WO2022214065A1 - Rna修饰嵌合蛋白及其应用 - Google Patents
Rna修饰嵌合蛋白及其应用 Download PDFInfo
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- WO2022214065A1 WO2022214065A1 PCT/CN2022/085760 CN2022085760W WO2022214065A1 WO 2022214065 A1 WO2022214065 A1 WO 2022214065A1 CN 2022085760 W CN2022085760 W CN 2022085760W WO 2022214065 A1 WO2022214065 A1 WO 2022214065A1
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- rna
- chimeric protein
- subunit
- functional fragment
- methyltransferase
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Definitions
- the present invention relates to the field of RNA synthesis, in particular to a chimeric protein for modifying RNA and its application.
- in vitro transcription to synthesize mRNA has obvious advantages in terms of biosafety, which is attributed to the difficulty of RNA integration into the genome, the shorter time window for RNA to express translated proteins, Without resorting to intracellular transcription steps, the expression initiation time and expression amount of the target protein can be better controlled.
- Therapeutic mRNA mainly mimics the natural structure of eukaryotic mRNA, which is mainly characterized by the presence of a cap structure at the 5-terminus, which consists of the first transcribed nucleotide of the RNA linked to reverse 7-methylguanosine (m7G) through a The triphosphate bridge is attached to the first nucleotide to form a structure called cap 0 (cap0, shown in Figure 10, C). Cap 0 is sufficient to recruit translation initiation factors and prevent mRNA degradation. Then, 2'-hydroxymethylation of the proximal nucleotide of the first cap forms cap 1 (cap1, shown in Figure 10, C).
- Cap0 cap structure The main functions of the Cap0 cap structure include: regulating splicing; nuclear export; stabilizing mRNA structure by protecting mRNA from 5'-exonuclease; assisting innate immunity to recognize "self RNA", which acts as an anchor for recruiting initiators to initiate 5' to 3' cyclization of mRNA during protein synthesis and translation.
- the cap1 structure is closely related to recognition by the innate immune system, and host immune proteins recognize abnormally uncapped RNAs.
- RNA generated by IVT requires different strategies to achieve RNA capping modification.
- there are two main production strategies for preparing capped mRNA one is the co-transcriptional capping method of cap analogs, and the other is the post-transcriptional capping enzyme-modified RNA capping method.
- RNA polymerases with relaxed substrate specificity e.g. T3, T7 or SP6 RNA polymerases
- T3, T7 or SP6 RNA polymerases are capable of 5' of RNA at transcription initiation
- the end-incorporation of cap analogs directly produces the corresponding 5' cap mRNA.
- Limitations of co-transcriptional capping include that GTP competes with cap analogs resulting in only partial capping of mRNAs obtained from IVT, and that capped/uncapped mRNAs are often difficult to separate and ratiometrically determine.
- m7GpppG as a co-transcriptional cap analog is that it leads to an extension of the RNA in the "wrong" direction, i.e. at the 3'-OH of m7G, resulting in an mRNA cap in the wrong direction.
- the post-transcriptional capping enzyme-modified RNA capping method refers to the enzymatic capping reaction of the RNA product generated by IVT with a specific capping enzyme and/or an mRNA-modifying enzyme in a suitable reaction system.
- the enzymatic reaction of cap0 consists of three sequential reaction steps: first, 5'-triphosphatase (TPase) hydrolyzes the ⁇ -phosphate of RNA; next, guanosyltransferase (GTase) converts the resulting 5'- The ⁇ -phosphate at the diphosphate terminus is coupled to GMP to form a 5'-5'-linked Gppp-RNA; finally, RNA(guanine-N7) methyltransferase (N7-MTase) uses S-adenosine- L-methionine (AdoMet) was used as a substrate to catalyze the methylation of the cap structure at the N7 position. Finally, m7G cap-specific 2
- capping enzymes and mRNA modifying enzymes derived from eukaryotes or viruses have been reported to have cap modification functions, there are very few enzymes that can actually be applied to mass production of cap-structured mRNAs. There remains a need in the art for capping enzymes that combine high efficiency and low cost.
- the object of the present invention is to provide proteins or protein subunits for capping modification of RNA and methods for modifying RNA therewith.
- the technical scheme of the present invention simplifies the production process of mRNA production-related modification enzymes, and reduces the addition of raw materials for mRNA production.
- a first aspect of the present invention provides a chimeric protein subunit comprising (a) a D12 subunit of an RNA capping enzyme or a functional fragment thereof linked to each other, or having at least 90% sequence identity therewith and having D12 subunit activity and (b) RNA cap structure 2'-O-methyltransferase or functional fragment thereof, or with at least 90% sequence identity therewith and having RNA cap structure 2'-O-methyltransferase activity Variants, and optionally (c) a linker between (a) and (b).
- the RNA capping enzyme is a vaccinia virus RNA capping enzyme.
- the RNA cap 2'-O-methyltransferase is a vaccinia virus 2'-O-methyltransferase.
- the carboxy terminus of (a) is linked to the amino terminus of (b).
- (b) further has a His tag and/or an MBP tag at its N-terminal or C-terminal.
- the His-tag is set forth in SEQ ID NO:8.
- the MBP tag is set forth in SEQ ID NO:9.
- amino acid sequence of the D12 subunit of the RNA capping enzyme is set forth in SEQ ID NO: 1, 1-287.
- the variant of the D12 subunit or functional fragment thereof of the RNA capping enzyme has a mutation selected from one or more of the following: N42A, Y43A, L61A, K62A, F245A, L246A, K111A, R112A , N120A, N121A, N126A, N127A, F141A, R142A, K223A, D224A, H260A, S261A, E275A, N276A, R280A, R281A.
- the amino acid sequence of the RNA cap 2'-O-methyltransferase is set forth in SEQ ID NO: 1, paragraphs 303-635.
- the variant of the RNA cap 2'-O-methyltransferase or functional fragment thereof is characterized by:
- the variant of the RNA cap structure 2'-O-methyltransferase or functional fragment thereof is set forth in SEQ ID NO: 1, paragraphs 303-635 and has a mutation selected from the group consisting of One or more of: K41D, C178S, A201R, A201K, C272S, one or more amino acids selected from R, K, H, Y, C, D, E are mutated to A.
- amino acid sequence of the linker is set forth in amino acids 288-302 of SEQ ID NO:1.
- amino acid sequence of the chimeric protein subunit is set forth in SEQ ID NO:1.
- the second aspect of the present invention provides a chimeric protein, comprising: (1) the chimeric protein subunit described in any one of the embodiments of the first aspect herein, and (2) the RNA capping enzyme D1 subunit or a functional fragment thereof , or a variant thereof having at least 90% sequence identity and corresponding activity.
- the protein is a heterodimeric protein of (1) and (2).
- the RNA capping enzyme is a vaccinia virus RNA capping enzyme.
- the functional fragment of the RNA capping enzyme D1 subunit comprises:
- N7-MTase N7 methyltransferase domain or its functional fragment
- TPase 5'-triphosphatase
- N7-MTase N7 methyltransferase domain or functional fragment thereof and guanosyltransferase (GTase) domain or functional fragment thereof;
- N7 methyltransferase N7-MTase domain or its functional fragment
- TPase 5'-triphosphatase
- GTase guanosyltransferase
- the RNA capping enzyme D1 subunit is set forth in SEQ ID NO:3.
- the functional fragment of the RNA capping enzyme D1 subunit comprises amino acids 498-844 or amino acids 540-844 of SEQ ID NO:3.
- the 5'-triphosphatase (TPase) domain is shown in amino acids 1-225 of SEQ ID NO:3.
- the guanosyltransferase (GTase) domain is set forth as amino acids 226-530 of SEQ ID NO:3.
- N7 methyltransferase (N7-MTase) domain is shown in amino acids 531-844 of SEQ ID NO:3.
- the variant of the N7-methyltransferase domain or functional fragment thereof has a mutation selected from one or more of the following: D545A, R548A, N550D, Y555F, R560K, R794A, R808A, Y683S , Y684A, Y684F, D598A, G600A, G602A, I681A, S684A, F685A, T571A, L575A, L576A, M579A, F585A, L586A, D587A, , D784A, N785A, R794A, F798A, M805A, E806A.
- the present invention also provides a nucleic acid molecule comprising a sequence selected from the group consisting of:
- the nucleic acid molecule has the sequence set forth in SEQ ID NO: 2, or a variant thereof having at least 80% sequence identity thereto, or a degenerate variant thereof encoding the same amino acid sequence.
- the present invention also provides a nucleic acid molecule comprising a sequence selected from the group consisting of:
- the RNA capping enzyme is a vaccinia virus RNA capping enzyme.
- the functional fragment of the RNA capping enzyme D1 subunit comprises:
- N7-MTase N7 methyltransferase domain or its functional fragment
- TPase 5'-triphosphatase
- N7-MTase N7 methyltransferase domain or functional fragment thereof and guanosyltransferase (GTase) domain or functional fragment thereof;
- N7 methyltransferase N7-MTase domain or its functional fragment
- TPase 5'-triphosphatase
- GTase guanosyltransferase
- the RNA capping enzyme D1 subunit is set forth in SEQ ID NO:3.
- the functional fragment of the RNA capping enzyme D1 subunit comprises amino acids 498-844 or amino acids 540-844 of SEQ ID NO:3.
- the 5'-triphosphatase (TPase) domain is shown in amino acids 1-225 of SEQ ID NO:3.
- the guanosyltransferase (GTase) domain is set forth as amino acids 226-530 of SEQ ID NO:3.
- N7 methyltransferase (N7-MTase) domain is shown in amino acids 531-844 of SEQ ID NO:3.
- the coding sequence of the RNA capping enzyme D1 subunit or functional fragment thereof is set forth in SEQ ID NO: 4, or a variant having at least 80% sequence identity thereto, or encoding the same amino acid Degenerate variants of sequences.
- the coding sequence for the chimeric protein subunit is set forth in SEQ ID NO:2.
- the nucleic acid construct contains an expression cassette for the chimeric protein subunit and an expression cassette for the RNA capping enzyme D1 subunit or functional fragment thereof; or the nucleic acid construct is an expression cassette box, wherein the coding sequence of the chimeric protein subunit and the coding sequence of the RNA capping enzyme D1 subunit or functional fragment thereof are in the expression box.
- the nucleic acid construct is a cloning vector or an expression vector.
- Another aspect of the present invention provides a host cell comprising, expressing and/or secreting a chimeric protein subunit or chimeric protein described herein.
- the host cell comprises the nucleic acid molecules, nucleic acid constructs described herein.
- the present invention also provides a method for modifying a target RNA into a capped RNA or a method for preparing a capped target RNA, comprising: combining the target RNA with the chimeric protein described herein under conditions that allow the RNA to be catalytically capped contacting, or contacting the target RNA with a cell expressing the chimeric protein described herein.
- the conditions that allow capping of the RNA are incubation at 37°C for at least 20 minutes.
- contacting a target RNA with a chimeric protein described herein comprises expressing the target RNA in a cell described herein expressing a chimeric protein described herein, or contacting the target RNA with a chimeric protein described herein Chimeric protein mix.
- the method comprises the step of introducing into the host cell a DNA sequence expressing the RNA of interest.
- the method comprises the steps of:
- RNA incubating the RNA with the chimeric protein described herein at 37°C for at least 20 minutes, such as 30-90 minutes, to obtain capped RNA
- the incubated mixture further comprises one or more reagents selected from the group consisting of GTP, SAM, buffer.
- the target RNA is selected from the group consisting of 5'-triphosphorylated RNA, 5'-diphosphorylated RNA, and RNA with a 5'-Gppp cap structure.
- the method modifies the target RNA to a capped RNA having a 5'-m7Gppp structure
- the target RNA is selected from the group consisting of 5'-triphosphorylated RNA, 5'-diphosphorylated RNA RNAs and RNAs with a 5'-Gppp cap structure, the chimeric proteins comprising the N7 methyltransferase domain of the RNA capping enzyme D1 subunit or a functional fragment thereof, a 5'-triphosphatase domain or a function thereof Fragments, guanosyltransferase domains or functional fragments thereof, and said chimeric protein subunits.
- the chimeric protein comprises an RNA capping enzyme D1 subunit as set forth in SEQ ID NO:3 and a chimeric protein subunit as set forth in SEQ ID NO:1.
- the method modifies the target RNA to a capped RNA having a 5'-m7GpppNmp structure
- the target RNA is selected from 5'-triphosphorylated RNA, 5'-diphosphorylated RNA RNA and RNA with 5'-Gppp cap structure
- the chimeric protein comprises the N7 methyltransferase (N7-MTase) domain of the RNA capping enzyme D1 subunit or a functional fragment thereof, a 5'-triphosphatase (TPase) domain or a functional fragment thereof and a guanosyltransferase (GTase) domain or functional fragment thereof, and the chimeric protein subunit.
- the chimeric protein comprises an RNA capping enzyme D1 subunit as set forth in SEQ ID NO:3 and a chimeric protein subunit as set forth in SEQ ID NO:1.
- the method is a method of capping a target RNA selected from the group consisting of 5'-bisphosphorylated RNAs and RNAs having a 5'-Gppp structure, the chimeric
- the protein comprises an N7 methyltransferase (N7-MTase) domain or a functional fragment thereof and a guanosyltransferase (GTase) domain or a functional fragment thereof of the D1 subunit of the RNA capping enzyme, and the chimeric protein subunit.
- N7-MTase N7 methyltransferase
- GTase guanosyltransferase
- the chimeric protein subunit is shown in SEQ ID NO: 1.
- the method is a method of capping a target RNA, the target RNA being an RNA having a 5'-Gppp structure, the chimeric protein comprising N7 of an RNA capping enzyme D1 subunit A methyltransferase (N7-MTase) domain or a functional fragment thereof, and the chimeric protein subunit.
- the chimeric protein subunit is shown in SEQ ID NO: 1.
- the 5'-triphosphatase (TPase) domain is shown in amino acids 1-225 of SEQ ID NO:3.
- the guanosyltransferase (GTase) domain is set forth as amino acids 226-530 of SEQ ID NO:3.
- N7 methyltransferase (N7-MTase) domain is shown in amino acids 531-844 of SEQ ID NO:3.
- the present invention also provides a method for methylating RNA of 5'-m7Gppp structure, comprising: contacting the RNA with a chimeric protein subunit described herein, or contacting the RNA with a subunit expressing a chimeric protein described herein Cell contacts of chimeric protein subunits.
- contacting the RNA with a chimeric protein subunit described herein comprises expressing the RNA in a cell described herein that expresses a chimeric protein subunit described herein.
- a third aspect of the present invention provides a fusion protein, comprising
- RNA cap 2'-O-methyltransferase or a functional fragment thereof or a variant thereof having at least 90% sequence identity therewith and having RNA cap 2'-O-methyltransferase activity (a) an RNA cap 2'-O-methyltransferase or a functional fragment thereof or a variant thereof having at least 90% sequence identity therewith and having RNA cap 2'-O-methyltransferase activity, and
- the RNA cap 2'-O-methyltransferase is a vaccinia virus 2'-O-methyltransferase.
- the amino acid sequence of the RNA cap 2'-O-methyltransferase is set forth in SEQ ID NO: 1, paragraphs 303-635.
- the variant of the RNA cap 2'-O-methyltransferase or functional fragment thereof is characterized by:
- the variant of the RNA cap structure 2'-O-methyltransferase or functional fragment thereof is set forth in SEQ ID NO: 1, paragraphs 303-635 and has a mutation selected from the group consisting of One or more of: K41D, C178S, A201R, A201K, C272S, one or more amino acids selected from R, K, H, Y, C, D, E are mutated to A.
- the His-tag is set forth in SEQ ID NO:8.
- the MBP tag is set forth in SEQ ID NO:9.
- the present invention also provides nucleic acid sequences encoding the fusion proteins described in the third aspect herein, nucleic acid constructs or host cells comprising said nucleic acid sequences.
- Figure 9 Comparison of cell biological functions of wild-type enzyme and chimeric enzyme to prepare eGFP mRNA respectively. Among them, 1, vaccinia virus capping enzyme and vaccinia virus 2-O-methyltransferase co-enzyme modified to prepare eGFP mRNA; 2, chimeric enzyme alone modified eGFP mRNA; 3, uncapped eGFP mRNA unmodified control group.
- FIG. 10 A, schematic diagram of RNA capping modification enzymatic reaction; B, schematic diagram of an exemplary embodiment of the present invention; C, schematic diagram of cap0 and cap1 cap structures.
- the inventors propose a novel RNA-modifying chimeric enzyme that combines capping enzymes with RNA triphosphatase, guanosyltransferase, guanine methyltransferase activity and mRNA cap structure 2'-O-methyl
- the methyltransferase activity of transferase is applied to mRNA production to achieve process simplification.
- the inventors found that the expression of 2'-O-methyltransferase native protein alone is relatively unstable, and it often has to be difficult during fermentation. to or only a relatively small amount of target protein can be obtained. If the N-terminal of 2'-O-methyltransferase is fused with the MBP solubilization tag, the 2'-O-methyltransferase fusion protein can be expressed stably and in high yield during the fermentation process.
- the inventors also found that when the double-promoter vector is used to express the capping enzyme of vaccinia virus, the expression levels of the D1 protein subunit and the D12 protein subunit cannot be balanced to reach the optimal ratio of 1:1, which is not conducive to the formation of more D1 :D12 complex, in which the expression level of the D12 protein subunit is much greater than that of the D1 protein subunit, which greatly reduces the expression yield of the complete capping enzyme D1:D12 complex structure.
- the applicant linked 2'-O-methyltransferase to the C-terminus of the D12 protein subunit (Fig. 10, B), one is to balance the reduction of D12 expression, and the other is to stabilize 2' The role of -O-methyltransferase.
