CN115197327A - RNA modified chimeric protein and application thereof - Google Patents

RNA modified chimeric protein and application thereof Download PDF

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Publication number
CN115197327A
CN115197327A CN202110379093.3A CN202110379093A CN115197327A CN 115197327 A CN115197327 A CN 115197327A CN 202110379093 A CN202110379093 A CN 202110379093A CN 115197327 A CN115197327 A CN 115197327A
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rna
subunit
chimeric protein
functional fragment
methyltransferase
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张平静
董颖颖
蒋婷
邵梅琪
孙娟娟
仇凯军
刘韬
高海霞
钱其军
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Shanghai Cell Therapy Group Co Ltd
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Shanghai Cell Therapy Group Co Ltd
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Priority to CN202110379093.3A priority Critical patent/CN115197327A/en
Priority to PCT/CN2022/085760 priority patent/WO2022214065A1/en
Publication of CN115197327A publication Critical patent/CN115197327A/en
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Abstract

The present invention provides a chimeric protein subunit comprising, linked to each other, (a) a D12 subunit of RNA capping enzyme or a functional fragment thereof, or a variant having at least 90% sequence identity thereto and having D12 subunit activity, and (b) an RNA cap structure 2 '-O-methyltransferase or a functional fragment thereof, or a variant having at least 90% sequence identity thereto and having RNA cap structure 2' -O-methyltransferase activity. The invention also provides chimeric proteins comprising the chimeric protein subunit and an RNA capping enzyme D1 subunit or a functional fragment thereof.

Description

RNA modified chimeric protein and application thereof
Technical Field
The invention relates to the field of RNA synthesis, in particular to a chimeric protein for modifying RNA and application thereof.
Background
In Vitro Transcription (IVT) of synthetic mRNA has a significant advantage in biosafety over DNA-based therapeutics due to the difficulty of integrating RNA into the genome, the short time window for RNA to express translated proteins, and the better control of the initiation time and amount of expression of the protein of interest without resorting to intracellular transcription steps.
Therapeutic mRNAs mimic primarily the natural structure of eukaryotic mRNAs and are characterized primarily by the presence of a cap at the 5-terminus, which consists of the first transcribed nucleotide of the RNA linked to inverted 7-methylguanosine (m 7G) and linked to the first nucleotide by a triphosphate bridge to form a structure called cap0 (cap 0, shown in FIG. 10, C). Cap0 is sufficient to recruit translation initiation factors and prevent mRNA degradation. Then, 2' -hydroxymethyl of the first cap proximal nucleotide forms cap1 (cap 1, shown in FIG. 10, C). The Cap0 structure has the main functions of: modulation of splicing; outputting a core; stabilizing the mRNA structure by protecting the mRNA from 5' -exonuclease; assisting innate immunity in recognizing "self-RNA" serves as an anchor for the recruitment of promoters, initiating protein synthesis and 5 'to 3' circularization of mRNA during translation. The cap1 structure is closely related to the recognition of the innate immune system, and the host immune protein can recognize abnormal non-capped RNA.
Because IVT does not directly produce eukaryotic capped mRNA with biological functions, but only 5' -triphosphorylated RNA, the RNA produced by it requires different strategies to effect capping of the RNA. At present, two production strategies for preparing mRNA with a cap mainly exist, one is a cap analogue co-transcription capping mode, and the other is a post-transcription capping enzyme modified RNA capping mode.
In the cap analog co-transcription capping mode, where the cap analog is added directly to the IVT, an RNA polymerase with relaxed substrate specificity (e.g., T3, T7 or SP6 RNA polymerase) can incorporate the cap analog at the 5 'end of the RNA at the start of transcription, directly producing the corresponding 5' cap mRNA. Limitations of co-transcriptional capping include: competition for GTP with cap analogs results in only partial capping of mRNA obtained from IVT, and capped/uncapped mRNA is often difficult to isolate and ratioed. Another problem with m7GpppG as a co-transcribed cap analog is that it results in RNA extension in the "wrong" direction, i.e., at the 3' -OH of m7G, resulting in a misdirected mRNA cap.
Post-transcriptional capping enzyme-modified RNA capping means that the RNA product generated by IVT is subjected to enzymatic capping reaction with a specific capping enzyme and/or mRNA modifying enzyme in a suitable reaction system. The enzymatic reaction of cap0 comprises three successive reaction steps: first, 5' -triphosphatase (TPase) hydrolyzes the gamma-phosphate of RNA; next, guanosine transferase (GTase) couples the resulting 5' -diphosphate terminated β -phosphate with GMP using GTP as a substrate to form 5' -5' -linked Gppp-RNA; finally, RNA (guanine-N7) methyltransferases (N7-MTases) catalyze the methylation of the cap structure at the N7 position using S-adenosyl-L-methionine (AdoMet) as a substrate. Finally, m7G cap-specific 2' -O-methyltransferase (2-O-MTase) modifies the ribose on the first nucleotide to generate the cap1 structure. As shown in figure 10, a.
Although many capping enzymes and mRNA modifying enzymes derived from eukaryotes or viruses have been reported in the literature as having a cap modifying function, there are few enzymes that can be applied to mass production of cap-structured mRNA. There remains a need in the art for capping enzymes that combine the properties of high efficiency and low cost.
Disclosure of Invention
The present invention aims to provide a protein or protein subunit for capping modification of RNA and a method for modifying RNA using the same. The technical scheme of the invention simplifies the production process of the mRNA production related modified enzyme and reduces the mRNA production added raw materials.
In a first aspect, the invention provides a chimeric protein subunit comprising, linked to each other, (a) the D12 subunit of an RNA capping enzyme or a functional fragment thereof, or a variant having at least 90% sequence identity thereto and having D12 subunit activity, and (b) an RNA cap structure 2 '-O-methyltransferase or a functional fragment thereof, or a variant having at least 90% sequence identity thereto and having RNA cap structure 2' -O-methyltransferase activity, and optionally (c) a linker between (a) and (b).
In one or more embodiments, the RNA capping enzyme is a vaccinia virus RNA capping enzyme.
In one or more embodiments, the RNA cap structure 2 '-O-methyltransferase is a vaccinia virus 2' -O-methyltransferase.
In one or more embodiments, the carboxy terminus of (a) is linked to the amino terminus of (b).
In one or more embodiments, (b) further has a His-tag and/or a MBP-tag at its N-or C-terminus. In one or more embodiments, the His tag is set forth in SEQ ID NO 8. In one or more embodiments, the MBP tag is set forth in SEQ ID NO 9.
In one or more embodiments, the amino acid sequence of the D12 subunit of the RNA capping enzyme is shown in SEQ ID NO 1, items 1-287.
In one or more embodiments, the variant of the D12 subunit of the RNA capping enzyme, or a functional fragment thereof, has a mutation selected from one or more of: N42A, Y43A, L61A, K62A, F245A, L246A, K111A, R112A, N120A, N121A, N126A, N127A, F141A, R142A, K223A, D224A, H260A, S261A, E275A, N276A, R280A, R281A.
In one or more embodiments, the amino acid sequence of the RNA cap structure 2' -O-methyltransferase is shown in SEQ ID NO. 1, items 303-635.
In one or more embodiments, the variant of the RNA cap structure 2' -O-methyltransferase or a functional fragment thereof has the following characteristics:
(1) A mutation selected from one or more of: 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.
In one or more embodiments, a variant of the RNA cap structure 2' -O-methyltransferase or a functional fragment thereof is set forth in SEQ ID No. 1, items 303-635, and has a mutation selected from 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.
In one or more embodiments, the amino acid sequence of the linker is as set forth in amino acids 288-302 of SEQ ID NO 1.
In one or more embodiments, the amino acid sequence of a subunit of the chimeric protein is set forth in SEQ ID NO 1.
