNA转录模板条件下，其转录介导的检测信号是野生型商品T7酶对照的2-4倍，或产生相同信号值所需的时间显著缩短。因此我们认为本发明的融合型突变RNA聚合酶对提高RNA转录介导的诊断学方法的灵敏度和效率有非常重要的意义。
实施例9：DNA结合域融合不同RNA聚合酶对单位酶活性的影响
除T7RNA聚合酶以外，发明人还构建表达了Sac7d DNA融合域与T3，sp6和sny5RNA聚合酶的融合蛋白，分别检测其酶活并与野生型酶活进行比较，每种融合RNA聚合酶酶活均可以达到野生型RNA聚合酶的2-2.5倍(表7)。
表7各RNA聚合酶酶活的酶活提高幅度
实施例10：共转录合成加帽RNA
1.实验步骤及条件：
(1)转录：
取1.5ml的无菌离心管，在室温条件下，依次加入注射用水、10×IVT Buffer、100mM ATP、100mM CTP、100mM Pseudo-UTP、100mM GTP、100Mm帽子类似物GAG(江苏申基生物科技)、MgCl2(1.5M)、线性化模板质粒、T7RNA聚合酶。
对于NTP浓度为10mM、NTP/GAG比例为5:4的实验组，在30μl的转录体系中，10×IVT Buffer的加入量为1.5μl，ATP、CTP、GTP、Pseudo-UTP的加入量均为3μl，帽子类似物GAG的加入量为2.4μl，MgCl2(1.5M)加入量为0.4μl，模板加入量为1.5μg，RI(200U/μl)的加入量为0.15μl，Ippase(0.6U/μl)的加入量为0.03μl，T7RNA聚合酶(1KU/μl)的加入量为1.5μl，补H2O到30μl。
本实验的研究变量包括酶的种类、NTP浓度、NTP/GAG的比例。其中酶的种类包括：WT T7酶、M4和M5；NTP的浓度包括10mM、7.5mM、5mM；NTP/GAG的比例包括(5:4)、(5:3)、(5:2)、(5:1)、(10:1)。所有组分加入完毕后，轻摇离心管，混匀后，将离心管放入金属浴内，设置金属浴温度为37℃，转录时间4h。
(2)模板消化：
对于25μl的转录产物，依次加入12.5μl的10×DNaseI Buffer，加入1μl的DNase I、86.5μl的注射用水，总体积为125μl。轻摇离心管，混匀后，放入37℃的恒温水浴锅 孵育30min。
(3)LiCl沉淀法纯化mRNA
模板消化后体系加入62.5μl的LiCl(7.5M)，-20℃放置30min，在4℃条件下13000rpm离心12min，弃上清，75％乙醇洗涤沉淀两次(离心2min)，离心浓缩仪中干燥沉淀，每管加入50μl注射水复溶，Nanodrop测浓度并计算转录产量。
(4)采用Agilent 5200测纯度。
(5)采用高分辨质谱进行加帽效率的检测。
2.实验结果及分析
设置各NTP的浓度为10mM，调整GAG的浓度，验证RNA聚合酶在不同GAG浓度下的转录产量，结果如表8所示。在GTP的浓度为10mM时，随着GTP/GAG的比例由5:4逐渐提高到10:1，对应的GAG添加量逐渐由8mM降低到1mM，此时WT T7的转录产量在5:2的比例下迅速降低到3.27mg/ml；当比例为10:1时，WT T7的转录产量再次降低到1.56mg/ml；M4的转录产量仅在比例为10:1时有较大幅度的下降，但仍高于WT T7。而M5对应的转录产量在各种NTP/GAG比例下基本没有变化，产量始终维持在7.5mg/ml附近。说明本发明的RNA聚合酶可以降低共转录时GAG的用量。
表8 RNA聚合酶在不同GAG浓度下的转录产量
设置各NTP的浓度为10mM和7.5mM，调整GAG的浓度，验证RNA聚合酶不同GAG浓度下的转录纯度，结果如表9所示。不同实验组的纯度差别不大，均在80％左右。
表9 RNA聚合酶在不同条件下的转录纯度
设置各NTP的浓度为10mM，调整GAG的浓度，验证RNA聚合酶在不同条件下的加帽率，结果如表10所示。在NTP/GAG的比例逐渐由5:4升高到10:1时，WT T7、M4、M5三种酶对应的加帽率均略有降低，但均具有96％以上的高加帽率。
表10 RNA聚合酶在不同GAG浓度下的加帽率