- the above designed chimeric enzymes completely retained the natural activities of 5'-triphosphatase, guanosyltransferase, N7 methyltransferase, 2-O-methyltransferase.
- a first aspect of the present invention provides a chimeric protein subunit comprising (a) the D12 subunit of an RNA capping enzyme or a functional fragment thereof, or a D12 subunit having at least 90% sequence identity therewith. base-active variants, and (b) an RNA cap 2'-O-methyltransferase or a functional fragment thereof, or having at least 90% sequence identity therewith and having an RNA cap 2'-O-methyltransferase Active variants, and optionally (c) a linker between (a) and (b).
- the present invention also provides a chimeric protein comprising the chimeric protein subunit and the RNA capping enzyme D1 subunit or a functional fragment thereof.
- the chimeric protein is a heterodimer.
- a “chimeric enzyme” refers to a non-native enzyme that does not occur in nature, a chimeric enzyme may comprise catalytic domains derived from different sources (eg, from different enzymes), or from the same source (eg, from the same enzymes) but the catalytic domains are arranged in a manner different from that found in nature.
- a chimeric enzyme can be a protein in which one (ie, single subunit) or multiple (ie, multi-subunit) catalytic domains or proteins are linked covalently or non-covalently.
- Catalytic domain refers to a protein domain that is necessary and sufficient (especially in terms of its three-dimensional structure) to ensure enzyme function.
- Oleomerase refers to a multi-subunit enzyme consisting of at least two polypeptide chains linked together either covalently or non-covalently.
- Oligonomerase includes homo-oligomerases and hetero-oligomerases. Homo-oligomerases are multi-subunit enzymes composed of only one type of monomer (subunit), and hetero-oligomerases are composed of different types of monomers (subunits), such as heterodimers.
- the RNA capping enzyme described herein is vaccinia virus RNA capping enzyme (VVCE).
- VVCE vaccinia virus RNA capping enzyme
- the vaccinia virus capping enzyme is a heterodimer (D1:D12) of two viral proteins, D1 (844aa) and D12 (287aa).
- the chimeric proteins herein may comprise the RNA capping enzyme D1 subunit or a variant thereof.
- the D1 subunit has three catalytic domains and thus contains three biological enzymatic activities that perform all three steps in m7GpppRNA synthesis.
- the three catalytic domains are combined in the 97kDa D1 protein, in which the catalytic domains of RNA 5'-triphosphatase (TPase) and guanosyltransferase (GTase) are located in the N-terminal part, while the N7 methyltransferase ( The catalytic domain of N7-MTase) is located in the C-terminal part of the D1 protein.
- TPase RNA 5'-triphosphatase
- GTase guanosyltransferase
- the catalytic domain of N7-MTase is located in the C-terminal part of the D1 protein.
- the vaccinia virus RNA capping enzyme D1 subunit is shown in SEQ ID NO:3, the 5'-triphosphatase domain is shown in amino acids 1-225 of SEQ ID NO:3, and the guanosyltransferase structure The domain is shown in amino acids 226-530 of SEQ ID NO:3, and the N7 methyltransferase domain is shown in amino acids 531-844 of SEQ ID NO:3.
- the chimeric proteins herein may also include a truncated form of the RNA capping enzyme D1 subunit or a variant thereof, preferably comprising an N7-MTase domain.
- Truncated vaccinia capping enzymes comprising an N7-MTase domain and having biological enzymatic activity are known in the art, e.g. Shuman Z S et al (Shuman Z S, RNA, 2008; Higman M A et al, Journal of Biological Chemistry, 1994 ), including but not limited to the 498-844 amino acid fragment of the D1 protein or the 540-844 amino acid fragment of the D1 protein.
- the chimeric proteins herein also include functional fragments of the RNA capping enzyme D1 subunit, comprising: (1) an N7 methyltransferase domain or a functional fragment thereof; (2) an N7 methyltransferase domain or a function thereof fragment and 5'-triphosphatase domain or functional fragment thereof; (3) N7 methyltransferase domain or functional fragment thereof and guanosyltransferase domain or functional fragment thereof; or (4) N7 methyltransferase A domain or a functional fragment thereof, a 5'-triphosphatase domain or a functional fragment thereof, and a guanosyltransferase domain or a functional fragment thereof.
- the functional fragment of the RNA capping enzyme D1 subunit comprises amino acids 498-844 or amino acids 540-844 of SEQ ID NO:3.
- D12 subunit (33kDa) itself has no methyltransferase catalytic activity, but it can activate and enhance the methyltransferase activity of D1 protein.
- the amino acid sequence of the D12 subunit of the vaccinia virus RNA capping enzyme is shown in SEQ ID NO: 1, 1-287.
- the RNA cap 2'-O-methyltransferase described herein is a vaccinia virus 2'-O-methyltransferase (2-O-MTase).
- the 39 kDa 2'-O-methyltransferase also known as the VP39 protein, enables cap-specific mRNA (nucleoside 2'-O-)-methyl transfer, converting the cap-0 structure to the cap-1 structure.
- the amino acid sequence of the vaccinia virus RNA cap structure 2'-O-methyltransferase is shown in SEQ ID NO: 1, 303-635.
- the present invention also provides a 2'-O-methyltransferase protein fused with an MBP tag for improving the stability of protein expression, which is equivalent to the native vaccinia virus 2-O-methyltransferase or more The best biological enzyme functional activity.
- the fusion protein comprises (a) an RNA cap structure 2'-O-methyltransferase or a functional fragment thereof or a variant thereof having at least 90% sequence identity and having RNA cap structure 2'-O-methyl transfer activity; body, and (b) a His tag and/or MBP tag at (a) the N- or C-terminus, and optionally a linker between (a) and (b).
- the RNA cap 2'-O-methyltransferase was described previously.
- the His-tags described herein are short peptides containing one or more consecutive histidine residues.
- the MBP tags described herein have conventional meanings in the art.
- polypeptides or proteins also include polypeptides or proteins that have at least 70% sequence identity with it and retain the same mutants of 5'-triphosphorylation activity, guanosine transmethylation activity, N7 transmethylation activity, 2-O-methyl transfection activity.
- Said mutants include those having at least 70%, at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 97% sequence identity to the reference sequence and retaining the biological activity of the reference sequence amino acid sequence.
- Sequence identity between two aligned sequences can be calculated using, eg, NCBI's BLASTp.
- Mutants also include amino acid sequences that have one or more mutations (insertion, deletion or substitution) in the amino acid sequence while still retaining the biological activity of the reference sequence.
- the number of mutations generally refers to within 1-50, such as 1-20, 1-10, 1-8, 1-5 or 1-3. Substitutions are preferably conservative substitutions.
- mutations can occur in the CDR regions (including the mutations in the CDR regions described above) or in the FR regions, as long as the biological activity of the reference sequence is retained after the mutation.
- amino acids with similar or similar properties include, for example, families of amino acid residues with similar side chains, including amino acids with basic side chains (eg, lysine, arginine, histidine), chain amino acids (eg aspartic acid, glutamic acid), amino acids with uncharged polar side chains (eg glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine amino acids), amino acids with non-polar side chains (e.g.
- substitution of one or several positions in a polypeptide of the invention with another amino acid residue from the same class of side chains will not substantially affect its activity.
- variants of the D12 subunit of RNA capping enzymes known in the art may have mutations selected from one or more of the following and retain the activity of the D12 subunit: N42A, Y43A, L61A, K62A, F245A, L246A, K111A, R112A, N120A, N121A, N126A, N127A, F141A, R142A, K223A, D224A, H260A, S261A, E275A, N276A, R280A, R281A.
- mutants include, but are not limited to, C178S, C272S, K41D, A201R, A210K mutants of 2'-O-methyltransferase; the 2'-O-methyltransferase charged amino acids R, K, H, Y , C, D, E are randomly replaced by A.
- mutants include, but are not limited to, D545A, R548A, N550D, Y555F, R560K, R794A, R808A, Y683S, Y684A, Y684F, D598A, G600A, G602A, I681A, S684A, F685A, T571A, L575A, L576A, M579A L586A, D587A, D784A, N785A, R794A, F798A, M805A, E806A. (Schnierle BS et al, J Biol Chem. 1994; Zheng S et al, RNA.
- the polypeptides or proteins described herein further comprise a signal peptide capable of directing it to subcellular structures.
- the signal peptide can be located at the N-terminus or the C-terminus of the polypeptide.
- subcellular structures include, but are not limited to, the Golgi apparatus or endoplasmic reticulum, proteasomes, cell membranes, or lysosomes.
- a linker is a polypeptide fragment that connects different proteins or polypeptides, and its purpose is to keep the connected proteins or polypeptides in their respective spatial conformations, so as to maintain the function or activity of the proteins or polypeptides.
- exemplary linkers include G and/or S containing linkers.
- linkers contain one or more motifs that are repeated in sequence. Preferably, the motifs are contiguous in the linker sequence, with no intervening amino acid residues between repeats.
- the linker sequence may consist of 1, 2, 3, 4 or 5 repeating motifs.
- the length of the linker can be 3-25 amino acid residues, eg, 3-15, 5-15, 10-20 amino acid residues.
- the linker sequence is a polyglycine linker sequence.
- the number of glycines in the linker sequence is not particularly limited, and is usually 2-20, such as 2-15, 2-10, 2-8.
- the linker can also contain other known amino acid residues, such as alanine (A), leucine (L), threonine (T), glutamic acid (E), phenylalanine amino acid (F), arginine (R), glutamine (Q), etc.
- different proteins or polypeptides of the present invention are linked by (GGGGS)n, wherein n is an integer from 1 to 5.
- the amino acid sequence of the linker is set forth in amino acids 288-302 of SEQ ID NO:1.
- polypeptide linkers that can be used herein without affecting the folding of the catalytic domain of the enzyme and can achieve the effect of the chimeric enzyme of the present invention, including but not limited to GGGGIAPSMVGGGGGS (Turner, Ritter et al.
- the present invention includes polynucleotides encoding the chimeric protein subunits or chimeric proteins of the present invention.
- the polynucleotides of the present invention may be in the form of DNA or RNA.
- DNA forms include cDNA, genomic DNA or synthetic DNA.
- DNA can be single-stranded or double-stranded.
- DNA can be the coding or non-coding strand.
- the present invention also includes degenerate variants of polynucleotides encoding polypeptides or proteins, ie, polynucleotides that encode the same amino acid sequence but differ in nucleotide sequence.
- polynucleotides described herein include codon-optimized sequences that vary, so long as the amino acid sequence encoded by the polynucleotide is unchanged. Codon-optimized sequences may exhibit more suitable expressivity for specific species. Methods for codon optimization of polynucleotide sequences are well known in the art.
- the polynucleotide of the present invention can be the coding sequence of the chimeric protein subunit and the coding sequence of the RNA capping enzyme D1 subunit or its functional fragment, or the expression cassette of the chimeric protein subunit and the RNA capping enzyme D1 Expression cassettes for subunits or functional fragments thereof.
- a coding sequence refers to the portion of a nucleic acid sequence that directly determines the amino acid sequence of its protein product (eg, a chimeric protein subunit, a polypeptide of an RNA capping enzyme D1 subunit or a functional fragment thereof, etc.).
- Coding sequences can include, but are not limited to, DNA, cDNA, and recombinant nucleic acid sequences.
- the expression cassette refers to the complete elements required to express the gene of interest, including the promoter, the gene coding sequence and the PolyA tailed signal sequence.
- the polynucleotides described herein can be two independent nucleic acid molecules, each containing a coding sequence for a chimeric protein subunit and a coding sequence for an RNA capping enzyme D1 subunit or a functional fragment thereof, such as a chimeric protein subunit, respectively
- the expression cassette and the expression cassette of the RNA capping enzyme D1 subunit or its functional fragment; alternatively, the coding sequence of the chimeric protein subunit and the coding sequence of the RNA capping enzyme D1 subunit or its functional fragment can be connected via a linker
- a nucleic acid molecule such as the coding sequence of the chimeric protein subunit and the coding sequence of the RNA capping enzyme D1 subunit or its functional fragment are in the same expression frame, or the two expression frames are connected to the same nucleic acid molecule via a suitable linker .
- the polynucleotide of the present invention is a nucleic acid molecule in which the coding sequence of the chimeric protein subunit and the coding sequence of the RNA capping enzyme D1 subunit or functional fragment thereof are co-located in the same expression frame, which contains a promoter , coding sequences encoding chimeric protein subunits and RNA capping enzyme D1 subunits or functional fragments thereof, and PolyA tailing signals.
- the polynucleotide further comprises an optional signal peptide.
- the polynucleotide comprises SEQ ID NO: 2 or SEQ ID NO: 2 and 4.
- the coding sequence or expression cassette is integrated into the genome of the cell.
- cells described herein have stably integrated into the genome of the cells described herein, expression cassettes comprising the encoding chimeric protein subunits described herein and the RNA capping enzyme D1 subunit, or functional fragments thereof.
- the present invention also relates to nucleic acid constructs comprising the polynucleotides described herein, and one or more regulatory sequences operably linked to these sequences.
- the polynucleotides of the present invention can be manipulated in a variety of ways to ensure expression of the chimeric protein subunit or chimeric protein.
- the nucleic acid construct can be manipulated according to the expression vector or requirements before inserting the nucleic acid construct into the vector. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.
- the promoter sequence is usually operably linked to the coding sequence for the protein to be expressed.
- the promoter can be any nucleotide sequence that exhibits transcriptional activity in the host cell of choice, including mutated, truncated and hybrid promoters, and can be derived from extracellular coding homologous or heterologous to the host cell. Or the gene acquisition of intracellular polypeptides.
- the regulatory sequence may also be a suitable transcription terminator sequence, a sequence recognized by the host cell to terminate transcription. A terminator sequence is operably linked to the 3' end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the host cell of choice can be used in the present invention.
- the regulatory sequence may also be a suitable leader sequence, an untranslated region of an mRNA that is important for translation by the host cell.
- the leader sequence is operably linked to the 5' end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the host cell of choice can be used in the present invention.
- the nucleic acid construct is a vector.
- the vector can be a cloning vector, an expression vector, or a homologous recombination vector.
- the polynucleotides of the present invention can be cloned into many types of vectors, eg, plasmids, phagemids, phage derivatives, animal viruses, and cosmids.
- Cloning vectors can be used to provide the coding sequences for the therapeutic proteins and polypeptides of the invention, eg, a nucleic acid molecule comprising the coding sequences for the therapeutic proteins and polypeptides.
- Expression vectors can be provided to cells in the form of viral vectors.
- Expression of the polynucleotides of the present invention is typically accomplished by operably linking the polynucleotides of the present invention to a promoter and incorporating the construct into an expression vector.
- the vector may be suitable for replication and integration in eukaryotic cells.
- Typical cloning vectors contain transcriptional and translational terminators, initiation sequences, and promoters that can be used to regulate the expression of the desired nucleic acid sequence.
- Homologous recombination vectors are used to integrate the expression cassettes described herein into the host genome.
- suitable vectors contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction enzyme sites, and one or more selectable markers.
- the present invention uses a lentiviral vector containing an origin of replication, a 3'LTR, a 5'LTR, a polynucleotide described herein, and optionally a selectable mark.
- a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence.
- the promoter sequence is a strong constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operably linked thereto.
- Another example of a suitable promoter is elongation growth factor-1 ⁇ (EF-1 ⁇ ).
- EF-1 ⁇ elongation growth factor-1 ⁇
- other constitutive promoter sequences known in the art to be suitable for prokaryotic or eukaryotic cells can also be used.
- the expression vector introduced into the cell may also contain either or both of a selectable marker gene or a reporter gene to facilitate transfection or infection from a viral vector seeking Identify and select expressing cells from a population of cells.
- the selectable marker can be carried on a single piece of DNA and used in co-transfection procedures. Both the selectable marker and the reporter gene can be flanked by appropriate regulatory sequences to enable expression in the host cell.
- Useful selectable markers include, for example, antibiotic resistance genes and the like.
- Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase or green fluorescent protein genes.
- Suitable expression systems are well known and can be prepared using known techniques or obtained commercially.
- the polynucleotides described herein can generally be obtained by PCR amplification.
- primers can be designed according to the nucleotide sequences disclosed herein, especially the open reading frame sequences, and a commercially available cDNA library or a cDNA library prepared by conventional methods known to those skilled in the art is used as a template, Amplified sequences were obtained. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splicing the amplified fragments together in the correct order.
- the nucleic acid molecules described herein can also be synthesized directly.
- Vectors can be readily introduced into host cells, eg, mammalian, bacterial, yeast or insect cells, by any method known in the art.
- an expression vector can be transferred into a host cell by physical, chemical or biological means.
- Physical methods of introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like.
- Biological methods for introducing polynucleotides of interest into host cells include the use of DNA vectors, RNA vectors, or viral vectors, such as lentiviral vectors.
- Chemical means of introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and lipids plastid.
- the host cell contains, expresses and/or secretes the protein subunits or proteins described herein.
- containing means that the molecule is contained in or on the cell;
- expressing means that the cell produces the molecule ;
- secretion means that the cell secretes the expressed molecule out of the cell.
- Host cells include not only cells that are ultimately used to secrete chimeric proteins or subunits thereof, but also cells that produce RNA, as well as various cells used in the production of these cells, such as E. coli cells, for purposes such as providing the present invention.
- the coding sequences for the inventive proteins or the vectors described herein are provided. After the host cell expresses the protein subunits or proteins described herein, the protein subunits or proteins can be purified by conventional protein purification methods in the art (eg, chromatography, including affinity chromatography, ion exchange chromatography, etc.).