In a second aspect, the present invention provides a chimeric protein comprising: (1) A chimeric protein subunit according to any one of the embodiments of the first aspect herein, and (2) an RNA capping enzyme D1 subunit or a functional fragment thereof, or a variant thereof having at least 90% sequence identity thereto and corresponding activity.
In one or more embodiments, the protein is a heterodimeric protein of (1) and (2).
In one or more embodiments, the RNA capping enzyme is a vaccinia virus RNA capping enzyme.
In one or more embodiments, the functional fragment of a D1 subunit of an RNA capping enzyme comprises:
(1) An N7 methyltransferase (N7-MTase) domain or a functional fragment thereof;
(2) An N7 methyltransferase (N7-MTase) domain or a functional fragment thereof and a5' -triphosphatase (TPase) domain or a functional fragment thereof;
(3) An N7 methyltransferase (N7-MTase) domain or a functional fragment thereof and a guanosine transferase (GTase) domain or a functional fragment thereof; or
(4) An N7 methyltransferase (N7-MTase) domain or a functional fragment thereof, a5' -triphosphatase (TPase) domain or a functional fragment thereof, and a guanosine transferase (GTase) domain or a functional fragment thereof.
In one or more embodiments, the RNA capping enzyme D1 subunit is set forth in SEQ ID NO 3.
In one or more embodiments, a functional fragment of the D1 subunit of the RNA capping enzyme comprises amino acids 498-844 or 540-844 of SEQ ID NO 3.
In one or more embodiments, the 5' -triphosphatase (TPase) domain is represented by amino acids 1-225 of SEQ ID NO 3.
In one or more embodiments, the guanosine transferase (GTase) domain is shown as amino acids 226-530 of SEQ ID NO: 3.
In one or more embodiments, the N7 methyltransferase (N7-MTase) domain is depicted as amino acids 531-844 of SEQ ID NO: 3.
In one or more embodiments, the variant of the N7-methyltransferase domain, or functional fragment thereof, has a mutation selected from one or more of: 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 invention also provides a nucleic acid molecule comprising a sequence selected from:
(1) A coding sequence for a subunit of a chimeric protein as described in the first aspect herein,
(2) A variant having at least 80% sequence identity to (1),
(3) The complementary sequence of (1) or (2).
In one or more embodiments, the nucleic acid molecule has the sequence shown 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 invention also provides a nucleic acid molecule comprising a sequence selected from
(1) The coding sequence for a subunit of a chimeric protein as described in the first aspect herein, and the coding sequence for a subunit of RNA capping enzyme D1, or a functional fragment or variant thereof,
(2) A variant having at least 80% sequence identity to (1),
(3) The complementary sequence of (1) or (2).
In one or more embodiments, the RNA capping enzyme is a vaccinia virus RNA capping enzyme.
In one or more embodiments, the functional fragment of the D1 subunit of the RNA capping enzyme comprises:
(1) An N7 methyltransferase (N7-MTase) domain or a functional fragment thereof;
(2) An N7 methyltransferase (N7-MTase) domain or a functional fragment thereof and a5' -triphosphatase (TPase) domain or a functional fragment thereof;
(3) An N7 methyltransferase (N7-MTase) domain or a functional fragment thereof and a guanosine transferase (GTase) domain or a functional fragment thereof; or
(4) An N7 methyltransferase (N7-MTase) domain or a functional fragment thereof, a5' -triphosphatase (TPase) domain or a functional fragment thereof, and a guanosine transferase (GTase) domain or a functional fragment thereof.
In one or more embodiments, the RNA capping enzyme D1 subunit is set forth in SEQ ID NO 3.
In one or more embodiments, a functional fragment of the D1 subunit of the RNA capping enzyme comprises amino acids 498-844 or 540-844 of SEQ ID NO 3.
In one or more embodiments, the 5' -triphosphatase (TPase) domain is shown as amino acids 1-225 of SEQ ID NO 3.
In one or more embodiments, the guanosine transferase (GTase) domain is as set forth in SEQ ID NO 3 amino acids 226-530.
In one or more embodiments, the N7 methyltransferase (N7-MTase) domain is set forth in amino acids 531-844 of SEQ ID NO: 3.
In one or more embodiments, the coding sequence for the D1 subunit of an RNA capping enzyme, or a functional fragment thereof, is as set forth in SEQ ID NO. 4, or a variant having at least 80% sequence identity thereto, or a degenerate variant encoding the same amino acid sequence therewith.
In one or more embodiments, the coding sequences for the subunits of the chimeric protein are set forth in SEQ ID NO 2.
In another aspect, the invention provides a nucleic acid construct comprising:
(1) Expressing a subunit of the chimeric protein of the first aspect, or the chimeric protein of the second aspect,
(2) Comprising a nucleic acid molecule as described herein.
In one or more embodiments, the nucleic acid construct comprises an expression cassette for the chimeric protein subunit and an expression cassette for a D1 subunit of RNA capping enzyme, or a functional fragment thereof; or the nucleic acid construct is an expression cassette, wherein the coding sequence for the chimeric protein subunit and the coding sequence for the RNA capping enzyme D1 subunit or a functional fragment thereof are in the expression cassette.
In one or more embodiments, the nucleic acid construct is a cloning vector or an expression vector.
In another aspect, the invention provides a host cell comprising, expressing and/or secreting a chimeric protein subunit or a chimeric protein as described herein.
In one or more embodiments, the host cell comprises a nucleic acid molecule, nucleic acid construct, as described herein.
The present invention also provides a method of modifying a target RNA to a capped RNA or a method of preparing a capped target RNA, comprising: contacting a target RNA with a chimeric protein described herein, or contacting a target RNA with a cell expressing a chimeric protein described herein, under conditions that allow the RNA to be catalytically capped.
In one or more embodiments, the conditions that allow RNA capping are incubation at 37 ℃ for at least 20 minutes.
In one or more embodiments, contacting the RNA of interest with the chimeric proteins described herein comprises expressing the RNA of interest in cells expressing the chimeric proteins described herein, or mixing the RNA of interest with the chimeric proteins described herein.
In one or more embodiments, the method comprises the step of introducing a DNA sequence expressing a target RNA into the host cell.
In one or more embodiments, the method comprises the steps of:
optionally (1) denaturing the target RNA, e.g.by incubation at 50-70 ℃ for 1-60 minutes, preferably at 65 ℃ for 5-20 minutes,
optionally (2) renaturing the target RNA of the product of (1), e.g., incubating at 0 ℃ for 2-10 minutes
(3) Incubating the RNA with the chimeric protein described herein for at least 20 minutes, e.g., 30-90 minutes, at 37 deg.C to obtain capped RNA,
optionally (4) purifying the capped RNA.
In one or more embodiments, the incubated mixture further comprises one or more agents selected from the group consisting of: GTP, SAM and buffer solution.
In one or more embodiments, the target RNA is selected from the group consisting of 5' -triphosphorylated RNA, 5' -diphosphonated RNA, and RNA having a5' -Gppp cap structure.
In one or more embodiments, the method modifies a target RNA selected from the group consisting of 5' -m7 Gppp-structured capped RNA, 5' -diphosphonated RNA, and RNA having a5' -Gppp cap structure into a capped RNA having a5' -m7Gppp structure, and the chimeric protein comprises an N7 methyltransferase domain of an RNA capping enzyme D1 subunit or a functional fragment thereof, a5' -triphosphatase domain or a functional fragment thereof, a guanosine transferase domain or a functional fragment thereof, and the chimeric protein subunit. Preferably, the chimeric protein comprises an RNA capping enzyme D1 subunit shown as SEQ ID NO. 3 and a chimeric protein subunit shown as SEQ ID NO. 1.