设置各NTP的浓度为10mM，调整GAG的浓度及RNA聚合酶的加入量，并以市售RNA聚合酶作为对比，验证M5的转录效果，转录产量如表11和图11所示。当酶的添加量为0.6ug时，采用M5进行共转录，GTP/GAG比例为5:1和10:1时转录产量没有显著降低，达到7mg/ml以上，而此时Roche的酶转录产量仅仅为1.1mg/ml；近岸的酶对应的转录产量更低，仅仅为0.39mg/ml。通过对比可知，在低GAG添加量条件下(1mM)，RNA聚合酶M5仍然具有较高的转录产量。转录产物琼脂糖凝胶电泳结果如图12所示，电泳图表明在相同上样体积时，M5转录产量最高，且无明显非特异性条带。
表11 M5和市售RNA聚合酶转录产量
虽然以上描述了本发明的具体实施方式，但是本领域的技术人员应当理解，这些仅是举例说明，在不背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改。因此，本发明的保护范围由所附权利要求书限定。
T7 RNA聚合酶的氨基酸序列(SEQ ID NO:1)

T3 RNA聚合酶的氨基酸序列(SEQ ID NO:2)
SP6 RNA聚合酶的氨基酸序列(SEQ ID NO:3)
Syn5 RNA聚合酶的氨基酸序列(SEQ ID NO:4)

KP34 RNA聚合酶的氨基酸序列(SEQ ID NO:5)
Pseudomonas phage VSW-3 RNA聚合酶的氨基酸序列(SEQ ID NO:6)

Claims (14)