- Chemical modification of one or more steps in the RNA capping process can be performed using the chimeric protein subunits or chimeric proteins of the invention. Accordingly, the present invention provides a method of modifying a target RNA to a capped RNA or a method of preparing a capped target RNA, comprising: contacting the target RNA with a chimeric protein described herein, or contacting the target RNA with an expression of the herein described Cell contact of the chimeric protein described.
- target RNA is the RNA of interest obtained in vivo or in vitro.
- the target RNA is uncapped or incompletely capped RNA.
- the target RNA may be derived from any source, such as in vitro transcription (IVT), chemical synthesis, extraction from in vivo, and the like.
- IVT in vitro transcription
- In vitro transcription refers to the use of DNA transcription templates with T7, T3, and SP6 promoters and corresponding DNA-dependent RNA polymerases in a suitable buffer system for enzymatic reaction to synthesize RNA.
- the buffer system includes but is not limited to NTP, Mg 2+ , ribonuclease inhibitor (RI), inorganic pyrophosphatase (iPPase) and other components.
- the target RNA described herein is an in vitro transcribed uncapped single-stranded RNA (ssRNA) having all the characteristics of mRNA except the cap structure, including but not limited to the 5'-UTR, 3'-UTR, protein or polypeptide translation coding region.
- ssRNA in vitro transcribed uncapped single-stranded RNA
- RNA substrates Methods for in vitro transcription of RNA substrates are well known in the art, and exemplary polymerase (eg, T7, T3, SP6) transcription methods include incubating a mixture of transpolymerase, rNTPs, and a DNA transcription template (containing a promoter recognized by the polymerase) .
- the reaction system also includes one or more reagents selected from the following: MgCl 2 , buffer solution, iPPase, inhibitor, nuclease-free water, and the like.
- the DNA used to transcribe the template can be obtained by conventional methods in the art, such as synthesis, hybridization, PCR, etc., and the required reagents in these methods are also well known in the art.
- target RNAs herein include 5'-triphosphorylated RNAs, 5'-diphosphorylated RNAs, and RNAs with a 5'-Gppp structure.
- capped RNA includes capped RNA with 5'-m7Gppp structure (capO) or capped RNA with 5'-m7GpppNmp structure (cap1).
- 5' indicates that the group is located at the 5' end of the RNA
- m indicates methylation
- the number “7” indicates the methylation position
- G is guanosine
- p is Phosphate group
- N is any nucleotide located at the 5' end of the target RNA.
- the target RNA can be purified and/or denatured and renatured RNA.
- Reagents and procedures for purifying and denaturing, renaturing RNA are known in the art, eg, incubating RNA samples at 50-70°C for 1-60 minutes (eg, 65°C for 5-20 minutes), followed by 0°C for 2-10 minutes.
- the capping of RNA can be achieved even without the process of purification, denaturation and renaturation.
- the target RNA can be expressed in a cell expressing the chimeric protein described herein (eg, by introducing a DNA sequence expressing the target RNA into a host cell described herein).
- the target RNA can be expressed in a cell by a suitable method known in the art, for example, DNA encoding the RNA is introduced into the cell, the DNA is transcribed in the cell to form the target RNA, and the target RNA is expressed in the chimeric protein or chimeric protein expressed by the cell. Catalytic capping of protein subunits.
- the target RNA eg, RNA obtained from in vitro transcription
- the RNA can be mixed in solution with the chimeric protein described herein to effect contact.
- the RNA can be purified and/or renatured prior to mixing.
- Procedures and conditions for RNA capping in solution are known in the art, such as mixing and incubating target RNA in nuclease-free water with capping buffer, GTP, SAM, and the chimeric protein herein. Incubation steps such as incubation at 37°C for 30-90 minutes.
- the modified capped RNA product can be purified, eg, by magnetic beads.
- the molar ratio of target RNA to the chimeric protein is less than 865:1, preferably 86.5-173:1.
- chimeric proteins can also contain different domains. If the target RNA is 5'-triphosphorylated RNA, the chimeric protein needs to have the activities of 5'-triphosphatase, guanosyltransferase, N7 methyltransferase, and 2-O-methyltransferase, that is, the The chimeric protein comprises an N7 methyltransferase (N7-MTase) domain or a functional fragment thereof of the D1 subunit of an RNA capping enzyme, a 5'-triphosphatase (TPase) domain or a functional fragment thereof, and a guanosyl transfer Enzyme (GTase) domain or functional fragment thereof, and said chimeric protein subunit.
- N7-MTase N7 methyltransferase
- TPase 5'-triphosphatase
- GTase guanosyl transfer Enzyme
- the chimeric protein needs to have the activities of guanosyltransferase, N7 methyltransferase, and 2-O-methyltransferase, that is, the chimeric protein An N7 methyltransferase (N7-MTase) domain or a functional fragment thereof and a guanosyltransferase (GTase) domain or a functional fragment thereof comprising an RNA capping enzyme D1 subunit, and the chimeric protein subunit.
- N7-MTase N7 methyltransferase
- GTase guanosyltransferase
- the chimeric protein needs to have the activities of N7-methyltransferase (N7-MTase) and 2-O-methyltransferase, that is, the chimeric protein contains The N7 methyltransferase (N7-MTase) domain of the RNA capping enzyme D1 subunit or a functional fragment thereof and the chimeric protein subunit.
- N7-MTase N7-methyltransferase domain of the RNA capping enzyme D1 subunit or a functional fragment thereof and the chimeric protein subunit.
- the present invention can also modify a capped RNA (cap0) with a 5'-m7Gppp structure into a capped RNA (cap1) with a 5'-m7GpppNmp structure, comprising the step of: making the RNA with the structure described herein chimeric protein subunits of the touch.
- a chimeric protein subunit comprising fused (a) the D12 subunit of an RNA capping enzyme or a functional fragment thereof, or a variant thereof having at least 90% sequence identity therewith and having D12 subunit activity, and (b) RNA cap 2'-O-methyltransferase or a functional fragment thereof, or a variant thereof having at least 90% sequence identity therewith and having RNA cap 2'-O-methyltransferase activity, and any Selected linker between (c)(a) and (b),
- RNA capping enzyme is a vaccinia virus RNA capping enzyme
- RNA cap structure 2'-O-methyltransferase is vaccinia virus 2'-O-methyltransferase
- amino acid sequence of the D12 subunit of the RNA capping enzyme is set forth in SEQ ID NO: 1, paragraphs 1-287, and/or
- RNA cap 2'-O-methyltransferase The amino acid sequence of the RNA cap 2'-O-methyltransferase is set forth in SEQ ID NO: 1, paragraphs 303-635, and/or
- Variants of the D12 subunit of RNA capping enzymes or functional fragments thereof have mutations selected from one or more of the following: N42A, Y43A, L61A, K62A, F245A, L246A, K111A, R112A, N120A, N121A, N126A, F141A, R142A, K223A, D224A, H260A, S261A, E275A, N276A, R280A, R281A, and/or
- a variant of the RNA cap structure 2'-O-methyltransferase or a functional fragment thereof has the following characteristics: (1) has a mutation selected from one or more of the following: K41D, C178S, A201R, A201K, C272S; and/or (2) wherein one or more amino acids selected from R, K, H, Y, C, D or E are mutated to A, and/or
- (b) also has a His tag and/or an MBP tag at its N-terminus or C-terminus,
- amino acid sequence of the linker is shown in the 288-302nd amino acid of SEQ ID NO: 1,
- amino acid sequence of the chimeric protein subunit is shown in SEQ ID NO: 1.
- a chimeric protein comprising: (1) the chimeric protein subunit described in item 1 or 2, and (2) the RNA capping enzyme D1 subunit or a functional fragment thereof, or having at least 90% sequence therewith Variants that are identical and have corresponding activities,
- the chimeric protein is a heterodimeric protein, and/or
- RNA capping enzyme is a vaccinia virus RNA capping enzyme, and/or
- RNA capping enzyme D1 subunit Functional fragments of the RNA capping enzyme D1 subunit include:
- the 5'-triphosphatase domain is shown in amino acids 1-225 of SEQ ID NO:3, and/or
- the guanosyltransferase domain is shown in amino acids 226-530 of SEQ ID NO:3, and/or
- N7 methyltransferase domain is shown in amino acids 531-844 of SEQ ID NO:3, and/or
- RNA capping enzyme D1 subunit is shown in SEQ ID NO:3, and/or
- a functional fragment of the RNA capping enzyme D1 subunit comprises amino acids 498-844 or amino acids 540-844 of SEQ ID NO: 3, and/or
- Variants of the N7-methyltransferase domain or functional fragment thereof have mutations selected from one or more of the following: D545A, R548A, N550D, Y555F, R560K, R794A, R808A, Y683S, Y684A, Y684F, D598A, G600A, G602A, I681A, S684A, F685A, T571A, L575A, L576A, M579A, F585A, L586A, D587A, , D784A, N785A, R794A, F798A, M805A, E806A.
- a nucleic acid molecule comprising a sequence selected from the group consisting of:
- the coding sequence of the chimeric protein subunit is shown in SEQ ID NO: 2, or a variant having at least 80% sequence identity thereto, or a degenerate variant thereof that encodes the same amino acid sequence, and/or
- the coding sequence of the chimeric protein the coding sequence of the RNA capping enzyme D1 subunit or its functional fragment is shown in SEQ ID NO: 4, or a variant with at least 80% sequence identity thereto, or the same coding Degenerate variants of amino acid sequences.
- nucleic acid construct the nucleic acid construct:
- the nucleic acid construct contains the expression cassette of the chimeric protein subunit and the expression cassette of the RNA capping enzyme D1 subunit or a functional fragment thereof; or the nucleic acid construct is an expression cassette, wherein the chimeric protein subunit is The coding sequence of the base and the coding sequence of the RNA capping enzyme D1 subunit or functional fragment thereof are in the expression frame, and/or
- the nucleic acid construct is a cloning vector or an expression vector.
- a host cell comprising, expressing and/or secreting the chimeric protein subunit of item 1 or 2, or the chimeric protein of item 3 or 4,
- the host cell comprises the nucleic acid molecule described in item 5 and/or the nucleic acid construct described in item 6.
- a method for modifying a target RNA into a capped RNA or a method for preparing a capped target RNA comprising: under conditions that allow the RNA to be capped, submerging the target RNA with the chimeric protein described in item 1 or 2
- the base or the chimeric protein described in item 3 or 4 is contacted, or the target RNA is made to comprise, express and/or secrete the chimeric protein subunit described in item 1 or 2 or the chimeric protein described in item 3 or 4. cell contact,
- Contacting the target RNA with the chimeric protein subunit or chimeric protein comprises expressing the target RNA in a cell expressing the chimeric protein subunit or chimeric protein, or contacting the target RNA with the chimeric protein subunit or chimeric protein egg whites, and/or
- the method includes the step of introducing into the cell a DNA sequence expressing the RNA of interest.
- the conditions that allow capping of RNA are incubation at 37°C for at least 20 minutes, and/or
- the conditions allowing capping of the RNA further comprise the presence of one or more reagents selected from the group consisting of GTP, SAM, buffer, and/or
- the target RNA is selected from 5'-triphosphorylated RNA, 5'-diphosphorylated RNA and RNA with 5'-Gppp cap structure.
- the method modifies the target RNA into a capped RNA with a 5'-m7Gppp structure
- the target RNA is selected from the group consisting of 5'-triphosphorylated RNA, 5'-diphosphorylated RNA and capped with 5'-Gppp
- a structural RNA comprising the N7 methyltransferase domain or a functional fragment thereof, a 5'-triphosphatase domain or a functional fragment thereof, a guanosyltransferase domain or a functional fragment thereof of an RNA capping enzyme D1 subunit, the chimeric protein Its functional fragment, and said chimeric protein subunit; preferably, said chimeric protein comprises RNA capping enzyme D1 subunit as shown in SEQ ID NO:3 and chimeric as shown in SEQ ID NO:1 protein subunit,
- the method modifies the target RNA into a capped RNA with a 5'-m7GpppNmp structure
- the target RNA is selected from the group consisting of 5'-triphosphorylated RNA, 5'-diphosphorylated RNA and capped with 5'-Gppp Structural RNA
- the chimeric protein comprises an N7 methyltransferase (N7-MTase) domain or a functional fragment thereof, a 5'-triphosphatase (TPase) domain or a functional fragment thereof of the D1 subunit of the RNA capping enzyme and a guanosyltransferase (GTase) domain or a functional fragment thereof, and the chimeric protein subunit;
- the chimeric protein comprises an RNA capping enzyme D1 subunit as shown in SEQ ID NO: 3 and A chimeric protein subunit as shown in SEQ ID NO: 1,
- the method is a method for capping a target RNA selected from the group consisting of 5'-diphosphorylated RNA and RNA with a 5'-Gppp structure, and the chimeric protein comprises an RNA capping enzyme D1 subunit
- the N7 methyltransferase (N7-MTase) domain or its functional fragment and the guanosyltransferase (GTase) domain or its functional fragment, and the chimeric protein subunit preferably, the chimeric protein subunit base as shown in SEQ ID NO: 1, or
- the method is a method for capping a target RNA, the target RNA being an RNA having a 5'-Gppp structure, and the chimeric protein comprises an N7 methyltransferase (N7-MTase) of the RNA capping enzyme D1 subunit A structural domain or a functional fragment thereof, and the chimeric protein subunit; preferably, the chimeric protein subunit is shown in SEQ ID NO: 1.
- N7-MTase N7 methyltransferase
- a fusion protein comprising
- RNA cap 2'-O-methyltransferase or a functional fragment thereof or a variant thereof having at least 90% sequence identity therewith and having RNA cap 2'-O-methyltransferase activity (a) an RNA cap 2'-O-methyltransferase or a functional fragment thereof or a variant thereof having at least 90% sequence identity therewith and having RNA cap 2'-O-methyltransferase activity, and
- RNA cap structure 2'-O-methyltransferase is vaccinia virus 2'-O-methyltransferase
- RNA cap structure 2'-O-methyltransferase The amino acid sequence of the RNA cap structure 2'-O-methyltransferase is shown in SEQ ID NO: 1 Nos. 303-635,
- the variant of the RNA cap structure 2'-O-methyltransferase or a functional fragment thereof has the following characteristics: (1) having a mutation selected from one or more of the following: K41D, C178S, A201R, A201K, C272S; and /or (2) wherein one or more amino acids selected from R, K, H, Y, C, D or E are mutated to A, and/or
- His tag is as shown in SEQ ID NO: 8, and/or
- the MBP tag is shown in SEQ ID NO:9.
- a chimeric enzyme with both capping enzyme and 2′-O-methyltransferase enzymatic biological activities can reduce the production process and production cost
- Cap0 capping and cap1 capping are continuous reactions, and the covalent coupling of the two enzymes by the chimeric enzyme is more conducive to continuous contact with the RNA substrate and improves the modification efficiency.
- Example 1 Expression and purification of native vaccinia virus capping enzymes, 2-O-methyltransferases, and chimeric enzymes
- D1 D12, 2-O-methyltransferase 2-O-MTase
- D1 D12-(2-O-MTase) chimeric enzymes in E.
- the DNA sequences corresponding to the above proteins were optimized and cloned into commercial prokaryotic expression vectors of pET28a (Novagen), pMAL-c5X (NEB) and pRSF-Duet1 (Novagen).
- the amino acid and DNA sequences of the enzymes are shown in SEQ ID NOs: 1-4, and the prokaryotic expression vector diagram is shown in Figure 1.
- a single His-tag was added to the N-terminus of the D1 enzyme to facilitate purification of intact D1:D12 or D1:D12- (2-O-MTase) protein complex.
- a His tag and/or an MBP tag was added to the N-terminal of the 2-O-methyltransferase.
- the plasmids were transformed into Escherichia coli BL21 (DE3) expressing bacteria according to the instructions of commercial expression vectors, and a single colony was picked and cultured in LB at 37 degrees to logarithmic growth phase, and IPTG inducer with a final concentration of 1 mM was added to continue the induction and culture at 25 degrees for 16 hours.
- all enzyme proteins were purified using two standard purification steps, namely Ni affinity chromatography, Capto SP ImpRes or Capto Q ImpRes (Cytiva) ion exchange chromatography to obtain the final enzyme protein.
- SDS-PAGE polyacrylamide gel electrophoresis
- Figure 2 shows the addition of His and/or MBP tags to the N-terminus of 2-O-methyltransferase.
- SDS-PAGE identification of bacterial cell lysate Figure 3 is the SDS-PAGE identification of bacterial lysate of vaccinia virus capping enzyme and chimeric enzyme
- Figure 4 is MBP-(2-O-MTase), vaccinia virus after purification
- SDS-PAGE identification of capping enzyme D1:D12 and chimeric enzyme D1:D12-((2-O-MTase) was performed on the cell lysate before and after induction and the final purified enzyme protein
- Figure 2 shows the addition of His and/or MBP tags to the N-terminus of 2-O-methyltransferase.
- the 2-O-methyltransferase when the 2-O-methyltransferase has no MBP tag, the protein expression is unstable and the yield is low, so the 2-O-methyltransferase used in the present invention is in the form of MBP tag
- the 2-O-methyltransferase is MBP-(2-O-MTase).
- the expression level of the D12 protein subunit when the natural vaccinia virus capping enzyme is expressed, the expression level of the D12 protein subunit is much higher than that of the D1 protein subunit, which is not conducive to the formation of a stable 1:1 D1:D12 complex.
- the expression level of the fusion protein subunit is closer to that of D1, which is beneficial to the formation of the protein complex D1:D12-(2-O-MTase).