In one or more embodiments, the methods modify a target RNA selected from the group consisting of 5' -m7 gppnmp-structured capped RNA, 5' -diphosphonated RNA, and RNA having a5' -Gppp cap structure into a capped RNA having a5' -m7 gppnmp structure, the chimeric protein comprising an N7 methyltransferase (N7-MTase) domain of a D1 subunit of RNA capping enzyme or a functional fragment thereof, a5' -triphosphatase (TPase) domain or a functional fragment thereof, and a guanosine transferase (GTase) domain or a functional fragment thereof, and the chimeric protein subunit. Preferably, the chimeric protein comprises an RNA capping enzyme D1 subunit shown as SEQ ID NO. 3 and a chimeric protein subunit shown as SEQ ID NO. 1.
In one or more embodiments, the method is a method of capping a target RNA selected from the group consisting of 5 '-diphosphonated RNA and RNA having a5' -Gppp structure, the chimeric protein comprises an N7 methyltransferase (N7-MTase) domain of the D1 subunit of RNA capping enzyme or a functional fragment thereof and a guanosine transferase (GTase) domain or a functional fragment thereof, and the chimeric protein subunits. Preferably, the subunit of the chimeric protein is shown in SEQ ID NO 1.
In one or more embodiments, the method is a method of capping a target RNA that is an RNA having a5' -Gppp structure, the chimeric protein comprises an N7 methyltransferase (N7-MTase) domain of an RNA capping enzyme D1 subunit, or a functional fragment thereof, and the chimeric protein subunit. Preferably, the subunit of the chimeric protein is shown in SEQ ID NO 1.
In one or more embodiments, the 5' -triphosphatase (TPase) domain is represented by amino acids 1-225 of SEQ ID NO 3.
In one or more embodiments, the guanosine transferase (GTase) domain is as set forth in SEQ ID NO 3 amino acids 226-530.
In one or more embodiments, the N7 methyltransferase (N7-MTase) domain is depicted as amino acids 531-844 of SEQ ID NO: 3.
The invention also provides a method for methylating RNA with a5' -m7Gppp structure, which comprises the following steps: contacting the RNA with a chimeric protein subunit described herein, or contacting the RNA with a cell expressing a chimeric protein subunit described herein.
In one or more embodiments, contacting the RNA with a chimeric protein subunit described herein comprises expressing the RNA in a cell described herein that expresses the chimeric protein subunit described herein.
In a third aspect, the invention provides a fusion protein comprising
(a) An RNA cap structure 2 '-O-methyltransferase or a functional fragment thereof or a variant having at least 90% sequence identity thereto and having RNA cap structure 2' -O-methyltransferase activity, and
(b) His tag and/or MBP tag at (a) N-or C-terminus.
In one or more embodiments, there is a linker between (a) and (b).
In one or more embodiments, the RNA cap structure 2 '-O-methyltransferase is a vaccinia virus 2' -O-methyltransferase.
In one or more embodiments, the amino acid sequence of the RNA cap structure 2' -O-methyltransferase is shown in SEQ ID NO. 1, items 303-635.
In one or more embodiments, the variant of the RNA cap structure 2' -O-methyltransferase or a functional fragment thereof has the following characteristics:
(1) A mutation selected from one or more of: 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.
In one or more embodiments, the variant of the RNA cap structure 2' -O-methyltransferase, or a functional fragment thereof, is set forth in SEQ ID No. 1, items 303-635, and has a mutation selected from 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.
In one or more embodiments, the His tag is set forth in SEQ ID NO 8.
In one or more embodiments, the MBP tag is set forth in SEQ ID NO 9.
The invention also provides a nucleic acid sequence encoding a fusion protein as described in the third aspect herein, a nucleic acid construct or a host cell comprising said nucleic acid sequence.
Drawings
FIG. 1 shows vector diagrams of 2-O-MTase, MBP- (2-O-MTase), vaccinia capping enzyme D1: D12, and chimerical enzyme D1: D12- ((2-O-MTase)).
FIG. 2 shows SDS-PAGE identification of cell lysates in which MBP and/or His tag was added to the N-terminus of 2-O-methyltransferase.
FIG. 3 is a SDS-PAGE identification of cell lysates of vaccinia virus capping enzyme and chimeric enzyme.
FIG. 4, SDS-PAGE, a map identifying wild-type vaccinia virus capping enzyme, 2-O-methyltransferase, and chimeric enzyme proteins.
FIG. 5, HPLC-MS analysis of vaccinia virus capping enzyme VVCE modified RNA triphosphate activity of 30 units.
FIG. 6, HPLC-MS analysis of the activity of 30 units of 2-O-methyltransferase-modified cap0 RNA.
FIG. 7 HPLC-MS analysis of vaccinia virus capping enzyme and 2-O-methyltransferase synergistically modify RNA triphosphate activity by 30 units.
FIG. 8, HPLC-MS analysis of chimeric enzyme-modified RNA triphosphate 30 units activity.
FIG. 9 shows a comparison of the biological functions of cells modified with wild-type and chimeric enzymes, respectively, to prepare eGFP mRNA. Wherein, 1, the vaccinia virus capping enzyme and the vaccinia virus 2-O-methyltransferase cooperate with enzyme modification to prepare eGFP mRNA;2, preparing eGFP mRNA by modifying chimeric enzyme with single enzyme; 3, capless eGFP mRNA unmodified control group.
FIG. 10, A, schematic representation of the RNA capping modification enzymatic reaction; b, schematic diagram of the exemplary embodiment of the invention; c, cap0 and cap1 are schematic structural diagrams.
Detailed Description
The inventors propose a novel RNA-modified chimeric enzyme having both the activities of a capping enzyme RNA triphosphatase, guanosine transferase, guanine methyltransferase and the methyltransferase of an mRNA cap structure 2' -O-methyltransferase, and realizing a simplified process when applied to mRNA production.
In the process of researching prokaryotic expression of mRNA cap structure 2 '-O-methyltransferase, the inventor finds that when the natural protein of the 2' -O-methyltransferase is expressed independently, the expression is unstable, and the target protein can not be obtained or can be obtained only in a relatively small amount in the fermentation process. If the MBP solubilizing promotion label is fused at the N end of the 2 '-O-methyltransferase, the 2' -O-methyltransferase fusion protein can be stably expressed in high yield in the fermentation process. In addition, the inventor also finds that when the vaccinia virus capping enzyme is expressed by using a double-promoter vector, the expression amounts of the D1 protein subunit and the D12 protein subunit cannot be evenly expressed to reach 1:1, which is detrimental to the formation of more D1: D12 complexes, wherein 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.
In view of the above findings, applicants have linked 2 '-O-methyltransferases to the C-terminus of the D12 protein subunit (FIG. 10, B), in order to balance the reduction in the expression level of D12, and in order to stabilize the 2' -O-methyltransferase. Surprisingly, the chimeric enzymes designed above completely retained the native activities of 5' -triphosphatase, guanosine transferase, N7 methyltransferase, 2-O-methyltransferase.
Accordingly, in a first aspect the present invention provides a chimeric protein subunit comprising, linked to each other, (a) the D12 subunit of RNA capping enzyme or a functional fragment thereof, or a variant thereof having at least 90% sequence identity thereto and having D12 subunit activity, and (b) an RNA cap structure 2 '-O-methyltransferase or a functional fragment thereof, or a variant thereof having at least 90% sequence identity thereto and having RNA cap structure 2' -O-methyltransferase activity, and optionally (c) a linker between (a) and (b). The invention also provides chimeric proteins comprising the chimeric protein subunit and an RNA capping enzyme D1 subunit or a functional fragment thereof. The chimeric protein is a heterodimer.