  1. 一种RNA聚合酶融合蛋白，其特征在于，其包括：
    (i)依赖于DNA模板的噬菌体RNA聚合酶；
    (ii)非特异性的DNA结合域多肽和/或RNA结合域多肽；
    其中，所述RNA聚合酶融合蛋白比所述噬菌体RNA聚合酶具有更高的单位酶活性。
  2. 如权利要求1所述的RNA聚合酶融合蛋白，其特征在于，所述噬菌体RNA聚合酶的氨基酸序列如SEQ ID NO:1～6所示，或为与其具有至少90％序列同一性的突变体。
  3. 如权利要求2所述的RNA聚合酶融合蛋白，其特征在于，所述DNA结合域多肽和/或RNA结合域多肽融合于所述噬菌体RNA聚合酶的N端；和/或，所述DNA结合域多肽和/或RNA结合域多肽与所述噬菌体RNA聚合酶之间通过连接子连接；
    优选地，所述连接子的序列如SEQ ID NO:7或8的基序所示，或由n个所述基序组成，其中n为0～10的自然数。
  4. 如权利要求2所述的RNA聚合酶融合蛋白，其特征在于，所述突变体为在如SEQ ID NO:1～6所示的氨基酸序列上具有选自以下的一个或多个氨基酸取代：S430P、S633P、F849I、F880Y、P266L和Q744R/L/P；
    优选地，所述突变体包括S430P、S633P、F849I和F880Y突变；
    更优选地，所述突变体还包括Q744R和/或P266L突变；例如，所述突变体包括S430P、S633P、Q744R、F849I和F880Y突变，或P266L、S430P、S633P、Q744R、F849I和F880Y突变。
  5. 如权利要求1～4任一项所述的RNA聚合酶融合蛋白，其特征在于，所述DNA结合域多肽选自源自古细菌(Archaea)的Sul7d蛋白家族中的一种或多种；
    优选地，所述DNA结合域多肽的氨基酸序列如SEQ ID NO:9～14任一所示，或与所述氨基酸序列具有至少90％同一性。
  6. 如权利要求5所述的RNA聚合酶融合蛋白，其特征在于，
    (i)中，所述噬菌体RNA聚合酶的氨基酸序列如SEQ ID NO:1所示；或在如SEQ ID NO:1所示的氨基酸序列上具有S430P、S633P、F849I和F880Y突变，S430P、S633P、Q744R、F849I和F880Y突变，或P266L、S430P、S633P、Q744R、F849I和F880Y突变；
    或，所述噬菌体RNA聚合酶的氨基酸序列如SEQ ID NO:2所示；或在如SEQ ID NO:2所示的氨基酸序列上具有S430P、S633P、F849I和F880Y突变，S430P、S633P、Q744R、F849I和F880Y突变，或P266L、S430P、S633P、Q744R、F849I和F880Y突变；
    或，所述噬菌体RNA聚合酶的氨基酸序列如SEQ ID NO:3所示；或在如SEQ ID NO:3 所示的氨基酸序列上具有S430P、S633P、F849I和F880Y突变，S430P、S633P、Q744R、F849I和F880Y突变，或P266L、S430P、S633P、Q744R、F849I和F880Y突变；
    或，所述噬菌体RNA聚合酶的氨基酸序列如SEQ ID NO:4所示；或在如SEQ ID NO:4所示的氨基酸序列上具有S430P、S633P、F849I和F880Y突变，S430P、S633P、Q744R、F849I和F880Y突变，或P266L、S430P、S633P、Q744R、F849I和F880Y突变；
    或，所述噬菌体RNA聚合酶的氨基酸序列如SEQ ID NO:5所示；或在如SEQ ID NO:5所示的氨基酸序列上具有S430P、S633P、F849I和F880Y突变，S430P、S633P、Q744R、F849I和F880Y突变，或P266L、S430P、S633P、Q744R、F849I和F880Y突变；
    或，所述噬菌体RNA聚合酶的氨基酸序列如SEQ ID NO:6所示；或在如SEQ ID NO:6所示的氨基酸序列上具有S430P、S633P、F849I和F880Y突变，S430P、S633P、Q744R、F849I和F880Y突变，或P266L、S430P、S633P、Q744R、F849I和F880Y突变；
    (ii)中，所述DNA结合域多肽的氨基酸序列如SEQ ID NO:9～14任一所示；
    优选地，(i)中，所述噬菌体RNA聚合酶的氨基酸序列如SEQ ID NO:1所示；或在如SEQ ID NO:1所示的氨基酸序列上具有S430P、S633P、F849I和F880Y突变，S430P、S633P、Q744R、F849I和F880Y突变，或P266L、S430P、S633P、Q744R、F849I和F880Y突变；(ii)中，所述DNA结合域多肽的氨基酸序列如SEQ ID NO:9所示；
    或，(i)中，所述噬菌体RNA聚合酶在如SEQ ID NO:2所示的氨基酸序列上具有S430P、S633P、Q744R、F849I和F880Y突变；(ii)中，所述DNA结合域多肽的氨基酸序列如SEQ ID NO:10所示；
    或，(i)中，所述噬菌体RNA聚合酶在如SEQ ID NO:3所示的氨基酸序列上具有S430P、S633P、Q744R、F849I和F880Y突变；(ii)中，所述DNA结合域多肽的氨基酸序列如SEQ ID NO:11所示；
    或，(i)中，所述噬菌体RNA聚合酶在如SEQ ID NO:4所示的氨基酸序列上具有S430P、S633P、Q744R、F849I和F880Y突变；(ii)中，所述DNA结合域多肽的氨基酸序列如SEQ ID NO:12所示；