- the above three expression schemes all obtained proteins or protein subunits of target size after purification, wherein the vaccinia virus capping enzyme contains 33kD D12 protein subunit and 100kD D1 protein subunit; chimeric enzymes include The D12-(2-O-MTase) fusion protein subunit of 73kD and the D1 protein subunit of 100kD meet the expected protein size; the size of the MBP-(2-O-MTase) fusion protein is 83kD, which is equal to 44kD his-MBP The sum of the protein tag and the 39kD 2-O-MTase protein also matched the expected protein size.
- Example 2 in vitro transcription of RNA substrates and enzymatic modification to synthesize cap0mRNA and cap1mRNA
- RNA30-T7F 5'-gataatacgactcactataGGGAAGGAGAGGAAGGAAAGGGAAGAAAGAA-3' (SEQ ID NO:5); RNA30-R: 5'-TTCTTTCTTCCCTTTCCTTCCTCTCCTTCCCtatagtgagtcgtattatc-3' (SEQ ID NO:6).
- the primers were mixed according to the ratio in Table 1 for annealing reaction.
- the annealing procedure was as follows: 95°C pre-denaturation for 10min, gradient temperature drop to 20°C within 2 hours, and then taken out after keeping at 20°C for one hour. After purification by magnetic beads, the 30nt RNA (RNA30) transcribed DNA template.
- reagent volume 10 ⁇ Annealing Buffer 5 ⁇ L RNA30-T7F (100 ⁇ M) 20 ⁇ L RNA30-R (100 ⁇ M) 20 ⁇ L RNase-free water to 50 ⁇ L
- Transcribe and synthesize RNA prepare a suitable T7 RNA polymerase reaction system at room temperature and add the reaction components in the order shown in the following table.
- the reaction system can be expanded or reduced in equal proportions. Incubate at 37°C for 6-16 hours. In this example, the transcribed RNA was 30nt RNA. After 2h, the reaction was completed, 2U DNase 1 was added to digest at 37°C for 15min, and then magnetic bead purification was performed for the next step of RNA capping modification enzymatic reaction.
- Capped modified RNA Take 10ug RNA after purification into a 1.5ml centrifuge tube, dilute to 14ul with nuclease-free water; heat at 65°C for 10 minutes, remove the centrifuge tube and place on ice for 5 minutes; Table 3-6 Add the following components in sequence and incubate at 37°C for 30-90 minutes. This step is suitable for the capping reaction of 10ugRNA, and the volume of the reaction substrate can be scaled up according to the experimental needs. After the reaction, the final modified RNA product can be obtained by magnetic bead purification.
- RNA component volume Denatured capless RNA 10 ⁇ g 10* capping buffer 2.0 ⁇ l GTP (10mM) 1.0 ⁇ l SAM (32mM) 1.0 ⁇ l Vaccinia virus capping enzyme D1:D12 X ⁇ g (as shown in Figure 7) MBP-(2-O-MTase) Y ⁇ g (as shown in Figure 7) RNase-free water to 20 ⁇ l
- RNA component volume Denatured capless RNA 10 ⁇ g 10* capping buffer 2.0 ⁇ l GTP (10mM) 1.0 ⁇ l SAM (32mM) 1.0 ⁇ l Chimeric enzyme D1: D12-(2-O-MTase) X ⁇ g (as shown in Figure 8) RNase-free water to 20 ⁇ l
- Embodiment 3 the method of HPLC-MS detects the efficiency of different RNA modification enzymes catalyzing RNA 5' capping
- the expected molecular weights of the transcriptional or enzymatically modified intermediates or final products in Example 2 can be calculated, and these sample distributions were submitted for HPLC-MS (liquid chromatography-mass spectrometry). ) detection.
- HPLC-MS liquid chromatography-mass spectrometry
- Figure 7 shows HPLC-MS analysis of vaccinia virus cappingase and 2-O-methyltransferase synergistically modifying 30 units of RNA triphosphate activity. Based on the detection result that capping enzyme modifies triphosphate RNA30 to cap0 RNA30, each group in this experimental group uniformly added 0.2 ⁇ g of vaccinia capping enzyme to ensure complete conversion of triphosphate RNA30 to cap0 RNA30, and 2-O-methyltransferase as cap0 RNA30 Gradient addition of 0.25 ⁇ g, 0.5 ⁇ g, 1 ⁇ g, 2 ⁇ g.
- cap0 when the molar ratio of substrate RNA: capping enzyme is less than 665:1, cap0 can be completely capped, and when the molar ratio of RNA:2-O-methyltransferase is between 81-162:1, it can be The complete modification of cap0 to cap1 caps is achieved, which indicates that the amount of enzyme used in the synergistic capping reaction system and the stepwise capping reaction system is similar.
- cap0 RNA30 When the dose of chimeric enzyme reaches 2 ⁇ g, it can continue to completely modify the cap0 RNA30 that has been converted into 10903Da and convert it into cap1 RNA30 with the expected molecular weight of 10918Da. This indicated that the chimeric enzyme not only completely retained the activity of the catalytic domain of 2-O-methyltransferase, but also catalyzed cap1 RNA30 more efficiently and thoroughly. That is, in the chimeric enzyme capping system, when the molar ratio of substrate RNA:chimeric enzyme is less than 865:1, cap0 can be completely capped; when the RNA:chimeric molar ratio is between 86.5-173:1, cap0 can be completely capped. Modified as cap1 cap.
- the above indicators are all equivalent to the molar ratio indicators of the mixed enzyme system of 665:1 and 81-162:1, indicating that the chimeric enzyme can achieve the same function of the mixed enzyme under the same molar concentration, that is, it proves that the chimeric enzyme does not change each function.
- the catalytic activity of the domain is beneficial to simplify the production and use of the enzyme (compared with using two enzymes, only one enzyme needs to be produced, which saves the cost of steps, and only one enzyme needs to be added during the reaction).
- Example 4 Comparison of cell biological functions of wild-type enzyme and chimeric enzyme modified to prepare eGFP mRNA respectively
- the identified fully capped mRNA samples were respectively taken for transfection test, and the cap1 eGFP mRNA prepared by natural vaccinia virus capping enzyme combined with 2-O-methyltransferase modified RNA was numbered as sample No. 1, and the chimeric enzyme modified RNA was numbered as sample No. 1.
- the prepared cap1 eGFP mRNA was numbered as sample No. 2, and the capless eGFP mRNA that was not modified with the enzyme was numbered as sample No. 3.
- the above samples were transfected into Chinese hamster ovary (CHO) cells according to the following cell transfection procedure to verify the cellular biological function of modified cap1 eGFP mRNA.
- Transfection procedure (1) One day before transfection, CHO cells were digested and plated in a 24-well plate with a plating density of 1 ⁇ 105/well. (2) Take two 1.5ml EP tubes respectively during transfection, add 50ulopti-MEM culture medium to each tube, add 2.25ul PEI transfection reagent to one tube, and add 1.5ug dose of mRNA to the other tube, After standing for 5 minutes, the PEI solution was added to the mRNA solution, mixed evenly, and then allowed to stand for 20 minutes. (3) The prepared PEI-mRNA solution was added to CHO cells containing serum-free medium, placed in a 37-degree incubator, and after culturing for 4 hours, it was replaced with a complete medium to continue culturing. (4) After culturing for 24 hours, take a fluorescence photograph, detect flow cytometry, and detect the positive rate of EGFP. All three samples were used as a parallel test group of multiple wells.
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Abstract
本发明提供一种嵌合蛋白亚基,包含相互连接的(a)RNA加帽酶的D12亚基或其功能片段,或与之具有至少90%序列相同性并具有D12亚基活性的变体,和(b)RNA帽结构2'-O-甲基转移酶或其功能片段,或与之具有至少90%序列相同性并具有RNA帽结构2'-O-甲基转移活性的变体。本发明还提供包含所述嵌合蛋白亚基和RNA加帽酶D1亚基或其功能片段的嵌合蛋白。
Description
本发明涉及RNA合成领域,具体涉及用于修饰RNA的嵌合蛋白及其应用。
与基于DNA的治疗剂相比,体外转录(IVT,in vitro transcription)合成mRNA在生物安全性方面具有明显优势,这归因于RNA难以整合到基因组中、RNA表达翻译蛋白的时间窗较短、不必借助于细胞内转录步骤,可以更好地控制目的蛋白的表达起始时间和表达量。