"chimeric enzyme" refers to a non-natural enzyme that does not occur in nature, and a chimeric enzyme may comprise catalytic domains derived from different sources (e.g., from different enzymes), or catalytic domains derived from the same source (e.g., from the same enzyme) but arranged in a manner different than that found in nature. Chimeric enzymes can be proteins in which one (i.e., a single subunit) or more (i.e., multiple subunits) 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. "oligomeric enzyme" refers to a multi-subunit enzyme consisting of at least two polypeptide chains linked together, either covalently or non-covalently. "Oligomers" include homo-and hetero-oligomerases, a multi-subunit enzyme consisting of only one type of monomer (subunit), and hetero-oligomerases consisting of different types of monomer (subunit), e.g., heterodimers.
In some embodiments, the RNA capping enzyme described herein is vaccinia virus RNA capping enzyme (VVCE). Vaccinia virus capping enzyme is a heterodimer of two viral proteins, D1 (844 aa) and D12 (287 aa) (D1: D12).
The chimeric proteins herein may comprise an RNA capping enzyme D1 subunit or variant thereof. The D1 subunit has three catalytic domains and therefore contains three biological enzyme activities, and all three steps can be performed in m7 gpppprna synthesis. The three catalytic domains are bound together in the 97kDa D1 protein, with the catalytic domains of RNA5' -triphosphatase (TPase) and guanosine transferase (GTase) being located in the N-terminal portion, and the catalytic domain of N7 methyltransferase (N7-MTase) being located in the C-terminal portion of the D1 protein. Illustratively, the D1 subunit of vaccinia virus RNA capping enzyme is shown in SEQ ID NO. 3, the 5' -triphosphatase domain is shown in amino acids 1-225 of SEQ ID NO. 3, the guanylyl transferase 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 truncated forms of the D1 subunit of RNA capping enzyme or variants thereof, preferably comprising the N7-MTase domain. Truncated vaccinia capping enzymes comprising an N7-MTase domain and having Biological enzymatic activity are known in the art, for example, as described by 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. Accordingly, the chimeric proteins herein also include functional fragments of the D1 subunit of RNA capping enzyme comprising: (1) an N7 methyltransferase domain or a functional fragment thereof; (2) An N7 methyltransferase domain or a functional fragment thereof and a5' -triphosphatase domain or a functional fragment thereof; (3) An N7 methyltransferase domain or functional fragment thereof and a guanosine transferase domain or functional fragment thereof; or (4) an N7 methyltransferase domain or a functional fragment thereof, a5' -triphosphatase domain or a functional fragment thereof, and a guanosine transferase domain or a functional fragment thereof. In one or more embodiments, a functional fragment of the D1 subunit of the RNA capping enzyme comprises amino acids 498-844 or 540-844 of SEQ ID NO 3.
The D12 subunit (33 kDa) has no methyltransferase catalytic activity per se, but it can activate the methyltransferase activity of the enhanced D1 protein. The amino acid sequence of the D12 subunit of vaccinia virus RNA capping enzyme is shown in SEQ ID NO. 1, 1-287.
In some embodiments, the RNA cap structure 2 '-O-methyltransferase described herein is a vaccinia virus 2' -O-methyltransferase (2-O-MTase). The 39kDa 2 '-O-methyltransferase, also known as the VP39 protein, effects 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.
In some aspects, the present invention also provides a MBP tag fused 2' -O-methyltransferase protein for improving protein expression stability, which has comparable or better biological enzyme functional activity than native vaccinia virus 2-O-methyltransferase. 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 thereto and having RNA cap structure 2' -O-methyltransferase activity, and (b) a His-tag and/or an MBP-tag located (a) N-or C-terminally, and optionally a linker between (a) and (b). The RNA cap structure 2' -O-methyltransferase was as previously described. The His-tag described herein is a short peptide containing one or more consecutive histidine residues. The MBP tags described herein have the conventional meaning in the art.
In the present invention, a polypeptide or protein (e.g., vaccinia virus capping enzyme or subunit or domain thereof, RNA cap 2 '-O-methyltransferase) also includes a mutant having at least 70% sequence identity thereto and retaining the activity (e.g., 5' -triphosphorylation activity, guanosine transferase activity, N7 methyl transferase activity, 2-O-methyl transferase activity) of the polypeptide or protein. The mutant comprises: an amino acid sequence 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 a reference sequence and retaining the biological activity of the reference sequence. Sequence identity between two aligned sequences can be calculated using, for example, BLASTp from NCBI. Mutants also include amino acid sequences having one or several mutations (insertions, deletions or substitutions) in the amino acid sequence while still retaining the biological activity of the reference sequence. The number of mutations usually means within 1-50, such as 1-20, 1-10, 1-8, 1-5 or 1-3. The substitution is preferably a conservative substitution. For scFv, the mutations may occur in the CDR regions (including those described above) or in the FR regions, provided that the biological activity of the reference sequence is retained after the mutation. For example, conservative substitutions with amino acids that are similar or analogous in performance are not typically used in the art to alter the function (e.g., enzymatic activity) of a protein or polypeptide. "amino acids with similar or analogous properties" include, for example, families of amino acid residues with analogous side chains, including amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, substitution of one or more sites with another amino acid residue from the same side chain species in a polypeptide of the invention will not substantially affect its activity.
For example, it is known in the art that variants of the D12 subunit of RNA capping enzymes 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. As another example, schnierle BS et al in the paper disclose a series of effective mutated amino acid sequences of mRNA cap structure 2' -O-methyltransferase, which still retains higher enzymatic catalytic activity. These mutants include, but are not limited to, the C178S, C272S, K41D, a201R, a210K mutants of 2' -O-methyltransferase; the charged amino acids R, K, H, Y, C, D, E of 2' -O-methyltransferase were randomly replaced by A. Zheng S, colin PY and Mao X respectively disclose effective mutation amino acid sequences of a series of vaccinia virus capping enzyme N7-methyltransferase structural domains in the literature, and the mutants also retain higher enzyme catalytic activity. These 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, F585A, L586A, D587A, D784A, N785A, R794A, F798A, M805A, E806A. (Schnierle BS et al, J Biol chem.1994; zheng S et al, RNA.2008; mao X et al, biochemistry.1996; kyieleis OJ et al, structure.2014; colin PY. Sci Rep.2020; nayanendu Saha et al, virology,2001, nayanendu Saha et al, J.VIROL, 2003) the foregoing documents are incorporated herein by reference in their entirety.
In some embodiments, the polypeptides or proteins described herein further comprise a signal peptide capable of directing it to a subcellular structure. The signal peptide may be located at the N-terminus or C-terminus of the polypeptide. Such subcellular structures include, but are not limited to, the golgi or endoplasmic reticulum, proteasomes, cell membranes, or lysosomes.
Herein, a Linker (Linker) is a polypeptide segment that connects different proteins or polypeptides, with the purpose of maintaining the connected proteins or polypeptides in their respective spatial conformations to maintain the function or activity of the proteins or polypeptides. Exemplary linkers include linkers containing G and/or S. Typically, the linker contains one or more motifs which repeat back and forth. Preferably, the motifs are adjacent in the linker sequence with no intervening amino acid residues between the repeats. The linker sequence may comprise 1,2, 3, 4 or 5 repeat motifs. The length of the linker may be 3-25 amino acid residues, for example 3-15, 5-15, 10-20 amino acid residues. In certain embodiments, the linker sequence is a polyglycine linker sequence. The number of glycines in the linker sequence is not particularly limited, and is typically 2-20, e.g., 2-15, 2-10, 2-8. In addition to glycine and serine, other known amino acid residues may be contained in the linker, such as alanine (a), leucine (L), threonine (T), glutamic acid (E), phenylalanine (F), arginine (R), glutamine (Q), and the like. In certain embodiments, the different proteins or polypeptides of the invention are linked by (GGGGS) n, where n is an integer from 1 to 5. In one or more embodiments, the amino acid sequence of the linker is as set forth in amino acids 288-302 of SEQ ID NO 1.