    或，(i)中，所述噬菌体RNA聚合酶在如SEQ ID NO:5所示的氨基酸序列上具有S430P、S633P、Q744R、F849I和F880Y突变；(ii)中，所述DNA结合域多肽的氨基酸序列如SEQ ID NO:13所示；
    或，(i)中，所述噬菌体RNA聚合酶在如SEQ ID NO:6所示的氨基酸序列上具有S430P、S633P、Q744R、F849I和F880Y突变；(ii)中，所述DNA结合域多肽的氨基酸序列如SEQ ID NO:14所示。
  7. 一种生物材料，其特征在于，其选自以下组：
    (1)分离的核酸，其编码如权利要求1～6任一项所述的RNA聚合酶融合蛋白；
    (2)重组表达载体，其包含如(1)中的分离的核酸；
    (3)转化体，其包含如(2)中的重组表达载体。
  8. 一种组合物，其包括如权利要求1～6任一项中所述的RNA聚合酶融合蛋白；优选还包括：缓冲剂、修饰或无修饰的核糖核苷三磷酸、以及帽子类似物的一种或几种。
  9. 一种试剂盒，其包括如权利要求1～6任一项中所述的RNA聚合酶融合蛋白，较佳地，还包括：缓冲剂、修饰或无修饰的核糖核苷三磷酸、以及帽子类似物的一种或几种。
  10. 一种合成RNA分子的方法，包括：温育混合物以将模板DNA分子转录成RNA，所述混合物包含权利要求1～6任一项中所述的RNA聚合酶变体融合蛋白以及模板DNA分子。
  11. 如权利要求10所述的一种合成RNA分子的方法，其特征在于，所述方法满足如下一种或多种条件：
    (1)所述温育在25-45℃的中温，例如25℃、37℃或42℃的温度下进行；
    (2)所述转录在0.05～0.2M的盐浓度下进行；较佳地，所述盐为NaCl，所述盐浓度不高于0.1M；
    (3)所述混合物还包含帽子类似物，较佳地，所述帽子类似物的浓度≤8mM，例如8mM、6mM、4mM、2mM或1mM。
  12. 一种如权利要求1～6任一项中所述的RNA聚合酶融合蛋白、权利要求7所述的生物材料或权利要求8所述的组合物在制备扩增RNA的试剂或RNA转录介导的诊断和/或体外翻译试剂盒中的应用。
  13. 一种如权利要求1～6任一项中所述的RNA聚合酶融合蛋白、权利要求7所述的生物材料或权利要求8所述的组合物在制备中温条件下具有高活性的酶制剂中的应用；其中，所述中温为25-42℃，所述高活性指与野生型RNA聚合酶相比具有至少提高1倍的活性。
  14. 一种核酸诊断方法，其特征在于，其包括核酸转录和核酸确定的步骤；其中，所述核酸扩增使用如权利要求1～6任一项中所述的RNA聚合酶变体融合蛋白、权利要求7所述的组合物或权利要求9所述的试剂盒对核酸模板例如DNA进行转录或扩增；所述核酸确定使用测序的方法例如Sanger测序或者NGS测序进行；优选地，所述核酸诊断方法为非疾病诊断目的的方法。
PCT/CN2023/097436 2022-06-02 2023-05-31 一种rna聚合酶融合蛋白及其应用 WO2023232075A1 (zh)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1720324A (zh) * 2002-10-23 2006-01-11 Mj生物工程股份有限公司 改善的ss07－聚合酶偶联蛋白质
CN108779446A (zh) * 2016-01-13 2018-11-09 新英格兰生物实验室公司 T7 rna聚合酶的热稳定变体
US10465190B1 (en) * 2015-12-23 2019-11-05 Modernatx, Inc. In vitro transcription methods and constructs
CN112574971A (zh) * 2020-12-29 2021-03-30 益善生物技术股份有限公司 Taq DNA聚合酶突变体、PCR反应试剂和试剂盒
CN112921014A (zh) * 2019-12-05 2021-06-08 左炽健 T7RNA聚合酶突变体、mRNA、基因、表达载体及细胞
US20220056425A1 (en) * 2018-09-12 2022-02-24 Guardian Therapeutics, Llc Rna polymerase for synthesis of modified rna

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1720324A (zh) * 2002-10-23 2006-01-11 Mj生物工程股份有限公司 改善的ss07－聚合酶偶联蛋白质
US10465190B1 (en) * 2015-12-23 2019-11-05 Modernatx, Inc. In vitro transcription methods and constructs
CN108779446A (zh) * 2016-01-13 2018-11-09 新英格兰生物实验室公司 T7 rna聚合酶的热稳定变体
US20220056425A1 (en) * 2018-09-12 2022-02-24 Guardian Therapeutics, Llc Rna polymerase for synthesis of modified rna
CN112921014A (zh) * 2019-12-05 2021-06-08 左炽健 T7RNA聚合酶突变体、mRNA、基因、表达载体及细胞
CN112574971A (zh) * 2020-12-29 2021-03-30 益善生物技术股份有限公司 Taq DNA聚合酶突变体、PCR反应试剂和试剂盒

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