治疗用mRNA主要模拟真核mRNA天然结构,其主要特征是在5末端存在帽结构,该结构由RNA的第一个转录核苷酸连接反向7-甲基鸟苷(m7G)组成,通过一个三磷酸桥连接到第一个核苷酸上形成一个称为帽0(cap0,如图10,C所示)的结构。帽0足以招募翻译起始因子并防止mRNA降解。然后,第一个帽近端核苷酸的2′-羟基甲基化形成帽1(cap1,如图10,C所示)。Cap0帽结构主要功能包括:调节剪接;核输出;通过保护mRNA免受5’-核酸外切酶的影响从而稳定mRNA结构;辅助先天免疫识别“自身RNA”,作为招募启动因子的锚点,启动蛋白质合成以及翻译过程中mRNA的5'至3'环化。cap1结构则与先天免疫***的识别密切相关,宿主免疫蛋白能识别异常非加帽的RNA。
因为IVT不直接产生具有生物学功能的真核带帽mRNA,只产生5'-三磷酸化RNA,所以其生成的RNA需要不同策略实现RNA的加帽修饰。目前主要有两种制备带帽mRNA的生产策略,一种是帽子类似物共转录加帽方式,一种是转录后加帽酶修饰RNA加帽方式。
在帽子类似物共转录加帽方式中,帽类似物直接添加到IVT中,具有松弛底物特异性的RNA聚合酶(例如T3,T7或SP6 RNA聚合酶)能够在转录起始时在RNA5'末端掺入帽子类似物,直接产生相应的5'帽mRNA。共转录加帽的局限性包括:GTP与帽子类似物竞争导致从IVT获得的mRNA只有部分被加帽,而且加帽/未加帽的mRNA通常难以分离和进行比例测定。用m7GpppG作为共转录帽子类似物的另一个问题是导致RNA向“错误”方向的延伸,即在m7G的3'-OH处延伸,产生的mRNA帽方向错误。
转录后加帽酶修饰RNA加帽方式指IVT生成的RNA产物在合适的反应体系中与特异性加帽酶和/或mRNA修饰酶的酶学加帽反应。cap0的酶促反应包括三个连续反应步骤:首先,5'-三磷酸酶(TPase)水解RNA的γ-磷酸;接下来,鸟苷转移酶(GTase)使用GTP作为底物将所得5'-二磷酸酯末端的β-磷酸酯与GMP偶联,形成5'-5'-连接Gppp-RNA;最后,RNA(鸟嘌呤-N7)甲基转移酶(N7-MTase)使用S-腺苷-L-蛋氨酸(AdoMet)作为底物,催化N7位甲基化帽结构。最后,m7G帽特异性2′-O-甲基转移酶(2-O-MTase)修饰了第一个核苷酸上的核糖生成cap 1结构。如图10,A所示。
截止到目前为止,尽管文献已报道了多种真核生物或病毒来源的加帽酶和mRNA修饰酶具有帽子修饰功能,但是真正能应用于大量生产制备帽结构mRNA的酶寥寥无几。本领域仍需兼具高效和低成本特性的加帽酶。
发明内容
本发明目的在于提供用于对RNA进行加帽修饰的蛋白或蛋白亚基以及用其对RNA进行修饰的方法。本发明的技术方案简化了mRNA生产相关修饰酶的生产工艺,减少mRNA生产添加原料。
本发明第一方面提供一种嵌合蛋白亚基,包含相互连接的(a)RNA加帽酶的D12亚基或其功能片段,或与之具有至少90%序列相同性并具有D12亚基活性的变体,和(b)RNA帽结构2′-O-甲基转移酶或其功能片段,或与之具有至少90%序列相同性并具有RNA帽结构2′-O-甲基转移活性的变体,以及任选的(c)(a)和(b)之间的接头。
在一个或多个实施方案中,所述RNA加帽酶是牛痘病毒RNA加帽酶。
在一个或多个实施方案中,所述RNA帽结构2′-O-甲基转移酶是牛痘病毒2′-O-甲基转移酶。
在一个或多个实施方案中,(a)的羧基端与(b)的氨基端连接。
在一个或多个实施方案中,(b)还具有位于其N端或C端的His标签和/或MBP标签。在一个或多个实施方案中,His标签如SEQ ID NO:8所示。在一个或多个实施方案中,MBP标签如SEQ ID NO:9所示。
在一个或多个实施方案中,RNA加帽酶的D12亚基的氨基酸序列如SEQ ID NO:1第1-287所示。
在一个或多个实施方案中,RNA加帽酶的D12亚基或其功能片段的变体具有选自以下一个或多个的突变:N42A、Y43A、L61A、K62A、F245A、L246A、K111A、 R112A、N120A、N121A、N126A、N127A、F141A、R142A、K223A、D224A、H260A、S261A、E275A、N276A、R280A、R281A。
在一个或多个实施方案中,RNA帽结构2′-O-甲基转移酶的氨基酸序列如SEQ ID NO:1第303-635所示。
在一个或多个实施方案中,RNA帽结构2′-O-甲基转移酶或其功能片段的变体具有如下特征:
(1)具有选自以下一个或多个的突变:K41D、C178S、A201R、A201K、C272S;和/或
(2)其中的一个或多个选自R、K、H、Y、C、D或E的氨基酸突变为A。
在一个或多个实施方案中,RNA帽结构2′-O-甲基转移酶或其功能片段的变体如SEQ ID NO:1第303-635所示并具有突变,所述突变选自以下一个或多个:K41D、C178S、A201R、A201K、C272S、一个或多个选自R、K、H、Y、C、D、E的氨基酸突变为A。
在一个或多个实施方案中,接头的氨基酸序列如SEQ ID NO:1第288-302位氨基酸所示。
在一个或多个实施方案中,嵌合蛋白亚基的氨基酸序列如SEQ ID NO:1所示。
本发明第二方面提供一种嵌合蛋白,包含:(1)本文第一方面中任一实施方式所述的嵌合蛋白亚基,和(2)RNA加帽酶D1亚基或其功能片段、或与之具有至少90%序列相同性并具有相应活性的变体。
在一个或多个实施方案中,所述蛋白是(1)和(2)的异二聚体蛋白。
在一个或多个实施方案中,所述RNA加帽酶是牛痘病毒RNA加帽酶。
在一个或多个实施方案中,RNA加帽酶D1亚基的功能片段包含:
(1)N7甲基转移酶(N7-MTase)结构域或其功能片段;
(2)N7甲基转移酶(N7-MTase)结构域或其功能片段和5'-三磷酸酶(TPase)结构域或其功能片段;
(3)N7甲基转移酶(N7-MTase)结构域或其功能片段和鸟苷转移酶(GTase)结构域或其功能片段;或
(4)N7甲基转移酶(N7-MTase)结构域或其功能片段、5'-三磷酸酶(TPase)结构域或其功能片段和鸟苷转移酶(GTase)结构域或其功能片段。
在一个或多个实施方案中,RNA加帽酶D1亚基如SEQ ID NO:3所示。
在一个或多个实施方案中,RNA加帽酶D1亚基的功能片段包含SEQ ID NO:3 的第498-844位氨基酸或第540-844位氨基酸。
在一个或多个实施方案中,5'-三磷酸酶(TPase)结构域如SEQ ID NO:3第1-225位氨基酸所示。
在一个或多个实施方案中,鸟苷转移酶(GTase)结构域如SEQ ID NO:3第226-530位氨基酸所示。
在一个或多个实施方案中,N7甲基转移酶(N7-MTase)结构域如SEQ ID NO:3第531-844位氨基酸所示。
在一个或多个实施方案中,N7-甲基转移酶结构域或其功能片段的变体具有选自以下一个或多个的突变:D545A、R548A、N550D、Y555F、R560K、R794A、R808A、Y683S、Y684A、Y684F、D598A、G600A、G602A,I681A、S684A、F685A、T571A、L575A、L576A、M579A、F585A、L586A、D587A,、D784A、N785A、R794A、F798A、M805A、E806A。
本发明还提供一种核酸分子,包含选自以下的序列:
(1)本文第一方面所述的嵌合蛋白亚基的编码序列,
(2)与(1)具有至少80%序列相同性的变体,
(3)(1)或(2)的互补序列。
在一个或多个实施方案中,所述核酸分子具有SEQ ID NO:2所示序列,或与其具有至少80%序列相同性的变体,或与其编码相同氨基酸序列的简并变体。
本发明还提供一种核酸分子,包含选自以下的序列
(1)本文第一方面所述的嵌合蛋白亚基的编码序列,和RNA加帽酶D1亚基或其功能片段或变体的编码序列,
(2)与(1)具有至少80%序列相同性的变体,
(3)(1)或(2)的互补序列。
在一个或多个实施方案中,所述RNA加帽酶是牛痘病毒RNA加帽酶。
在一个或多个实施方案中,RNA加帽酶D1亚基的功能片段包含:
(1)N7甲基转移酶(N7-MTase)结构域或其功能片段;
(2)N7甲基转移酶(N7-MTase)结构域或其功能片段和5'-三磷酸酶(TPase)结构域或其功能片段;
(3)N7甲基转移酶(N7-MTase)结构域或其功能片段和鸟苷转移酶(GTase)结构域或其功能片段;或
(4)N7甲基转移酶(N7-MTase)结构域或其功能片段、5'-三磷酸酶(TPase)结构域或其功能片段和鸟苷转移酶(GTase)结构域或其功能片段。
在一个或多个实施方案中,RNA加帽酶D1亚基如SEQ ID NO:3所示。
在一个或多个实施方案中,RNA加帽酶D1亚基的功能片段包含SEQ ID NO:3的第498-844位氨基酸或第540-844位氨基酸。
在一个或多个实施方案中,5'-三磷酸酶(TPase)结构域如SEQ ID NO:3第1-225位氨基酸所示。
在一个或多个实施方案中,鸟苷转移酶(GTase)结构域如SEQ ID NO:3第226-530位氨基酸所示。
在一个或多个实施方案中,N7甲基转移酶(N7-MTase)结构域如SEQ ID NO:3第531-844位氨基酸所示。
在一个或多个实施方案中,RNA加帽酶D1亚基或其功能片段的编码序列如SEQ ID NO:4所示,或与其具有至少80%序列相同性的变体,或与其编码相同氨基酸序列的简并变体。
在一个或多个实施方案中,嵌合蛋白亚基的编码序列如SEQ ID NO:2所示。
本发明另一方面提供一种核酸构建物,所述核酸构建物:
(1)表达本文第一方面所述的嵌合蛋白亚基,或第二方面所述的嵌合蛋白,
(2)包含本文所述的核酸分子。
在一个或多个实施方案中,所述核酸构建物含有所述嵌合蛋白亚基的表达框和RNA加帽酶D1亚基或其功能片段的表达框;或所述核酸构建物为一表达框,其中所述嵌合蛋白亚基的编码序列和所述RNA加帽酶D1亚基或其功能片段的编码序列处于该表达框内。
在一个或多个实施方案中,所述核酸构建物是克隆载体或表达载体。
本发明另一方面提供一种宿主细胞,所述宿主细胞包含、表达和/或分泌本文所述的嵌合蛋白亚基或嵌合蛋白。
在一个或多个实施方案中,所述宿主细胞包含本文所述的核酸分子、核酸构建物。
本发明还提供一种将目标RNA修饰为加帽RNA的方法或制备加帽的目标RNA的方法,包括:在允许RNA被催化加帽的条件下,使目标RNA与本文所述的嵌合 蛋白接触,或使目标RNA与表达本文所述的嵌合蛋白的细胞接触。
在一个或多个实施方案中,所述允许RNA加帽的条件是37℃孵育至少20分钟。
在一个或多个实施方案中,使目标RNA与本文所述的嵌合蛋白接触包括在本文所述的表达本文所述嵌合蛋白的细胞中表达目标RNA,或将目标RNA与本文所述的嵌合蛋白混合。
在一个或多个实施方案中,所述方法包括将表达目标RNA的DNA序列引入所述宿主细胞的步骤。
在一个或多个实施方案中,所述方法包括步骤:
任选的(1)使目标RNA变性,例如在50-70℃孵育1-60分钟,优选65℃孵育5-20分钟,
任选的(2)将(1)的产物目标RNA复性,例如于0℃孵育2-10分钟
(3)使RNA与本文所述的嵌合蛋白于37℃孵育至少20分钟,例如30-90分钟,获得加帽的RNA,
任选的(4)纯化加帽的RNA。
在一个或多个实施方案中,所述孵育的混合物还包含选自以下的一种或多种试剂:GTP、SAM、缓冲液。
在一个或多个实施方案中,所述目标RNA选自5'-三磷酸化的RNA、5'-二磷酸酯化的RNA和具有5'-Gppp帽结构的RNA。
在一个或多个实施方案中,所述方法将目标RNA修饰为具有5'-m7Gppp结构的加帽RNA,所述目标RNA选自5'-三磷酸化的RNA、5'-二磷酸酯化的RNA和具有5'-Gppp帽结构的RNA,所述嵌合蛋白包含RNA加帽酶D1亚基的N7甲基转移酶结构域或其功能片段、5'-三磷酸酶结构域或其功能片段、鸟苷转移酶结构域或其功能片段,和所述嵌合蛋白亚基。优选地,所述嵌合蛋白包含如SEQ ID NO:3所示的RNA加帽酶D1亚基和如SEQ ID NO:1所示的嵌合蛋白亚基。
在一个或多个实施方案中,所述方法将目标RNA修饰为具有5'-m7GpppNmp结构的加帽RNA,所述目标RNA选自5'-三磷酸化的RNA、5'-二磷酸酯化的RNA和具有5'-Gppp帽结构的RNA,所述嵌合蛋白包含RNA加帽酶D1亚基的N7甲基转移酶(N7-MTase)结构域或其功能片段、5'-三磷酸酶(TPase)结构域或其功能片段和鸟苷转移酶(GTase)结构域或其功能片段,和所述嵌合蛋白亚基。优选地,所述嵌合蛋白包含如SEQ ID NO:3所示的RNA加帽酶D1亚基和如SEQ ID NO:1所示的嵌合蛋白亚基。
在一个或多个实施方案中,所述方法是将目标RNA加帽的方法,所述目标RNA 选自5'-二磷酸酯化的RNA和具有5'-Gppp结构的RNA,所述嵌合蛋白包含RNA加帽酶D1亚基的N7甲基转移酶(N7-MTase)结构域或其功能片段和鸟苷转移酶(GTase)结构域或其功能片段,和所述嵌合蛋白亚基。优选地,所述嵌合蛋白亚基如SEQ ID NO:1所示。
在一个或多个实施方案中,所述方法是将目标RNA加帽的方法,所述目标RNA是具有5'-Gppp结构的RNA,所述嵌合蛋白包含RNA加帽酶D1亚基的N7甲基转移酶(N7-MTase)结构域或其功能片段,和所述嵌合蛋白亚基。优选地,所述嵌合蛋白亚基如SEQ ID NO:1所示。
在一个或多个实施方案中,5'-三磷酸酶(TPase)结构域如SEQ ID NO:3第1-225位氨基酸所示。
在一个或多个实施方案中,鸟苷转移酶(GTase)结构域如SEQ ID NO:3第226-530位氨基酸所示。
在一个或多个实施方案中,N7甲基转移酶(N7-MTase)结构域如SEQ ID NO:3第531-844位氨基酸所示。
本发明还提供一种对5'-m7Gppp结构的RNA进行甲基化的方法,包括:使所述RNA与本文所述的嵌合蛋白亚基接触,或使所述RNA与表达本文所述的嵌合蛋白亚基的细胞接触。
在一个或多个实施方案中,使所述RNA与本文所述的嵌合蛋白亚基接触包括在本文所述的表达本文所述嵌合蛋白亚基的细胞中表达所述RNA。
本发明第三方面提供一种融合蛋白,包含
(a)RNA帽结构2′-O-甲基转移酶或其功能片段或与之具有至少90%序列相同性并具有RNA帽结构2′-O-甲基转移活性的变体,和
(b)位于(a)N端或C端的His标签和/或MBP标签。
在一个或多个实施方案中,(a)和(b)之间具有接头。
在一个或多个实施方案中,所述RNA帽结构2′-O-甲基转移酶是牛痘病毒2′-O-甲基转移酶。
在一个或多个实施方案中,RNA帽结构2′-O-甲基转移酶的氨基酸序列如SEQ ID NO:1第303-635所示。
在一个或多个实施方案中,RNA帽结构2′-O-甲基转移酶或其功能片段的变体具有如下特征:
(1)具有选自以下一个或多个的突变:K41D、C178S、A201R、A201K、C272S;和/或
(2)其中的一个或多个选自R、K、H、Y、C、D或E的氨基酸突变为A。
在一个或多个实施方案中,RNA帽结构2′-O-甲基转移酶或其功能片段的变体如SEQ ID NO:1第303-635所示并具有突变,所述突变选自以下一个或多个:K41D、C178S、A201R、A201K、C272S、一个或多个选自R、K、H、Y、C、D、E的氨基酸突变为A。
在一个或多个实施方案中,His标签如SEQ ID NO:8所示。
在一个或多个实施方案中,MBP标签如SEQ ID NO:9所示。
本发明还提供编码本文第三方面所述融合蛋白的核酸序列、包含所述核酸序列的核酸构建物或宿主细胞。
图1,2-O-MTase、MBP-(2-O-MTase)、牛痘加帽酶D1:D12、嵌合酶D1:D12-((2-O-MTase))载体图。
图2,2-O-甲基转移酶的N端添加MBP和/或His标签菌体裂解液SDS-PAGE鉴定图。
图3,牛痘病毒加帽酶和嵌合酶的菌体裂解液SDS-PAGE鉴定图。
图4,SDS-PAGE鉴定野生型牛痘病毒加帽酶、2-O-甲基转移酶和嵌合酶蛋白图。
图5,HPLC-MS分析牛痘病毒加帽酶VVCE修饰三磷酸RNA30单位活性。
图6,HPLC-MS分析2-O-甲基转移酶修饰cap0 RNA30单位活性。
图7,HPLC-MS分析牛痘病毒加帽酶和2-O-甲基转移酶协同修饰三磷酸RNA30单位活性。
图8,HPLC-MS分析嵌合酶修饰三磷酸RNA30单位活性。
图9,野生型酶与嵌合酶分别修饰制备eGFP mRNA的细胞生物学功能比较。其中,1,牛痘病毒加帽酶和牛痘病毒2-O-甲基转移酶协同酶修饰制备eGFP mRNA;2,嵌合酶单独酶修饰制备eGFP mRNA;3,无帽eGFP mRNA未修饰对照组。
图10,A,RNA加帽修饰酶学反应示意图;B,本发明示例性实施方式示意图;C,cap0和cap1帽结构示意图。
发明人提出了一种全新的RNA修饰嵌合酶,其兼具有加帽酶的RNA三磷酸酶、鸟苷转移酶、鸟嘌呤甲基转移酶活性和mRNA帽结构2′-O-甲基转移酶的甲基转移酶活性,应用于mRNA生产实现工艺简化。
本发明人在研究mRNA帽结构2′-O-甲基转移酶的原核表达的过程中发现,单独表达2′-O-甲基转移酶天然蛋白时表达比较不稳定,发酵过程中经常得不到或只能得到比较少量的目标蛋白。如果在2′-O-甲基转移酶N端融合MBP促溶标签,其发酵过程中则可以稳定高产表达2′-O-甲基转移酶融合蛋白。此外,本发明人还发现利用双启动子载体表达牛痘病毒加帽酶时,D1蛋白亚基和D12蛋白亚基表达量无法均衡表达达到1:1的最佳比例,不利于形成更多的D1:D12复合体,其中D12蛋白亚基的表达量远大于D1蛋白亚基表达量,这大大降低了完整加帽酶D1:D12复合体结构的表达产量。
鉴于上述发现,申请人将2′-O-甲基转移酶连接到D12蛋白亚基的C端(图10,B),一是为了平衡减少D12的表达量,二是可以起到稳定2′-O-甲基转移酶的作用。令人惊奇的是,上述设计嵌合酶完全保留了5'-三磷酸酶、鸟苷转移酶、N7甲基转移酶、2-O-甲基转移酶的天然活性。
因此,本发明第一方面提供一种嵌合蛋白亚基,包含相互连接的(a)RNA加帽酶的D12亚基或其功能片段,或与之具有至少90%序列相同性并具有D12亚基活性的变体,和(b)RNA帽结构2′-O-甲基转移酶或其功能片段,或与之具有至少90%序列相同性并具有RNA帽结构2′-O-甲基转移活性的变体,以及任选的(c)(a)和(b)之间的接头。本发明还提供包含所述嵌合蛋白亚基和RNA加帽酶D1亚基或其功能片段的嵌合蛋白。所述嵌合蛋白是异源二聚体。