Within the scope of the present invention, polypeptide linkers useful herein that do not affect the folding of the enzymatic domain and that can achieve the effect of the chimeric enzymes of the invention are well known to those skilled in the art and include, but are not limited to, GGGGIAPISMVGGGGS (Turner, ritter et al 1997), SPNGASNSGSAPDTSSAPGSQ (Hennecke, krebber et al 1998), EGKSSGSESKSTE (Bird, hardman et al 1988), EGKSSGSGSESKEF (Newton, xue et al 1996), GGGSGGGSGGGTGGGSGGGGG (Robinson and Sauer 1998), GSGSGGSGSEGKSKG (Bedzyk, weidner et al 1990), YPRSIYIRIIRRRHPSPSLTT (Tang, jiang et al 1996), GSGKPGSGEGS (Ting, kain et al 2001), SSADDAKKDAAKKDDKDDAKKDA (Pantoliano, bird et al 1991), GSADDAXXDAAXKDDAKKDDAKKDGS (Gregoire, lin et al 1996), LSADAKKDAAKKDDKDDAKKDL (Pavlikova, bersford et al 1999), AEAAAKEAAAKEAAAKA (Wickham, carrion et al 1995), GSTSGSGKPGSGSGGAGTGAGGGAGSTSTSGGKPSGEG (Ting, kain et al 2001), LSLEVAEEIARLEAEV (Ting, kain et al 2001), GTPTPTPTPTPTPTGEF (Gustavsson, lehtio et al 2001), GSGSGSGGSGSGGTKG (Whitlow, bell et al 1993) and GSHSGGK GK (Ting, kain et al 42334, or the universal linker described in US 20142334. The above documents are incorporated herein by reference in their entirety.
The invention includes polynucleotides encoding the chimeric protein subunits or chimeric proteins of the invention. The polynucleotide of the present invention may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand. The invention also includes degenerate variants of polynucleotides that encode a polypeptide or protein, i.e., polynucleotides that encode the same amino acid sequence but differ in nucleotide sequence.
Polynucleotides described herein include sequences that are altered by codon optimization, so long as the amino acid sequence encoded by the polynucleotide is not altered. Codon-optimized sequences may exhibit more favorable expression for a particular species. Methods for codon optimizing polynucleotide sequences are well known in the art.
The polynucleotide of the present invention may be the coding sequence of the chimeric protein subunit and the coding sequence of the RNA capping enzyme D1 subunit or a functional fragment thereof, or an expression cassette of the chimeric protein subunit and an expression cassette of the RNA capping enzyme D1 subunit or a functional fragment thereof. Herein, a coding sequence refers to a portion of a nucleic acid sequence that directly determines the amino acid sequence of its protein product (e.g., a chimeric protein subunit, a polypeptide of the RNA capping enzyme D1 subunit or a functional fragment thereof, etc.). The boundaries of the coding sequence are generally determined by a ribosome binding site (for prokaryotic cells) immediately upstream of the 5 'open reading frame of the mRNA and a transcription termination sequence immediately downstream of the 3' open reading frame of the mRNA. A coding sequence can include, but is not limited to, DNA, cDNA, and recombinant nucleic acid sequences. Herein, an expression cassette refers to the complete elements required for expression of a gene of interest, including the promoter, gene coding sequence, and PolyA tailing signal sequence. The polynucleotides described herein may be two separate nucleic acid molecules, each comprising a coding sequence for a chimeric protein subunit and a coding sequence for a D1 subunit of RNA capping enzyme or a functional fragment thereof, e.g., an expression cassette for a chimeric protein subunit and an expression cassette for a D1 subunit of RNA capping enzyme or a functional fragment thereof, respectively; alternatively, the coding sequence of the chimeric protein subunit and the coding sequence of the RNA capping enzyme D1 subunit or a functional fragment thereof may be linked as one nucleic acid molecule via a linker, e.g. the coding sequence of the chimeric protein subunit and the coding sequence of the RNA capping enzyme D1 subunit or a functional fragment thereof are in the same expression cassette, or the two expression cassettes are linked as the same nucleic acid molecule via a suitable linker. In certain embodiments, the polynucleotide of the invention is a nucleic acid molecule comprising a promoter, a coding sequence encoding the chimeric protein subunit and the RNA capping enzyme D1 subunit or a functional fragment thereof, and a PolyA tailing signal, wherein the coding sequence of the chimeric protein subunit and the coding sequence of the RNA capping enzyme D1 subunit or a functional fragment thereof are in one expression cassette. In one or more embodiments, the polynucleotide further comprises an optional signal peptide. In one or more embodiments, the polynucleotide comprises SEQ ID NO 2 or SEQ ID NO 2 and 4.
In certain embodiments, the coding sequence or expression cassette is integrated into the genome of the cell. Thus, in these embodiments, the cell described herein has stably integrated into its genome an expression cassette comprising a subunit encoding a chimeric protein described herein and a subunit of RNA capping enzyme D1, or a functional fragment thereof.
The invention also relates to nucleic acid constructs comprising a polynucleotide as described herein, and one or more control sequences operably linked to the sequences. The polynucleotides of the present invention may be manipulated in a variety of ways to ensure expression of the chimeric protein subunits or chimeric proteins. The nucleic acid construct may be manipulated prior to insertion into the vector, depending on the type of expression vector or requirements. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.
The control sequence may be an appropriate promoter sequence. The promoter sequence is typically operably linked to the coding sequence of the protein to be expressed. The promoter may be any nucleotide sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell. The control sequence may also be a suitable transcription terminator sequence, a sequence recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3' terminus of the nucleotide sequence encoding the polypeptide. Any terminator which is functional in the host cell of choice may be used in the present invention. The control sequence may also be a suitable leader sequence, a nontranslated region of an mRNA which is important for translation by the host cell. The leader sequence is operably linked to the 5' terminus of the nucleotide sequence encoding the polypeptide. Any terminator which is functional in the host cell of choice may be used in the present invention.
In certain embodiments, the nucleic acid construct is a vector. The vector may be a cloning vector, an expression vector, or a homologous recombinant vector. The polynucleotides of the present invention can be cloned into many types of vectors, for example, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Cloning vectors may be used to provide coding sequences for therapeutic proteins and polypeptides of the invention, such as a nucleic acid molecule comprising coding sequences for therapeutic proteins and polypeptides. The expression vector may be provided to the cell in the form of a viral vector. Expression of a polynucleotide of the invention is typically achieved by operably linking the polynucleotide of the invention to a promoter and incorporating the construct into an expression vector. The vector may be suitable for replication and integration into eukaryotic cells. Typical cloning vectors contain transcriptional and translational terminators, initiation sequences, and promoters useful for regulating the expression of the desired nucleic acid sequence. Homologous recombinant vectors are used to integrate the expression cassettes described herein into the host genome.
Generally, suitable vectors comprise an origin of replication functional in at least one organism, a promoter sequence, a convenient restriction enzyme site and one or more selectable markers. For example, in certain embodiments, the invention uses a lentiviral vector comprising a replication initiation site, a 3'LTR,5' LTR, a polynucleotide as described herein, and optionally a selectable marker.
An example of 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 α (EF-1 α). However, other constitutive promoter sequences known in the art to be suitable for prokaryotic or eukaryotic cells may also be used.
To assess the expression of a therapeutic protein, polypeptide, or portion thereof, the expression vector introduced into the cells may also contain either or both of a selectable marker gene or a reporter gene to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected by the viral vector. In other aspects, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in a host cell. Useful selectable markers include, for example, antibiotic resistance genes and the like. Suitable reporter genes may include genes encoding luciferase, β -galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein. 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. Specifically, primers can be designed based on the nucleotide sequences disclosed herein, particularly open reading frame sequences, and the relevant sequences can be amplified using commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art as templates. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice together the amplified fragments in the correct order. Alternatively, the nucleic acid molecules described herein can also be synthesized directly.
Methods for introducing and expressing genes into cells are known in the art. The vector may be readily introduced into a host cell by any method known in the art, e.g., mammalian, bacterial, yeast or insect cells. For example, the expression vector may be transferred into a host cell by physical, chemical or biological means.
Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Biological methods for introducing a polynucleotide of interest into a host cell 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 liposomes.
Herein, a host cell contains, expresses and/or secretes a protein subunit or protein described herein. Herein, when a cell is referred to as containing or comprising, expressing, secreting a molecule, such as a polypeptide, "containing" means that the molecule is contained within or on the surface of the cell; "expressing" means that the cell produces the molecule; "secreted" means that the cell secretes the expressed molecule outside the cell. Host cells include both cells that are ultimately used to secrete the chimeric protein or a subunit thereof, and cells that produce RNA, as well as various cells used in the production of such cells, such as e.coli cells, for use, e.g., in providing a coding sequence for a protein of the invention or in providing a vector as described herein. After the host cell expresses a protein subunit or protein described herein, the protein subunit or protein can be purified by methods conventional in the art for purifying proteins (e.g., chromatography, including affinity chromatography, ion exchange chromatography, and the like).
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 into a capped RNA or a method of preparing a capped target RNA, comprising: contacting a target RNA with a chimeric protein described herein, or contacting a target RNA with a cell expressing a chimeric protein described herein.
Herein, a "target RNA" is an RNA of interest obtained in vivo or in vitro. Typically, the target RNA is uncapped or incompletely capped RNA. The target RNA may be from any source, e.g., in Vitro Transcription (IVT), chemical synthesis, extraction from the body, etc. In vitro transcription refers to the enzymatic synthesis of RNA using a DNA transcription template with T7, T3, SP6 promoters and a corresponding DNA-dependent RNA polymerase in a suitable buffer system including, but not limited to, NTP, mg 2+ And components such as Ribonuclease Inhibitors (RI) and inorganic pyrophosphatase (iPPase). Compared with solid phase synthesis of RNA, in vitro transcription synthesis can prepare a large amount of long-chain RNA with high quality, and is suitable for industrial production of mRNA drugs. In one or more embodiments, the RNA of interest described herein is an in vitro transcribed cap-free single-stranded RNA (ssRNA) that has all the features of an mRNA other than a cap, including but not limited to a 5'-UTR, a 3' -UTR, a protein or polypeptide translation coding region.
Methods for in vitro transcription of RNA substrates are well known in the art, and exemplary methods for transcription of polymerases (e.g., T7, T3, SP 6) include mixed incubation of a polymerase, rNTP, and a DNA transcription template containing a promoter recognized by the polymerase. The reaction system also comprises one or more reagents selected from the following: mgCl 2 Buffer, iPPase, inhibitor, nuclease-free water, and the like. The DNA for transcription of the template can be obtained by methods conventional in the art, such as synthesis, hybridization, PCR, etc., and the reagents required for these methods are also well known in the art.
Without wishing to be bound by theory, target RNAs herein include 5' -triphosphorylated RNA, 5' -diphosphonated RNA, and RNA having a5' -Gppp structure. As used herein, "capped RNA" includes capped RNA having a structure of 5'-m7Gppp (cap 0) or capped RNA having a structure of 5' -m7 GppNmp (cap 1). In each of the above structures, "5'" indicates that the group is located at the 5' end of the RNA, "m" indicates methylated, "7" indicates the methylation position, "G" is guanylic acid, "p" is a phosphate group, and "N" is any nucleotide located at the 5' end of the target RNA. Optionally, the target RNA may be purified and/or denatured and renatured RNA. Reagents and procedures for purifying and denaturing, renaturing RNA are known in the art, for example, by incubating an RNA sample at 50-70 ℃ for 1-60 minutes (e.g., 65 ℃ for 5-20 minutes), followed by 0 ℃ for 2-10 minutes. However, in the method of the present invention, the capping of RNA can be achieved without purification and denaturation and renaturation.
In order to contact the RNA of interest with the chimeric proteins described herein, the RNA of interest can be expressed in a cell expressing the chimeric proteins described herein (e.g., by introducing a DNA sequence expressing the RNA of interest into a host cell described herein). The target RNA can be expressed in the cell by any suitable method known in the art, for example, by introducing into the cell DNA encoding the RNA which is transcribed in the cell to form the target RNA, which is capped catalyzed by the chimeric protein or chimeric protein subunit expressed by the cell.
Alternatively, the RNA of interest (e.g., RNA transcribed in vitro) can be mixed in solution with the chimeric proteins described herein to effect contact. The RNA may be purified and/or renatured prior to mixing. Procedures and conditions for RNA capping in solution are known in the art, for example, mixing and incubating target RNA in nuclease-free water with capping buffer, GTP, SAM, and the chimeric proteins herein. An incubation step such as incubation at 37 ℃ for 30-90 minutes. The modified capped RNA product can be purified, for example, by magnetic beads.
In the capping method described herein, the molar ratio of the target RNA to the chimeric protein is less than 865 1, preferably 86.5-173. The inventors have found that different capping RNAs can be obtained at different molar ratios. For example, if the molar ratio of the target RNA to the chimeric protein is less than 865 1, the method modifies the target RNA to a capped RNA having a5' -m7Gppp structure (cap 0). If the number of moles of the target RNA and the chimeric protein is less than 86.5-173, the method modifies the target RNA into a capped RNA (cap 1) having a5' -m7 GppNmp structure.
Furthermore, the chimeric proteins may comprise different domains for different target RNAs. If the target RNA is a5' -triphosphorylated RNA, it is desirable that the chimeric protein has the activity of 5' -triphosphatase, guanosine transferase, N7 methyltransferase, 2-O-methyltransferase, i.e.the chimeric protein comprises the N7 methyltransferase (N7-MTase) domain or functional fragment thereof, the 5' -triphosphatase (TPase) domain or functional fragment thereof and the guanosine transferase (GTase) domain or functional fragment thereof of the RNA capping enzyme D1 subunit, and the chimeric protein subunits.
If the target RNA is a5' -diphosphonated RNA, it is desirable that the chimeric protein has the activity of a guanosine transferase, an N7 methyltransferase, a 2-O-methyltransferase, i.e.the chimeric protein comprises the N7 methyltransferase (N7-MTase) domain or a functional fragment thereof and the guanosine transferase (GTase) domain or a functional fragment thereof of the D1 subunit of the RNA capping enzyme, and the subunits of the chimeric protein.
If the target RNA is an RNA having a5' -Gppp structure, it is desirable that the chimeric protein has the activity of N7 methyltransferase (N7-MTase) and 2-O-methyltransferase, i.e., the chimeric protein comprises the N7 methyltransferase (N7-MTase) domain of the D1 subunit of RNA capping enzyme or a functional fragment thereof and the chimeric protein subunit.
In another aspect, the present invention can also modify a capped RNA having a 5'-m7Gppp structure (cap 0) into a capped RNA having a5' -m7 gppnmp structure (cap 1), comprising the steps of: contacting the RNA with a chimeric protein subunit described herein (e.g., expressing the RNA in a cell expressing a chimeric protein subunit described herein), or contacting the RNA with a cell expressing a chimeric protein subunit described herein.