“嵌合酶”是指在自然界中不存在的非天然酶,嵌合酶可以包含源自不同来源(例如,源自不同酶)的催化结构域,或源自相同来源(例如,源自相同酶)但是以不同于自然界中发现的方式排列的催化结构域。嵌合酶可以是一个(即单亚基)或多个(即多亚基)催化结构域或蛋白质以共价或非共价连接的蛋白质。“催化结构域”是指对于确保酶功能而言是必需且足够(尤其是在其三维结构方面)的蛋白质结构域。“寡聚酶”是指由共价或非共价连接在一起的至少两条多肽链组成的多亚基酶。“寡聚酶”包括同源寡聚酶和异源寡聚酶,同源寡聚酶是仅由一种类型的单体(亚基)组成的多亚基酶,异源寡聚酶由不同类型的单体(亚基)组成,例如异源二聚体。
在一些实施方案中,本文所述RNA加帽酶是牛痘病毒RNA加帽酶(VVCE)。牛痘病毒加帽酶是D1(844aa)和D12(287aa)两种病毒蛋白的异二聚体(D1:D12)。
本文的嵌合蛋白可包含RNA加帽酶D1亚基或其变体。D1亚基具有三种催化 结构域,因此包含三种生物酶活性,可在m7GpppRNA合成中执行所有三个步骤。这三种催化结构域在97kDa D1蛋白中结合在一起,其中RNA 5'-三磷酸酶(TPase)和鸟苷转移酶(GTase)的催化结构域位于N端部分,而N7甲基转移酶(N7-MTase)的催化结构域则位于D1蛋白的C末端部分。示例性地,牛痘病毒RNA加帽酶D1亚基如SEQ ID NO:3所示,5'-三磷酸酶结构域如SEQ ID NO:3第1-225位氨基酸所示,鸟苷转移酶结构域如SEQ ID NO:3第226-530位氨基酸所示,N7甲基转移酶结构域如SEQ ID NO:3第531-844位氨基酸所示。
本文的嵌合蛋白还可包括截短形态的RNA加帽酶D1亚基或其变体,优选包含N7-MTase结构域。包含N7-MTase结构域并且具有生物学酶活性功能的截短牛痘加帽酶本领域已知,例如Shuman Z S等(Shuman Z S,RNA,2008;Higman M A等,Journal of Biological Chemistry,1994)所述,包括但不限于D1蛋白的498-844氨基酸片段或D1蛋白的540-844氨基酸片段。因此,本文的嵌合蛋白还包括RNA加帽酶D1亚基的功能片段,包含:(1)N7甲基转移酶结构域或其功能片段;(2)N7甲基转移酶结构域或其功能片段和5'-三磷酸酶结构域或其功能片段;(3)N7甲基转移酶结构域或其功能片段和鸟苷转移酶结构域或其功能片段;或(4)N7甲基转移酶结构域或其功能片段、5'-三磷酸酶结构域或其功能片段和鸟苷转移酶结构域或其功能片段。在一个或多个实施方案中,RNA加帽酶D1亚基的功能片段包含SEQ ID NO:3的第498-844位氨基酸或第540-844位氨基酸。
D12亚基(33kDa)本身没有甲基转移酶催化活性,但其可以激活增强D1蛋白的甲基转移酶活性。牛痘病毒RNA加帽酶的D12亚基的氨基酸序列如SEQ ID NO:1第1-287所示。
在一些实施方案中,本文所述RNA帽结构2′-O-甲基转移酶是牛痘病毒2′-O-甲基转移酶(2-O-MTase)。39kDa的2'-O-甲基转移酶也称为VP39蛋白,其可实现帽特异性mRNA(核苷2'-O-)-甲基转移,将cap-0结构转换为cap-1结构。牛痘病毒RNA帽结构2′-O-甲基转移酶的氨基酸序列如SEQ ID NO:1第303-635所示。
在一些方面,本发明还提供一种融合有MBP标签的2′-O-甲基转移酶蛋白用于提高蛋白表达稳定性,其具有与天然牛痘病毒2-O-甲基转移酶相当或更佳的生物酶功能活性。所述融合蛋白包含(a)RNA帽结构2′-O-甲基转移酶或其功能片段或与之具有至少90%序列相同性并具有RNA帽结构2′-O-甲基转移活性的变体,和(b)位于(a)N端或C端的His标签和/或MBP标签,以及任选的(a)和(b)之间具有接头。RNA帽结构2′-O-甲基转移酶如前所述。本文所述His标签是含有一个或多个连续组氨酸残基的短肽。本文所述MBP标签具有本领域常规含义。
本发明中,多肽或蛋白(例如牛痘病毒加帽酶或其亚基或结构域、RNA帽结构2′-O-甲基转移酶)也包括与其具有至少70%序列相同性并保留多肽或蛋白的活性(例如5'-三磷酸化活性、鸟苷转移活性、N7甲基转移活性、2-O-甲基转移活性)的突变体。所述突变体包括:与参照序列具有至少70%,至少80%,优选至少85%,优选至少90%,优选至少95%,优选至少97%的序列相同性并保留参照序列的生物学活性的氨基酸序列。可采用例如NCBI的BLASTp计算两条比对的序列之间的序列相同性。突变体还包括在所述氨基酸序列中具有一个或数个突变(***、缺失或取代)、同时仍保留该参照序列的生物学活性的氨基酸序列。所述数个突变通常指1-50个以内,例如1-20、1-10、1-8、1-5或1-3个。取代优选是保守性取代。对于scFv,突变可以发生在CDR区内(包括前文所述的CDR区内的突变),也可以发生在FR区内,只要突变后仍保留参照序列的生物学活性即可。例如,在本领域中,用性能相近或相似的氨基酸进行保守性取代时,通常不会改变蛋白质或多肽的功能(例如酶活性)。“性能相近或相似的氨基酸”包括例如,具有相似侧链的氨基酸残基的家族,这些家族包括具有碱性侧链的氨基酸(例如赖氨酸、精氨酸、组氨酸)、具有酸性侧链的氨基酸(例如天冬氨酸、谷氨酸)、具有不带电荷的极性侧链的氨基酸(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸)、具有非极性侧链的氨基酸(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸、色氨酸)、具有β-分支侧链的氨基酸(例如苏氨酸、缬氨酸、异亮氨酸)和具有芳香侧链的氨基酸(例如酪氨酸、苯丙氨酸、色氨酸、组氨酸)。因此,在本发明多肽中用来自同一侧链类的另一氨基酸残基替换一个或几个位点,将不会在实质上影响其活性。
例如,本领域已知RNA加帽酶的D12亚基的变体可具有选自以下一个或多个的突变并保留D12亚基的活性:N42A、Y43A、L61A、K62A、F245A、L246A、K111A、R112A、N120A、N121A、N126A、N127A、F141A、R142A、K223A、D224A、H260A、S261A、E275A、N276A、R280A、R281A。又如,Schnierle BS等在论文中披露了一系列mRNA帽结构2′-O-甲基转移酶的有效突变氨基酸序列,突变体仍保留了较高的酶催化活性。这些突变体包括但不局限于2′-O-甲基转移酶的C178S、C272S、K41D、A201R、A210K突变体;将2′-O-甲基转移酶带电荷氨基酸R、K、H、Y、C、D、E随机替换为A。Zheng S、Colin PY和Mao X则分别在文献中披露了一系列牛痘病毒加帽酶N7-甲基转移酶结构域的有效突变氨基酸序列,突变体也保留了较高的酶催化活性。这些突变体包括但不局限于D545A、R548A、N550D、Y555F、R560K、R794A、R808A、Y683S、Y684A、Y684F、D598A、G600A、G602A,I681A、S684A、F685A、T571A、L575A、L576A、M579A、F585A、L586A、D587A,、D784A、N785A、R794A、 F798A、M805A、E806A。(Schnierle BS等,J Biol Chem.1994;Zheng S等,RNA.2008;Mao X等,Biochemistry.1996;Kyrieleis OJ等,Structure.2014;Colin PY.Sci Rep.2020;Nayanendu Saha等,Virology,2001;Nayanendu Saha等,J.VIROL,2003)上述文献通过引用全文纳入本文。
在一些实施方案中,本文所述多肽或蛋白还包含能将其引导至亚细胞结构的信号肽。所述信号肽可位于多肽的N端或C端。所述亚细胞结构包括但不限于高尔基体或内质网、蛋白酶体、细胞膜或溶酶体。
本文中,接头(Linker)是连接不同蛋白或多肽之间的多肽片段,其目的是使所连接的蛋白或多肽保持各自的空间构象,以维持蛋白或多肽的功能或活性。示例性的接头包括含有G和/或S的接头。通常,接头含有一个或多个前后重复的基序。优选地,该基序在接头序列中是相邻的,在重复之间没有***氨基酸残基。接头序列可以包含1、2、3、4或5个重复基序组成。接头的长度可以是3-25个氨基酸残基,例如3-15、5-15、10-20个氨基酸残基。在某些实施方案中,接头序列是多甘氨酸接头序列。接头序列中甘氨酸的数量无特别限制,通常为2-20个,例如2-15、2-10、2-8个。除甘氨酸和丝氨酸来,接头中还可含有其它已知的氨基酸残基,例如丙氨酸(A)、亮氨酸(L)、苏氨酸(T)、谷氨酸(E)、苯丙氨酸(F)、精氨酸(R)、谷氨酰胺(Q)等。在某些实施方案中,本发明不同蛋白或多肽之间由(GGGGS)n连接,其中n为1~5的整数。在一个或多个实施方案中,接头的氨基酸序列如SEQ ID NO:1第288-302位氨基酸所示。
在本发明范围内,本领域内技术人员熟知可用于本文的不影响酶催化结构域折叠并可以达到本发明中嵌合酶效果的多肽接头,包括但不限于GGGGIAPSMVGGGGS(Turner,Ritter等1997)、SPNGASNSGSAPDTSSAPGSQ(Hennecke,Krebber等1998)、EGKSSGSGSESKSTE(Bird,Hardman等人1988)、EGKSSGSGSESKEF(Newton,Xue等人1996)、GGGSGGGSGGGTGGGSGGG(Robinson和Sauer 1998)、GSTSGSGKSSEGKG(Bedzyk,Weidner等人1990)、YPRSIYIRRRHPSPSLTT(Tang,Jiang等人1996)、STSGSGKPGSGEGS(Ting,Kain等人2001)、SSADDAKKDAAKKDDAKKDDAKKDA(Pantoliano,Bird等人1991)、GSADDAXXDAAXKDDAKKDDAKKDGS(Gregoire,Lin等人1996)、LSADDAKKDAAKKDDAKKDDAKKDL(Pavlinkova,Beresford等人1999)、AEAAAKEAAAKEAAAKA(Wickham,Carrion等人1995)、GSTSGSGKPGSGEGSTGAGGAGSTSGSGKPSGEG(Ting,Kain等人2001)、LSLEVAEEIARLEAEV(Ting,Kain等人2001)、GTPTPTPTPTGEF(Gustavsson,Lehtio 等人2001)、GSTSGSGKPGSGEGSTKG(Whitlow,Bell等人1993)和GSHSGSGKP(Ting,Kain等人2001)、或US20130042334中所描述的通用接头。上述文献通过引用全文纳入本文。
本发明包括编码本发明所述嵌合蛋白亚基或嵌合蛋白的多核苷酸。本发明的多核苷酸可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。本发明也包括编码多肽或蛋白的多核苷酸的简并变异体,即编码相同的氨基酸序列但核苷酸序列有所不同的多核苷酸。
本文所述多核苷酸包括经密码子优化而发生变化的序列,只要多核苷酸所编码的氨基酸序列不变即可。经密码子优化的序列可对具体物种表现出更适合的表达性。本领域周知对多核苷酸序列进行密码子优化的方法。
本发明的多核苷酸可以是所述嵌合蛋白亚基的编码序列和RNA加帽酶D1亚基或其功能片段的编码序列,或者是嵌合蛋白亚基的表达框和RNA加帽酶D1亚基或其功能片段的表达框。本文中,编码序列指核酸序列中直接确定其蛋白产物(例如嵌合蛋白亚基、RNA加帽酶D1亚基或其功能片段的多肽等)的氨基酸序列的部分。编码序列的边界通常是由紧邻mRNA 5’端开放读码框上游的核糖体结合位点(对于原核细胞)和紧邻mRNA 3’端开放读码框下游的转录终止序列确定。编码序列可以包括,但不限于DNA、cDNA和重组核酸序列。本文中,表达框指表达感兴趣基因所需的完整元件,包括启动子、基因编码序列和PolyA加尾信号序列。本文所述的多核苷酸可以是独立的两个核酸分子,分别含嵌合蛋白亚基的编码序列和RNA加帽酶D1亚基或其功能片段的编码序列,如分别是嵌合蛋白亚基的表达框和RNA加帽酶D1亚基或其功能片段的表达框;或者,所述嵌合蛋白亚基的编码序列和RNA加帽酶D1亚基或其功能片段的编码序列可经由接头连接为一个核酸分子,如嵌合蛋白亚基的编码序列和RNA加帽酶D1亚基或其功能片段的编码序列在同一表达框内,或者是两个表达框经由合适的接头连接为同一核酸分子。在某些实施方案中,本发明的多核苷酸为嵌合蛋白亚基的编码序列和RNA加帽酶D1亚基或其功能片段的编码序列同处一个表达框的核酸分子,其含有启动子、编码嵌合蛋白亚基和RNA加帽酶D1亚基或其功能片段的编码序列以及PolyA加尾信号。在一个或多个实施方案中,所述多核苷酸还包含任选的信号肽。在一个或多个实施方案中,多核苷酸包含SEQ ID NO:2或SEQ ID NO:2和4。
在某些实施方案中,所述编码序列或表达框整合到细胞的基因组中。因此,在这些实施方案中,本文所述的细胞的基因组中稳定整合了包含编码本文所述嵌合蛋白 亚基和RNA加帽酶D1亚基或其功能片段的表达框。
本发明也涉及核酸构建物,该核酸构建物含有本文所述的多核苷酸,以及与这些序列操作性连接的一个或多个调控序列。本发明所述的多核苷酸可以多种方式***作以保证所述嵌合蛋白亚基或嵌合蛋白的表达。在将核酸构建物***载体之前可根据表达载体的不同或要求而对核酸构建物进行操作。利用重组DNA方法来改变多核苷酸序列的技术是本领域已知的。
调控序列可以是合适的启动子序列。启动子序列通常与待表达蛋白的编码序列操作性连接。启动子可以是在所选择的宿主细胞中显示转录活性的任何核苷酸序列,包括突变的、截短的和杂合启动子,并且可以从编码与该宿主细胞同源或异源的胞外或胞内多肽的基因获得。调控序列也可以是合适的转录终止子序列,由宿主细胞识别以终止转录的序列。终止子序列与编码该多肽的核苷酸序列的3’末端操作性连接。在选择的宿主细胞中有功能的任何终止子都可用于本发明。调控序列也可以是合适的前导序列,对宿主细胞翻译重要的mRNA的非翻译区。前导序列与编码该多肽的核苷酸序列的5’末端可操作连接。在选择的宿主细胞中有功能的任何终止子都可用于本发明。
在某些实施方案中,所述核酸构建物是载体。载体可以是克隆载体,也可以是表达载体,或者是同源重组载体。本发明的多核苷酸可被克隆入许多类型的载体,例如,质粒、噬菌粒、噬菌体衍生物、动物病毒和粘粒。克隆载体可用于提供本发明治疗用蛋白与多肽的编码序列,如含治疗用蛋白的编码序列与多肽的编码序列的一个核酸分子。表达载体可以以病毒载体形式提供给细胞。通常通过可操作地连接本发明的多核苷酸至启动子,并将构建体并入表达载体,实现本发明多核苷酸的表达。该载体对于复制和整合真核细胞可为合适的。典型的克隆载体包含可用于调节期望核酸序列表达的转录和翻译终止子、起始序列和启动子。同源重组载体用于将本文所述的表达框整合到宿主基因组中。
通常,合适的载体包含在至少一种有机体中起作用的复制起点、启动子序列、方便的限制酶位点和一个或多个可选择的标记。例如,在某些实施方案中,本发明使用慢病毒载体,该慢病毒载体含有复制起始位点,3’LTR,5’LTR,本文所述的多核苷酸,以及任选的可选择的标记。
合适的启动子的一个例子为即时早期巨细胞病毒(CMV)启动子序列。该启动子序列是能够驱动可操作地连接至其上的任何多核苷酸序列高水平表达的强组成型启动子序列。合适的启动子的另一个例子为延伸生长因子-1α(EF-1α)。然而,也可使用本领域已知的适合原核或真核细胞的其他组成型启动子序列。
为了评估治疗用蛋白、多肽或其部分的表达,被引入细胞的表达载体也可包含可选择的标记基因或报道基因中的任一个或两者,以便于从通过病毒载体寻求被转染或感染的细胞群中鉴定和选择表达细胞。在其他方面,可选择的标记可被携带在单独一段DNA上并用于共转染程序。可选择的标记和报道基因两者的侧翼都可具有适当的调节序列,以便能够在宿主细胞中表达。有用的可选择标记包括例如抗生素抗性基因等。合适的报道基因可包括编码荧光素酶、β-半乳糖苷酶、氯霉素乙酰转移酶、分泌型碱性磷酸酶或绿色萤光蛋白基因的基因。合适的表达***是公知的并可利用已知技术制备或从商业上获得。
本文所述的多核苷酸通常可以用PCR扩增法获得。具体而言,可根据本文所公开的核苷酸序列,尤其是开放阅读框序列来设计引物,并用市售的cDNA库或按本领域技术人员已知的常规方法所制备的cDNA库作为模板,扩增而得有关序列。当序列较长时,常常需要进行两次或多次PCR扩增,然后再将各次扩增出的片段按正确次序拼接在一起。或者,也可直接合成本文所述的核酸分子。
将基因引入细胞和将基因表达入细胞的方法在本领域中是已知的。载体可通过在本领域中的任何方法容易地引入宿主细胞,例如,哺乳动物、细菌、酵母或昆虫细胞。例如,表达载体可通过物理、化学或生物学手段转移入宿主细胞。
将多核苷酸引入宿主细胞的物理方法包括磷酸钙沉淀、脂质转染法、粒子轰击、微注射、电穿孔等等。将感兴趣的多核苷酸引入宿主细胞的生物学方法包括使用DNA载体、RNA载体或病毒载体,例如慢病毒载体。将多核苷酸引入宿主细胞的化学手段包括胶体分散***,诸如大分子复合物、纳米胶囊、微球、珠;和基于脂质的***,包括水包油乳剂、胶束、混合胶束和脂质体。
本文中,宿主细胞含有、表达和/或分泌本文所述的蛋白亚基或蛋白。本文中,当提及细胞含有或包含、表达、分泌某种分子如多肽时,“含有”指所述所述分子含于所述细胞内或表面上;“表达”指该细胞生产所述分子;“分泌”指该细胞将所表达的分子分泌出细胞外。宿主细胞既包括最终用于分泌嵌合蛋白或其亚基的细胞,也包括生产RNA的细胞,还包括生产这些细胞过程中使用到的各种细胞,如大肠杆菌细胞,以用于如提供本发明蛋白的编码序列或提供本文所述的载体。宿主细胞表达本文所述的蛋白亚基或蛋白后,通过本领域常规纯化蛋白的方法(例如色谱法,包括亲和色谱、离子交换色谱等)可将所述蛋白亚基或蛋白纯化。