The invention has the advantages that:
1. the chimeric enzyme with the biological activities of the capping enzyme and the 2' -O-methyltransferase can reduce the production process and the production cost;
2. the cap0 and cap1 are continuous reactions, and the chimeric enzyme covalently couples the two enzymes together, so that the two enzymes are more favorable for continuous contact with an RNA substrate, and the modification efficiency is improved.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications and patents specifically mentioned herein are incorporated herein by reference in their entirety for all purposes including describing and disclosing the chemicals, equipment, statistical analyses and methods reported in the publications that could be used in connection with the invention. All references cited in this specification are to be considered as indicative of the level of skill in the art. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not noted in the following examples, are generally performed according to conventional conditions such as those described in J. SammBruk et al, molecular cloning protocols, third edition, scientific Press, 2002, or according to the manufacturer's recommendations.
Examples
Example 1 expression and purification of native vaccinia virus capping enzyme, 2-O-methyltransferase, chimeric enzyme
In order to be able to overexpress the vaccinia virus capping enzyme D1: d12, 2-O-methyltransferase 2-O-MTase and D1: D12- (2-O-MTase) chimeric enzyme, and DNA sequences were optimally designed according to the amino acid sequences corresponding to the above proteins and cloned into commercial prokaryotic expression vectors pET28a (Novagen), pMAL-c5X (NEB) and pRSF-Duet1 (Novagen). The amino acid and DNA sequences of the enzyme are shown in SEQ ID NO 1-4, and the prokaryotic expression vector diagram is shown in figure 1.
To ensure high level expression of the enzyme and to allow direct reproducible purification of the vaccinia virus capping enzyme or chimeric enzyme complex, a single His tag was added to the N-terminus of the D1 enzyme to facilitate purification of the complete D1: D12 or D1: D12- (2-O-MTase) protein complex. In order to make the 2-O-methyltransferase more stable and facilitate purification, a His tag and/or an MBP tag is added to the N-terminus of the 2-O-methyltransferase. The plasmids were transformed into E.coli BL21 (DE 3) expression bacteria according to the instructions of the commercial expression vectors, single colonies were picked up and cultured in LB at 37 ℃ to the logarithmic phase, and then added with 1mM IPTG inducer at the final concentration and cultured for further induction at 25 ℃ for 16 hours. After collection of the induced bacteria, all enzyme proteins were purified using two standard purification steps, ni affinity chromatography, capto SP ImpRes or Capto Q ImpRes (Cytiva corporation) ion exchange chromatography to isolate the final enzyme protein. The lysate before and after induction and the finally purified enzyme protein are respectively subjected to SDS-PAGE (polyacrylamide gel electrophoresis), wherein FIG. 2 is a SDS-PAGE identification picture of the lysate with His and/or MBP labels added at the N end of 2-O-methyltransferase, FIG. 3 is a SDS-PAGE identification picture of the lysate of vaccinia virus capping enzyme and chimeric enzyme, and FIG. 4 is a SDS-PAGE identification picture of purified MBP- (2-O-MTase), vaccinia virus capping enzyme D1: D12 and chimeric enzyme D1: D12- ((2-O-MTase).
As shown in FIG. 2, when the 2-O-methyltransferase does not have the MBP tag, protein expression is unstable and the yield is low, so that the 2-O-methyltransferases used in the present invention are all 2-O-methyltransferases in the form of MBP tags, i.e., MBP- (2-O-MTase). As shown in FIG. 3, 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 favorable for the formation of stable 1:1, D1: D12 complex in stable relationship; when 2-O-MTase and D12 are expressed in a fusion mode, the expression level of the fusion protein subunit is closer to that of D1, and the formation of a protein complex D1: D12- (2-O-MTase) is facilitated. As shown in FIG. 4, the three expression schemes described above all resulted in a protein or protein subunit of the desired size after purification, wherein the vaccinia virus capping enzyme comprises a 33kD protein subunit D12 and a 100kD protein subunit D1; the chimeric enzyme comprises 73kD D12- (2-O-MTase) fusion protein subunit and 100kD D1 protein subunit, and meets the expected protein size; the size of the MBP- (2-O-MTase) fusion protein is 83kD, and the sum of the his-MBP protein tag of 44kD and the 2-O-MTase protein of 39kD also meets the expected protein size.
Example 2 in vitro transcription and enzymatic modification of RNA substrates to synthesize Cap0 mRNA, cap1 mRNA
Primers containing the T7 promoter sequence were designed and synthesized as follows, RNA30-T7F:5' galatatacgcactcactataGGGAAGGAGAGGAAGAAGGAAAGGGAAGAAGGAAGAAGAAGAAGAAGAA-; RNA30-R: 5- 'TTCTTTCTTCCCTTCTTCCTCTCCTTCCttagtgagtgtctattatc-3' (SEQ ID NO: 6). Mixing the primers according to the proportion in the table 1 to carry out annealing reaction, wherein the annealing procedure is as follows: pre-denaturation at 95 ℃ for 10min, gradient cooling to 20 ℃ within 2 hours, keeping the temperature at 20 ℃ for one hour, and taking out. The magnetic beads are purified to obtain the 30nt RNA (RNA 30) transcription DNA template.
TABLE 1 primer annealing reaction System
Reagent Volume of
10 × annealing buffer 5μL
RNA30-T7F(100μM) 20μL
RNA30-R(100μM) 20μL
Water without ribonuclease To 50 μ L
Transcription synthesis of RNA: the reaction system for preparing the T7 RNA polymerase at room temperature is added with reaction components according to the sequence shown in the table below, and the reaction system can be expanded or reduced according to equal proportion. The reaction was carried out at 37 ℃ for 6 to 16 hours. In this example, the transcribed RNA was 30nt RNA. And 2h, after the reaction is finished, adding 2U DNase, digesting for 15min at the temperature of 1 ℃, and then carrying out magnetic bead purification for the next RNA capping modification enzymatic reaction.
TABLE 2 RNA transcription Synthesis System
Figure BDA0003012133870000201
Capping modified RNA: taking 10ug of the purified RNA to a 1.5ml centrifuge tube, and diluting the RNA to 14ul by using water without nuclease; heating at 65 ℃ for 10 minutes, taking out the centrifuge tube, and placing on ice for 5 minutes; the following components were added in sequence according to tables 3-6 for different purposes of RNA modification and incubated at 37 ℃ for 30-90 minutes. The step is suitable for capping reaction of 10ug RNA, and the volume of the reaction substrate can be amplified according to the experiment requirement. After the reaction is finished, a final modified RNA product can be obtained by adopting a magnetic bead purification mode.
TABLE 3 enzymatic modification of uncapped RNA capped as Cap0RNA Synthesis System
Figure BDA0003012133870000202
TABLE 4 enzymatic modification of cap0RNA as cap1 RNA Synthesis System
Figure BDA0003012133870000203
Figure BDA0003012133870000211
TABLE 5 enzymatic modification of uncapped RNA capped with Cap1 RNA Synthesis System (Natural enzyme)
Components Volume of
Denatured cap-free RNA 10μg
10 + capping buffer 2.0μl
GTP(10mM) 1.0μl
SAM(32mM) 1.0μl
Vaccinia virus capping enzyme D1: D12 X μ g (as shown in FIG. 7)
MBP-(2-O-MTase) Y μ g (as shown in FIG. 7)
Water without ribonuclease To 20. Mu.l
TABLE 6 enzymatic modification of uncapped RNA capped with Cap1 RNA Synthesis System (chimeric enzyme)
Components Volume of
Denatured cap-free RNA 10μg
10 + capping buffer 2.0μl
GTP(10mM) 1.0μl
SAM(32mM) 1.0μl
Chimeric enzyme D1: d12- (2-O-MTase) X μ g (as shown in FIG. 8)
Water without ribonuclease To 20. Mu.l
Example 3 detection of the efficiency of 5' capping of RNA catalyzed by different RNA-modifying enzymes by HPLC-MS
As shown in Table 7, the expected molecular weights of the transcribed or enzymatically modified intermediates or final products in example 2 were calculated and these sample distributions were subjected to HPLC-MS (liquid chromatography-Mass Spectrometry) detection. The proportion of target products generated after the RNA substrates are subjected to enzymatic catalytic reaction can be deduced by comparing the detected approximate molecular weights, and then the enzyme catalytic activities of different RNA modification enzymes are deduced. The results are shown in FIGS. 5 to 8.