利用本发明的嵌合蛋白亚基或嵌合蛋白可进行RNA加帽过程中一个或多个步骤的化学修饰。因此,本发明提供一种将目标RNA修饰为加帽RNA的方法或制备加帽的目标RNA的方法,包括:使目标RNA与本文所述的嵌合蛋白接触,或使目 标RNA与表达本文所述的嵌合蛋白的细胞接触。
本文中,“目标RNA”是体内或体外获得感兴趣的RNA。通常,目标RNA是未加帽或未完全加帽的RNA。目标RNA可来自任何来源,例如体外转录(IVT,in vitro transcription)、化学合成、由体内提取等。体外转录是指使用带有T7、T3、SP6启动子DNA转录模板和对应的依赖于DNA的RNA聚合酶在合适的缓冲液体系中酶学反应合成RNA,缓冲体系中包括但不局限与NTP、Mg
2+、核糖核酸酶抑制剂(RI)、无机焦磷酸酶(iPPase)等成分。与固相合成RNA相比,体外转录合成可以高质量制备大量的长链RNA,适合于工业化生产mRNA药物。在一个或多个实施方案中,本文所述目标RNA是体外转录的无帽结构的单链RNA(ssRNA),其具有mRNA除帽结构外的所有特征,包括但不局限于5’-UTR,3’-UTR,蛋白或多肽翻译编码区。
RNA底物的体外转录方法本领域周知,示例性的聚合酶(例如T7、T3、SP6)转录方法包括将转聚合酶、rNTP和DNA转录模板(含能被聚合酶识别的启动子)混合孵育。反应体系中还包含选自以下的一种或多种试剂:MgCl
2、缓冲液、iPPase、抑制剂、无核酸酶水等。用于转录模板的DNA可通过本领域常规方法获得,例如合成、杂交、PCR等,这些方法中所需试剂也是本领域周知的。
不希望受理论限制,本文的目标RNA包括5'-三磷酸化的RNA、5'-二磷酸酯化的RNA和具有5'-Gppp结构的RNA。本文中,“加帽RNA”包括具有5'-m7Gppp结构的加帽RNA(cap0)或具有5'-m7GpppNmp结构的加帽RNA(cap1)。上述各结构中,“5'”表示基团位于RNA的5'端,“m”表示经甲基化,数字“7”表示甲基化位置,“G”是鸟苷酸,“p”是磷酸基团,“N”是位于目标RNA的5’端的任意核苷酸。任选地,目标RNA可经纯化和/或变性和复性的RNA。本领域已知纯化和变性、复性RNA的试剂和过程,例如将RNA样品在50-70℃孵育1-60分钟(例如65℃孵育5-20分钟),然后0℃孵育2-10分钟。不过,本发明方法中,即使不经纯化和变性、复性的过程也可以实现RNA的加帽。
为了使目标RNA与本文所述的嵌合蛋白接触,可以在表达本文所述嵌合蛋白的细胞中表达目标RNA(例如将表达目标RNA的DNA序列引入本文所述宿主细胞)。可用本领域已知的适合方法在细胞中表达目标RNA,例如将编码该RNA的DNA导入细胞中,DNA在细胞中经转录形成目标RNA,该目标RNA在该细胞表达的嵌合蛋白或嵌合蛋白亚基的催化下加帽。
或者,可将目标RNA(例如体外转录得到的RNA)与本文所述的嵌合蛋白在溶液中混合以实现接触。混合前,所述RNA可经纯化和/或变复性。本领域已知在溶液中进行RNA加帽的过程和条件,例如将无核酸酶水中的目标RNA与加帽缓冲液、 GTP、SAM、和本文的嵌合蛋白混合并孵育。孵育步骤例如37℃孵育30-90分钟。经修饰的加帽RNA产物可经纯化,例如通过磁珠纯化。
在本文所述的加帽方法中,目标RNA与所述嵌合蛋白的摩尔数比小于865:1,优选86.5-173:1。发明人发现,不同的摩尔数比可以获得不同的加帽RNA。例如,若目标RNA与所述嵌合蛋白的摩尔数比小于865:1,则所述方法将目标RNA修饰为具有5'-m7Gppp结构的加帽RNA(cap0)。若目标RNA与所述嵌合蛋白的摩尔数小于86.5-173:1,则所述方法将目标RNA修饰为具有5'-m7GpppNmp结构的加帽RNA(cap1)。
此外,针对不同的目标RNA,嵌合蛋白包含的结构域也可不同。若目标RNA为5'-三磷酸化的RNA,则需要嵌合蛋白具有5'-三磷酸酶、鸟苷转移酶、N7甲基转移酶、2-O-甲基转移酶的活性,即所述嵌合蛋白包含RNA加帽酶D1亚基的N7甲基转移酶(N7-MTase)结构域或其功能片段、5'-三磷酸酶(TPase)结构域或其功能片段和鸟苷基转移酶(GTase)结构域或其功能片段,和所述嵌合蛋白亚基。
若所述目标RNA为5'-二磷酸酯化的RNA,则需要嵌合蛋白具有鸟苷转移酶、N7甲基转移酶、2-O-甲基转移酶的活性,即所述嵌合蛋白包含RNA加帽酶D1亚基的N7甲基转移酶(N7-MTase)结构域或其功能片段和鸟苷基转移酶(GTase)结构域或其功能片段,和所述嵌合蛋白亚基。
若所述目标RNA是具有5'-Gppp结构的RNA,则需要嵌合蛋白具有N7甲基转移酶(N7-MTase)和2-O-甲基转移酶的活性,即所述嵌合蛋白包含RNA加帽酶D1亚基的N7甲基转移酶(N7-MTase)结构域或其功能片段和所述嵌合蛋白亚基。
在另一方面,本发明还可将具有5'-m7Gppp结构的加帽RNA(cap0)修饰为具有5'-m7GpppNmp结构的加帽RNA(cap1),包括步骤:使所述RNA与本文所述的嵌合蛋白亚基接触(例如在本文所述的表达本文所述嵌合蛋白亚基的细胞中表达所述RNA),或使所述RNA与表达本文所述的嵌合蛋白亚基的细胞接触。
本发明包含如下具体实施方式:
1、一种嵌合蛋白亚基,包含融合的(a)RNA加帽酶的D12亚基或其功能片段,或与之具有至少90%序列相同性并具有D12亚基活性的变体,和(b)RNA帽结构2′-O-甲基转移酶或其功能片段,或与之具有至少90%序列相同性并具有RNA帽结构2′-O-甲基转移活性的变体,以及任选的(c)(a)和(b)之间的接头,
优选地,
所述RNA加帽酶是牛痘病毒RNA加帽酶,
所述RNA帽结构2′-O-甲基转移酶是牛痘病毒2′-O-甲基转移酶,
(a)的羧基端与(b)的氨基端连接。
2、如项目1所述的嵌合蛋白亚基,其特征在于,
RNA加帽酶的D12亚基的氨基酸序列如SEQ ID NO:1第1-287所示,和/或
RNA帽结构2′-O-甲基转移酶的氨基酸序列如SEQ ID NO:1第303-635所示,和/或
RNA加帽酶的D12亚基或其功能片段的变体具有选自以下一个或多个的突变:N42A、Y43A、L61A、K62A、F245A、L246A、K111A、R112A、N120A、N121A、N126A、N127A、F141A、R142A、K223A、D224A、H260A、S261A、E275A、N276A、R280A、R281A,和/或
RNA帽结构2′-O-甲基转移酶或其功能片段的变体具有如下特征:(1)具有选自以下一个或多个的突变:K41D、C178S、A201R、A201K、C272S;和/或(2)其中的一个或多个选自R、K、H、Y、C、D或E的氨基酸突变为A,和/或
(b)还具有位于其N端或C端的His标签和/或MBP标签,
接头的氨基酸序列如SEQ ID NO:1第288-302位氨基酸所示,
优选地,嵌合蛋白亚基的氨基酸序列如SEQ ID NO:1所示。
3、一种嵌合蛋白,包含:(1)项目1或2所述的嵌合蛋白亚基,和(2)RNA加帽酶D1亚基或其功能片段、或与之具有至少90%序列相同性并具有相应活性的变体,
优选地,
所述嵌合蛋白是异二聚体蛋白,和/或
所述RNA加帽酶是牛痘病毒RNA加帽酶,和/或
RNA加帽酶D1亚基的功能片段包含:
(1)N7甲基转移酶结构域或其功能片段;
(2)N7甲基转移酶结构域或其功能片段和5'-三磷酸酶结构域或其功能片段;
(3)N7甲基转移酶结构域或其功能片段和鸟苷转移酶结构域或其功能片段;或
(4)N7甲基转移酶结构域或其功能片段、5'-三磷酸酶结构域或其功能片段和鸟苷转移酶结构域或其功能片段。
4、如项目3所述的嵌合蛋白,其特征在于,
5'-三磷酸酶结构域如SEQ ID NO:3第1-225位氨基酸所示,和/或
鸟苷转移酶结构域如SEQ ID NO:3第226-530位氨基酸所示,和/或
N7甲基转移酶结构域如SEQ ID NO:3第531-844位氨基酸所示,和/或
RNA加帽酶D1亚基如SEQ ID NO:3所示,和/或
RNA加帽酶D1亚基的功能片段包含SEQ ID NO:3的第498-844位氨基酸或第540-844位氨基酸,和/或
N7-甲基转移酶结构域或其功能片段的变体具有选自以下一个或多个的突变:D545A、R548A、N550D、Y555F、R560K、R794A、R808A、Y683S、Y684A、Y684F、D598A、G600A、G602A,I681A、S684A、F685A、T571A、L575A、L576A、M579A、F585A、L586A、D587A,、D784A、N785A、R794A、F798A、M805A、E806A。
5、一种核酸分子,包含选自以下的序列:
(1)项目1或2所述的嵌合蛋白亚基的编码序列,或项目3或4所述的嵌合蛋白的编码序列,
(2)与(1)具有至少80%序列相同性的变体,
(3)(1)或(2)的互补序列,
优选地,
所述嵌合蛋白亚基的编码序列如SEQ ID NO:2所示,或与其具有至少80%序列相同性的变体,或与其编码相同氨基酸序列的简并变体,和/或
所述嵌合蛋白的编码序列中,RNA加帽酶D1亚基或其功能片段的编码序列如SEQ ID NO:4所示,或与其具有至少80%序列相同性的变体,或与其编码相同氨基酸序列的简并变体。
6、一种核酸构建物,所述核酸构建物:
(1)表达项目1或2所述的嵌合蛋白亚基,或项目3或4所述的嵌合蛋白,
(2)包含项目5所述的核酸分子,
优选地,
所述核酸构建物含有所述嵌合蛋白亚基的表达框和RNA加帽酶D1亚基或其功能片段的表达框;或所述核酸构建物为一表达框,其中所述嵌合蛋白亚基的编码序列和所述RNA加帽酶D1亚基或其功能片段的编码序列处于该表达框内,和/或
所述核酸构建物是克隆载体或表达载体。
7、一种宿主细胞,所述宿主细胞包含、表达和/或分泌项目1或2所述的嵌合蛋白亚基,或项目3或4所述的嵌合蛋白,
优选地,所述宿主细胞包含项目5所述的核酸分子和/或项目6所述的核酸构建物。
8、一种将目标RNA修饰为加帽RNA的方法或制备加帽的目标RNA的方法,包括:在允许RNA加帽的条件下,使目标RNA与项目1或2所述的嵌合蛋白亚基 或项目3或4所述的嵌合蛋白接触,或使目标RNA与包含、表达和/或分泌项目1或2所述的嵌合蛋白亚基或项目3或4所述的嵌合蛋白的细胞接触,
优选地,
使目标RNA与嵌合蛋白亚基或嵌合蛋白接触包括在表达所述嵌合蛋白亚基或嵌合蛋白的细胞中表达目标RNA,或将目标RNA与所述嵌合蛋白亚基或嵌合蛋白混合,和/或
所述方法包括将表达目标RNA的DNA序列引入所述细胞的步骤。
9、如项目8所述的方法,其特征在于,所述方法包括步骤:
任选的(1)使目标RNA变性,
任选的(2)使目标RNA复性,
(3)在允许RNA加帽的条件下,使RNA与项目1或2所述的嵌合蛋白亚基或项目3或4所述的嵌合蛋白孵育,获得加帽的RNA,
任选的(4)纯化加帽的RNA,
优选地,
所述允许RNA加帽的条件是37℃孵育至少20分钟,和/或
所述允许RNA加帽的条件还包含存在选自以下的一种或多种试剂:GTP、SAM、缓冲液,和/或
所述目标RNA选自5'-三磷酸化的RNA、5'-二磷酸酯化的RNA和具有5'-Gppp帽结构的RNA。
10、如项目8或9所述的方法,其特征在于,
所述方法将目标RNA修饰为具有5'-m7Gppp结构的加帽RNA,所述目标RNA选自5'-三磷酸化的RNA、5'-二磷酸酯化的RNA和具有5'-Gppp帽结构的RNA,所述嵌合蛋白包含RNA加帽酶D1亚基的N7甲基转移酶结构域或其功能片段、5'-三磷酸酶结构域或其功能片段、鸟苷转移酶结构域或其功能片段,和所述嵌合蛋白亚基;优选地,所述嵌合蛋白包含如SEQ ID NO:3所示的RNA加帽酶D1亚基和如SEQ ID NO:1所示的嵌合蛋白亚基,
所述方法将目标RNA修饰为具有5'-m7GpppNmp结构的加帽RNA,所述目标RNA选自5'-三磷酸化的RNA、5'-二磷酸酯化的RNA和具有5'-Gppp帽结构的RNA,所述嵌合蛋白包含RNA加帽酶D1亚基的N7甲基转移酶(N7-MTase)结构域或其功能片段、5'-三磷酸酶(TPase)结构域或其功能片段和鸟苷转移酶(GTase)结构域或其功能片段,和所述嵌合蛋白亚基;优选地,所述嵌合蛋白包含如SEQ ID NO:3所示的RNA加帽酶D1亚基和如SEQ ID NO:1所示的嵌合蛋白亚基,
所述方法是将目标RNA加帽的方法,所述目标RNA选自5'-二磷酸酯化的RNA和具有5'-Gppp结构的RNA,所述嵌合蛋白包含RNA加帽酶D1亚基的N7甲基转移酶(N7-MTase)结构域或其功能片段和鸟苷转移酶(GTase)结构域或其功能片段,和所述嵌合蛋白亚基;优选地,所述嵌合蛋白亚基如SEQ ID NO:1所示,或
所述方法是将目标RNA加帽的方法,所述目标RNA是具有5'-Gppp结构的RNA,所述嵌合蛋白包含RNA加帽酶D1亚基的N7甲基转移酶(N7-MTase)结构域或其功能片段,和所述嵌合蛋白亚基;优选地,所述嵌合蛋白亚基如SEQ ID NO:1所示。
11、一种融合蛋白,包含
(a)RNA帽结构2′-O-甲基转移酶或其功能片段或与之具有至少90%序列相同性并具有RNA帽结构2′-O-甲基转移活性的变体,和
(b)位于(a)N端或C端的His标签和/或MBP标签,
优选地,
(a)和(b)之间具有接头,
所述RNA帽结构2′-O-甲基转移酶是牛痘病毒2′-O-甲基转移酶,
所述RNA帽结构2′-O-甲基转移酶的氨基酸序列如SEQ ID NO:1第303-635所示,
所述RNA帽结构2′-O-甲基转移酶或其功能片段的变体具有如下特征:(1)具有选自以下一个或多个的突变:K41D、C178S、A201R、A201K、C272S;和/或(2)其中的一个或多个选自R、K、H、Y、C、D或E的氨基酸突变为A,和/或
His标签如SEQ ID NO:8所示,和/或
MBP标签如SEQ ID NO:9所示。
12、编码项目11所述融合蛋白的核酸序列、包含所述核酸序列的核酸构建物或宿主细胞。
本发明优点:
1、兼具加帽酶和2′-O-甲基转移酶两种酶生物学活性的嵌合酶可以减少生产工艺,降低生产成本;
2、cap0加帽和cap1加帽为连续反应,嵌合酶将两种酶共价偶联在一起更有利于与RNA底物的连续接触,提高修饰效率。
除非另外定义,否则,本文中所使用的所有技术和科学术语都具有本发明所属领域普通技术人员通常所理解的同样含义。虽然可采用与本文所述类似或等同的任何 方法和材料实施或测试本发明,但现在描述优选的方法和材料。本文具体提及的所有出版物和专利都通过引用全文纳入本文用于所有目的,包括描述和公开所述出版物报道的可与本发明关联使用的化学物质、设备、统计分析和方法。本说明书引用的所有参考文献都应看作对本领域技术水平的指示。本文中所有内容均不应解释为承认本发明不能凭借在先发明而先于这些公开内容。
下面结合具体实施例,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法,通常按照常规条件如J.萨姆布鲁克等编著,分子克隆实验指南,第三版,科学出版社,2002中所述的条件,或按照制造厂商所建议的条件。
实施例
实施例1,天然牛痘病毒加帽酶、2-O-甲基转移酶、嵌合酶的表达和纯化
为了能够在大肠杆菌中分别过表达牛痘病毒加帽酶D1:D12、2-O-甲基转移酶2-O-MTase、D1:D12-(2-O-MTase)嵌合酶,我们分别根据上述蛋白所对应的氨基酸序列优化设计了DNA序列并克隆到pET28a(Novagen公司)、pMAL-c5X(NEB公司)、pRSF-Duet1(Novagen公司)商业化原核表达载体中。酶的氨基酸和DNA序列如SEQ ID NO:1-4所示,原核表达载体图参见图1。
为确保酶的高水平表达,并允许直接可重复地纯化牛痘病毒加帽酶或嵌合酶复合物,在D1酶N端上添加单个His标签以方便纯化完整的D1:D12或D1:D12-(2-O-MTase)蛋白复合物。为使2-O-甲基转移酶更稳定和利于纯化,在2-O-甲基转移酶的N端添加His标签和/或MBP标签。按照商业化表达载体说明书分别将质粒转化于大肠杆菌BL21(DE3)表达菌,挑取单菌落LB培养37度至对数生长期,添加终浓度为1mM IPTG诱导剂继续25度诱导培养16小时。收集诱导菌后所有酶蛋白都使用两个标准的纯化步骤进行蛋白纯化,分别是Ni亲和色谱,Capto SP ImpRes或Capto Q ImpRes(Cytiva公司)离子交换色谱分离得到最终的酶蛋白。将诱导前后菌体裂解液和最终纯化到的酶蛋白分别进行SDS-PAGE(聚丙烯酰胺凝胶电泳),其中图2为2-O-甲基转移酶的N端添加His和/或MBP标签菌体裂解液SDS-PAGE鉴定图,图3为牛痘病毒加帽酶和嵌合酶的菌体裂解液SDS-PAGE鉴定图,图4为纯化后MBP-(2-O-MTase)、牛痘病毒加帽酶D1:D12、嵌合酶D1:D12-((2-O-MTase))的SDS-PAGE鉴定图。
如图2所示、当2-O-甲基转移酶无MBP标签时蛋白表达不稳定,产量较低,因此在本发明中所述使用的2-O-甲基转移酶均为MBP标签形式的2-O-甲基转移酶即 MBP-(2-O-MTase)。如图3所示,表达天然牛痘病毒加帽酶时,D12蛋白亚基远高于D1蛋白亚基的表达量,不利于形稳定的1:1的稳定关系的D1:D12复合体;当将2-O-MTase与D12融合表达后,融合蛋白亚基与D1表达水平更接近,利于蛋白复合体D1:D12-(2-O-MTase)的形成。如图4所示,上述三种表达方案经纯化后都得到目标大小的蛋白或蛋白亚基,其中牛痘病毒加帽酶包含33kD的D12蛋白亚基和100kD的D1蛋白亚基;嵌合酶包括73kD的D12-(2-O-MTase)融合蛋白亚基和100kD的D1蛋白亚基,符合预期蛋白大小;MBP-(2-O-MTase)融合蛋白大小则为83kD,等于44kD的his-MBP蛋白标签和39kD的2-O-MTase蛋白之和,也符合预期蛋白大小。
实施例2,RNA底物的体外转录及酶学修饰合成cap0mRNA、cap1mRNA
设计并合成含有T7启动子序列引物如下,RNA30-T7F:5’-gataatacgactcactataGGGAAGGAGAGGAAGGAAAGGGAAGAAAGAA-3’(SEQ ID NO:5);RNA30-R:5’-TTCTTTCTTCCCTTTCCTTCCTCTCCTTCCCtatagtgagtcgtattatc-3’(SEQ ID NO:6)。按表1中比例混合引物进行退火反应,退火程序为:95℃预变性10min,2小时内梯度降温度至20℃,保持20℃一小时后取出。磁珠纯化后即为30nt RNA(RNA30)转录DNA模板。
表1,引物退火反应体系
试剂 | 体积 |
10×退火缓冲液 | 5μL |
RNA30-T7F(100μM) | 20μL |
RNA30-R(100μM) | 20μL |
无核糖核酸酶水 | 至50μL |
转录合成RNA:室温配制合适的T7RNA聚合酶反应体系按下表示范所示顺序加入反应成分,反应体系可按等比例扩大或缩小反应。37℃温育条件下反应6-16小时。本实施例中转录RNA为30nt RNA。2h,反应结束,加入2U DNase 1 37℃消化15min,然后进行磁珠纯化用于下一步的RNA加帽修饰酶学反应。
表2,RNA转录合成体系
加帽修饰RNA:取上述纯化后10ug RNA到1.5ml离心管,使用无核酸酶的水稀释至14ul;65℃加热10分钟后取出离心管置于冰上5分钟;根据不同的RNA修饰目的按照表3-6依次加入以下组分后37℃孵育30-90分钟。本步骤适用10ugRNA的加帽反应,且可根据实验需要按比例放大反应底物体积。反应完毕后可以采用磁珠纯化的方式获得最终的修饰RNA产物。
表3,无帽RNA酶学修饰加帽为cap0 RNA合成体系
表4,cap0 RNA酶学修饰加帽为cap1 RNA合成体系
组分 | 体积 |
变性的cap0 RNA | 10μg |
10*加帽缓冲液 | 2.0μl |
GTP(10mM) | 1.0μl |
SAM(32mM) | 1.0μl |
无核糖核酸酶水 | 至20μl |
MBP-(2-O-MTase) | Xμg(如图6所示) |
表5,无帽RNA酶学修饰加帽为cap1 RNA合成体系(天然酶)
组分 | 体积 |
变性的无帽RNA | 10μg |
10*加帽缓冲液 | 2.0μl |
GTP(10mM) | 1.0μl |
SAM(32mM) | 1.0μl |
牛痘病毒加帽酶D1:D12 | Xμg(如图7所示) |
MBP-(2-O-MTase) | Yμg(如图7所示) |
无核糖核酸酶水 | 至20μl |
表6,无帽RNA酶学修饰加帽为cap1 RNA合成体系(嵌合酶)
组分 | 体积 |
变性的无帽RNA | 10μg |
10*加帽缓冲液 | 2.0μl |
GTP(10mM) | 1.0μl |
SAM(32mM) | 1.0μl |
嵌合酶D1:D12-(2-O-MTase) | Xμg(如图8所示) |
无核糖核酸酶水 | 至20μl |
实施例3,HPLC-MS的方法检测不同RNA修饰酶催化RNA 5’加帽的效率
如表7中所示,实施例2中转录或酶学修饰的中间产物或最终产物的预期分子量是可以计算出来的,将这些样品分布送检进行HPLC-MS(液相色谱-质谱联用技术)检测。通过对比检测出的近似分子量大小,可以推测出这些RNA底物经过酶学催化反应后所产生目标产物的比例,进而推论出这些不同RNA修饰酶的酶催化活性高低。 结果如图5-8所示。
表7,不同帽子结构RNA30的预期分子量表
*注:实际质谱检测分子量与预测分子量有时会有约<5Da的偏差,但不影响目的产物的判断
图5结果表明:牛痘病毒加帽酶(约133kDa)大于0.1μg条件下,能够将预期分子量为10623Da的无帽三磷酸RNA30修饰转换为预期分子量为10903Da的cap0 RNA30,当酶剂量小于0.05μg时无帽三磷酸RNA30修饰不完全,只有部分底物转化为cap0 RNA30,少量底物转化为G-cap RNA30。即底物RNA:加帽酶摩尔比小于1330时,可以实现cap0完全加帽。(注:将RNA约等于10kDa,利于快速计算)
图6结果表明:2-O-甲基转移酶(约81kDa)大于0.5μg条件下,能够将预期分子量为10903Da的cap0 RNA30修饰转换为预期分子量为10918Da的cap1 RNA30,当酶剂量小于0.25μg时cap0 RNA30修饰不完全,只有部分底物转化为cap1 RNA30。即底物RNA:2-O-甲基转移酶摩尔比小于162时,可以实现cap0完全修饰为cap1帽子。
图7显示HPLC-MS分析牛痘病毒加帽酶和2-O-甲基转移酶协同修饰三磷酸RNA30单位活性。基于加帽酶修饰三磷酸RNA30为cap0 RNA30的检测结果,本实验组中各组统一加入0.2μg的牛痘加帽酶确保三磷酸RNA30完全转化为cap0 RNA30,2-O-甲基转移酶则以0.25μg、0.5μg、1μg、2μg梯度添加。图7结果表明:2-O-甲基转移酶添加量0.25ug时,只有一半底物转化为cap1 RNA30;2-O-甲基转移酶添加量0.5ug时,仍然有少量的底物未转化为cap1 RNA30;2-O-甲基转移酶添加量大于1ug时,协同修饰RNA30才能完全转化为cap1 RNA。这表明一步法修饰RNA为cap1比分步修饰RNA为cap1结构需要更多的2-O甲基转移酶蛋白。协同加帽体系中,底 物RNA:加帽酶摩尔比小于665:1时,可以实现cap0完全加帽,RNA:2-O-甲基转移酶摩尔比介于81-162:1时,可以实现cap0完全修饰为cap1帽子,这表明协同加帽反应体系与分步加帽反应体系中的酶使用量是类似的。
图8结果表明:嵌合酶(173kDa,133kDa+39kDa)在0.1-0.2μg条件下,能够将预期分子量为10623Da的无帽三磷酸RNA30修饰完全转换为预期分子量为10903Da的cap0 RNA30;其中0.1μg条件下已能够将90%左右的RNA30完全加帽。这说明嵌合酶完整的保留了野生型牛痘病毒加帽酶中5'-三磷酸酶、鸟苷转移酶、N7-鸟嘌呤甲基转移酶三种酶催化结构域的活性,使用剂量与单独野生型酶使用剂量相当。当嵌合酶使用剂量达到2μg时,能够将已转化为10903Da的cap0 RNA30继续完全修饰转换为预期分子量为10918Da的cap1 RNA30。这表明嵌合酶不仅完整的保留了2-O-甲基转移酶催化结构域的活性,而且可以更高效彻底地催化获得cap1 RNA30。即嵌合酶加帽体系中,底物RNA:嵌合酶摩尔比小于865:1时,可以实现cap0完全加帽;RNA:嵌合摩尔比介于86.5-173:1时,可以实现cap0完全修饰为cap1帽子。上述指标皆与混合酶体系的摩尔数比指标665:1,81-162:1相当,表明在同样的摩尔浓度下嵌合酶可以实现混合酶的相同功能,即证明嵌合酶不改变各功能结构域的催化活性,有利于实现酶的简化生产和使用(使用嵌合酶与使用两种酶相比,只需生产一种酶,节约步骤成本,而且反应时只需添加一种酶)。
实施例4,野生型酶与嵌合酶分别修饰制备eGFP mRNA的细胞生物学功能比较
基因合成eGFPmRNA转录用模板DNA(SEQ ID NO:7)并亚克隆于商业化载体,例如pUC57等。将质粒转化于Top10大肠杆菌中,提取质粒然后用限制性内切酶进行线性化,磁珠回收DNA线性模板用于RNA转录合成试验。eGFP mRNA的转录合成体系和步骤参见实施例2中的表2所述,转录合成后酶学修饰制备cap1 eGFP mRNA的反应体系和步骤参见实施例2中的表5、6所述。分别取鉴定完全加帽修饰的mRNA样品进行转染试验,将天然牛痘病毒加帽酶联合2-O-甲基转移酶修饰RNA制备的cap1 eGFP mRNA编号为1号样品,将嵌合酶修饰RNA制备的cap1 eGFP mRNA编号为2号样品,将未用酶修饰的无帽eGFP mRNA编号为3号样品。将上述样品按如下细胞转染程序转染中国仓鼠卵巢(CHO)细胞以验证修饰cap1 eGFP mRNA的细胞生物学功能。转染程序:(1)转染前一天将CHO细胞进行消化,铺板,24孔板,铺板密度为1x105/孔。(2)转染时分别取2个1.5ml的EP管,每管分别加入50ulopti-MEM培养液,在一管中加入2.25ul的PEI转染试剂,另一管中加 入1.5ug剂量的mRNA,静置5min后,将PEI溶液加入到mRNA溶液中,混合均匀后静置20min。(3)将配置好的PEI-mRNA溶液加入到含有无血清培养液的CHO细胞中,放置于37度培养箱中,培养4小时后换成完全培养基继续培养。(4)培养24小时后,进行荧光拍照,检测流式,检测EGFP阳性率。三个样品均做复孔平行试验组。
结果如图9和表8所示,1号和2号eGFP mRNA样品转染CHO细胞的绿色荧光阳性率和荧光强度均处于同一水平,无明显差异。这说明单一使用嵌合酶修饰可以达到天然牛痘病毒加帽酶联合2-O-甲基转移酶修饰RNA的同样效果。
表8,不同mRNA样品转染细胞的EGFP阳性率及荧光强度
Claims (12)
- 一种嵌合蛋白亚基,包含融合的(a)RNA加帽酶的D12亚基或其功能片段,或与之具有至少90%序列相同性并具有D12亚基活性的变体,和(b)RNA帽结构2′-O-甲基转移酶或其功能片段,或与之具有至少90%序列相同性并具有RNA帽结构2′-O-甲基转移活性的变体,以及任选的(c)(a)和(b)之间的接头,优选地,所述RNA加帽酶是牛痘病毒RNA加帽酶,所述RNA帽结构2′-O-甲基转移酶是牛痘病毒2′-O-甲基转移酶,(a)的羧基端与(b)的氨基端连接。
- 如权利要求1所述的嵌合蛋白亚基,其特征在于,RNA加帽酶的D12亚基的氨基酸序列如SEQ ID NO:1第1-287所示,和/或RNA帽结构2′-O-甲基转移酶的氨基酸序列如SEQ ID NO:1第303-635所示,和/或RNA加帽酶的D12亚基或其功能片段的变体具有选自以下一个或多个的突变:N42A、Y43A、L61A、K62A、F245A、L246A、K111A、R112A、N120A、N121A、N126A、N127A、F141A、R142A、K223A、D224A、H260A、S261A、E275A、N276A、R280A、R281A,和/或RNA帽结构2′-O-甲基转移酶或其功能片段的变体具有如下特征:(1)具有选自以下一个或多个的突变:K41D、C178S、A201R、A201K、C272S;和/或(2)其中的一个或多个选自R、K、H、Y、C、D或E的氨基酸突变为A,和/或(b)还具有位于其N端或C端的His标签和/或MBP标签,接头的氨基酸序列如SEQ ID NO:1第288-302位氨基酸所示,优选地,嵌合蛋白亚基的氨基酸序列如SEQ ID NO:1所示。
- 一种嵌合蛋白,包含:(1)权利要求1或2所述的嵌合蛋白亚基,和(2)RNA加帽酶D1亚基或其功能片段、或与之具有至少90%序列相同性并 具有相应活性的变体,优选地,所述嵌合蛋白是异二聚体蛋白,和/或所述RNA加帽酶是牛痘病毒RNA加帽酶,和/或RNA加帽酶D1亚基的功能片段包含:(1)N7甲基转移酶结构域或其功能片段;(2)N7甲基转移酶结构域或其功能片段和5'-三磷酸酶结构域或其功能片段;(3)N7甲基转移酶结构域或其功能片段和鸟苷转移酶结构域或其功能片段;或(4)N7甲基转移酶结构域或其功能片段、5'-三磷酸酶结构域或其功能片段和鸟苷转移酶结构域或其功能片段。
- 如权利要求3所述的嵌合蛋白,其特征在于,5'-三磷酸酶结构域如SEQ ID NO:3第1-225位氨基酸所示,和/或鸟苷转移酶结构域如SEQ ID NO:3第226-530位氨基酸所示,和/或N7甲基转移酶结构域如SEQ ID NO:3第531-844位氨基酸所示,和/或RNA加帽酶D1亚基如SEQ ID NO:3所示,和/或RNA加帽酶D1亚基的功能片段包含SEQ ID NO:3的第498-844位氨基酸或第540-844位氨基酸,和/或N7-甲基转移酶结构域或其功能片段的变体具有选自以下一个或多个的突变:D545A、R548A、N550D、Y555F、R560K、R794A、R808A、Y683S、Y684A、Y684F、D598A、G600A、G602A,I681A、S684A、F685A、T571A、L575A、L576A、M579A、F585A、L586A、D587A,、D784A、N785A、R794A、F798A、M805A、E806A。
- 一种核酸分子,包含选自以下的序列:(1)权利要求1或2所述的嵌合蛋白亚基的编码序列,或权利要求3或4所述的嵌合蛋白的编码序列,(2)与(1)具有至少80%序列相同性的变体,(3)(1)或(2)的互补序列,优选地,所述嵌合蛋白亚基的编码序列如SEQ ID NO:2所示,或与其具有至少80%序列相同性的变体,或与其编码相同氨基酸序列的简并变体,和/或所述嵌合蛋白的编码序列中,RNA加帽酶D1亚基或其功能片段的编码序列如SEQ ID NO:4所示,或与其具有至少80%序列相同性的变体,或与其编码相同氨基酸序列的简并变体。
- 一种核酸构建物,所述核酸构建物:(1)表达权利要求1或2所述的嵌合蛋白亚基,或权利要求3或4所述的嵌合蛋白,(2)包含权利要求5所述的核酸分子,优选地,所述核酸构建物含有所述嵌合蛋白亚基的表达框和RNA加帽酶D1亚基或其功能片段的表达框;或所述核酸构建物为一表达框,其中所述嵌合蛋白亚基的编码序列和所述RNA加帽酶D1亚基或其功能片段的编码序列处于该表达框内,和/或所述核酸构建物是克隆载体或表达载体。
- 一种宿主细胞,所述宿主细胞包含、表达和/或分泌权利要求1或2所述的嵌合蛋白亚基,或权利要求3或4所述的嵌合蛋白,优选地,所述宿主细胞包含权利要求5所述的核酸分子和/或权利要求6所述的核酸构建物。
- 一种将目标RNA修饰为加帽RNA的方法或制备加帽的目标RNA的方法,包括:在允许RNA加帽的条件下,使目标RNA与权利要求1或2所述的嵌合蛋白亚基或权利要求3或4所述的嵌合蛋白接触,或使目标RNA与包含、表达和/或分泌权利要求1或2所述的嵌合蛋白亚基或权利要求3或4所述的嵌合蛋白的细胞接触,优选地,使目标RNA与嵌合蛋白亚基或嵌合蛋白接触包括在表达所述嵌合蛋白亚基或嵌合蛋白的细胞中表达目标RNA,或将目标RNA与所述嵌合蛋白亚基或嵌合蛋白混合,和/或所述方法包括将表达目标RNA的DNA序列引入所述细胞的步骤。
- 如权利要求8所述的方法,其特征在于,所述方法包括步骤:任选的(1)使目标RNA变性,任选的(2)使目标RNA复性,(3)在允许RNA加帽的条件下,使RNA与权利要求1或2所述的嵌合蛋白亚基或权利要求3或4所述的嵌合蛋白孵育,获得加帽的RNA,任选的(4)纯化加帽的RNA,优选地,所述允许RNA加帽的条件是37℃孵育至少20分钟,和/或所述允许RNA加帽的条件还包含存在选自以下的一种或多种试剂:GTP、SAM、缓冲液,和/或所述目标RNA选自5'-三磷酸化的RNA、5'-二磷酸酯化的RNA和具有5'-Gppp帽结构的RNA。
- 如权利要求8或9所述的方法,其特征在于,所述方法将目标RNA修饰为具有5'-m7Gppp结构的加帽RNA,所述目标RNA选自5'-三磷酸化的RNA、5'-二磷酸酯化的RNA和具有5'-Gppp帽结构的RNA,所述嵌合蛋白包含RNA加帽酶D1亚基的N7甲基转移酶结构域或其功能片段、5'-三磷酸酶结构域或其功能片段、鸟苷转移酶结构域或其功能片段,和所述嵌合蛋白亚基;优选地,所述嵌合蛋白包含如SEQ ID NO:3所示的RNA加帽酶D1亚基和如SEQ ID NO:1所示的嵌合蛋白亚基,所述方法将目标RNA修饰为具有5'-m7GpppNmp结构的加帽RNA,所述目标RNA选自5'-三磷酸化的RNA、5'-二磷酸酯化的RNA和具有5'-Gppp帽结构的RNA,所述嵌合蛋白包含RNA加帽酶D1亚基的N7甲基转移酶(N7-MTase)结构域或其功能片段、5'-三磷酸酶(TPase)结构域或其功能片段和鸟苷转移酶(GTase)结构域或其功能片段,和所述嵌合蛋白亚基;优选地,所述嵌合蛋白包含如SEQ ID NO:3所示的RNA加帽酶D1亚基和如SEQ ID NO:1所示的嵌合蛋白亚基,所述方法是将目标RNA加帽的方法,所述目标RNA选自5'-二磷酸酯化的RNA和具有5'-Gppp结构的RNA,所述嵌合蛋白包含RNA加帽酶D1亚基 的N7甲基转移酶(N7-MTase)结构域或其功能片段和鸟苷转移酶(GTase)结构域或其功能片段,和所述嵌合蛋白亚基;优选地,所述嵌合蛋白亚基如SEQ ID NO:1所示,或所述方法是将目标RNA加帽的方法,所述目标RNA是具有5'-Gppp结构的RNA,所述嵌合蛋白包含RNA加帽酶D1亚基的N7甲基转移酶(N7-MTase)结构域或其功能片段,和所述嵌合蛋白亚基;优选地,所述嵌合蛋白亚基如SEQ ID NO:1所示。
- 一种融合蛋白,包含(a)RNA帽结构2′-O-甲基转移酶或其功能片段或与之具有至少90%序列相同性并具有RNA帽结构2′-O-甲基转移活性的变体,和(b)位于(a)N端或C端的His标签和/或MBP标签,优选地,(a)和(b)之间具有接头,所述RNA帽结构2′-O-甲基转移酶是牛痘病毒2′-O-甲基转移酶,所述RNA帽结构2′-O-甲基转移酶的氨基酸序列如SEQ ID NO:1第303-635所示,所述RNA帽结构2′-O-甲基转移酶或其功能片段的变体具有如下特征:(1)具有选自以下一个或多个的突变:K41D、C178S、A201R、A201K、C272S;和/或(2)其中的一个或多个选自R、K、H、Y、C、D或E的氨基酸突变为A,和/或His标签如SEQ ID NO:8所示,和/或MBP标签如SEQ ID NO:9所示。
- 编码权利要求11所述融合蛋白的核酸序列、包含所述核酸序列的核酸构建物或宿主细胞。
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