TABLE 7 predicted molecular weight Scale for different cap structures RNA30
Figure BDA0003012133870000212
Figure BDA0003012133870000221
* Note: the actual mass spectrometric detection molecular weight and the predicted molecular weight sometimes have a deviation of less than 5Da, but do not affect the judgment of the target product
The results in FIG. 5 show that: the vaccinia virus capping enzyme (about 133 kDa) is capable of converting the modification of the cap-free triphosphate RNA30 with the expected molecular weight of 10623Da into the cap0RNA30 with the expected molecular weight of 10903Da under the condition that the amount of the capping enzyme is more than 0.1. Mu.g, and when the enzyme dosage is less than 0.05. Mu.g, the modification of the cap-free triphosphate RNA30 is incomplete, only part of the substrate is converted into the cap0RNA30, and a small amount of the substrate is converted into the G-cap RNA30. Namely, the substrate RNA: when the molar ratio of the capping enzyme is less than 1330, cap0 can be completely capped. (Note: RNA approximately equal to 10kDa, facilitating rapid calculation)
The results in FIG. 6 show that: 2-O-methyltransferase (about 81 kDa), at greater than 0.5. Mu.g, was able to convert the modification of cap0RNA30 with the expected molecular weight of 10903Da to cap1 RNA30 with the expected molecular weight of 10918Da, and at enzyme doses less than 0.25. Mu.g, the modification of cap0RNA30 was incomplete and only a portion of the substrate was converted to cap1 RNA30. Namely, the substrate RNA: when the molar ratio of the 2-O-methyltransferase is less than 162, the cap0 can be completely modified into the cap1 cap.
FIG. 7 shows HPLC-MS analysis of vaccinia virus capping enzyme and 2-O-methyltransferase synergistically modifying RNA triphosphate activity of 30 units. Based on the detection result of the capping enzyme modified RNA triphosphate 30 as cap0RNA30, 0.2. Mu.g of vaccinia capping enzyme was added to each group in this experimental group to ensure that the RNA triphosphate 30 was completely converted into cap0RNA30, and the 2-O-methyltransferase was added in a gradient of 0.25. Mu.g, 0.5. Mu.g, 1. Mu.g, and 2. Mu.g. The results in FIG. 7 show that: when the addition amount of the 2-O-methyltransferase is 0.25ug, only half of the substrate is converted into cap1 RNA30; when the addition amount of the 2-O-methyltransferase is 0.5ug, a small amount of substrate is still not converted into cap1 RNA30; when the addition amount of the 2-O-methyltransferase is more than 1ug, the RNA30 can be modified cooperatively to be completely converted into cap1 RNA. This indicates that more 2-O methyltransferase protein is required to modify RNA to cap1 in one step than to modify RNA to cap1 structure in steps. In the synergistic capping system, the substrate RNA: capping enzyme molar ratio less than 665:1, cap0 can be completely capped, RNA: the molar ratio of the 2-O-methyltransferase is between 81 and 162:1, a complete cap0 modification to cap1 can be achieved, indicating that the enzyme usage in the synergistic capping reaction system and the stepwise capping reaction system is similar.
The results in FIG. 8 show that: the chimeric enzyme (173kDa, 133kDa + 39kDa) can completely convert the uncapped triphosphate RNA30 with the expected molecular weight of 10623Da into cap0RNA30 with the expected molecular weight of 10903Da under the condition of 0.1-0.2 mu g; of these, about 90% of RNA30 was completely capped at 0.1. Mu.g. This indicates that the chimeric enzyme retains the activity of the three enzymatic domains of 5' -triphosphatase, guanosine transferase and N7-guanine methyltransferase in the wild-type vaccinia virus capping enzyme in the same dose as the wild-type enzyme alone. When the chimeric enzyme is used in a dose of 2. Mu.g, the cap0RNA30 converted to 10903Da can be further completely modified to convert it to a cap1 RNA30 of the expected molecular weight of 10918 Da. This shows that the chimeric enzyme not only completely retains the activity of the catalytic domain of 2-O-methyltransferase, but also can more efficiently and thoroughly catalyze to obtain cap1 RNA30. I.e., chimeric enzyme capping system, substrate RNA: when the mole ratio of the chimeric enzyme is less than 865, cap0 can be completely capped; RNA: when the mosaic molar ratio is between 86.5 and 173, the cap0 can be completely modified into a cap1 cap. The indexes are all equivalent to the mole ratio index 665, 81-162 of the mixed enzyme system, and show that the chimeric enzyme can realize the same function of the mixed enzyme under the same molar concentration, namely the chimeric enzyme is proved not to change the catalytic activity of each functional structural domain, and the simplified production and use of the enzyme are favorably realized (compared with the use of two enzymes, only one enzyme is required to be produced, the step cost is saved, and only one enzyme is required to be added during the reaction).
Example 4 comparison of cellular biological function of eGFP mRNA prepared by separately modifying wild-type and chimeric enzymes
A template DNA (SEQ ID NO: 7) for gene synthesis eGFPmRNA transcription was subcloned into a commercial vector such as pUC57 and the like. The plasmid is transformed into Top10 colibacillus, the plasmid is extracted and then linearized by restriction endonuclease, and a DNA linear template is recovered by magnetic beads and used for RNA transcription synthesis experiment. The transcription synthesis system and procedure of eGFP mRNA are described in Table 2 of example 2, and the reaction system and procedure for enzymatically modifying the prepared cap1 eGFP mRNA after transcription synthesis are described in tables 5 and 6 of example 2. Respectively taking and identifying mRNA samples which are completely modified by capping for carrying out transfection tests, numbering a cap1 eGFP mRNA prepared by combining natural vaccinia virus capping enzyme and 2-O-methyltransferase modified RNA as a No. 1 sample, numbering a cap1 eGFP mRNA prepared by chimeric enzyme modified RNA as a No. 2 sample, and numbering a non-capped eGFP mRNA which is not modified by enzyme as a No. 3 sample. The above samples were transfected into Chinese Hamster Ovary (CHO) cells following the following cell transfection procedure to verify the cellular biological function of the modified cap1 eGFP mRNA. Transfection procedure: (1) The day before transfection, CHO cells were digested, plated in 24-well plates at a density of 1 × 105/well. (2) When in transfection, 2 EP tubes with the volume of 1.5ml are respectively taken, 50ul of opti-MEM culture solution is respectively added into each tube, 2.25ul of PEI transfection reagent is added into one tube, 1.5ug of mRNA is added into the other tube, after standing for 5min, PEI solution is added into mRNA solution, and after uniform mixing, the mixture is left standing for 20min. (3) Adding the prepared PEI-mRNA solution into CHO cells containing serum-free culture solution, placing the CHO cells in a 37-degree incubator, culturing for 4 hours, and then replacing the culture medium with a complete culture medium for continuous culture. (4) After 24 hours of culture, fluorescence photographing is carried out, and the EGFP positive rate is detected by detecting the flow type. Duplicate well replicates were made for all three samples.
As shown in FIG. 9 and Table 8, the green fluorescence positive rate and fluorescence intensity of the eGFP mRNA samples No. 1 and No. 2 transfected CHO cells were at the same level, and there was no significant difference. This demonstrates that the same effect of natural vaccinia virus capping enzyme in combination with 2-O-methyltransferase modification of RNA can be achieved with a single modification using chimeric enzyme.
TABLE 8 EGFP positivity and fluorescence intensity of cells transfected with different mRNA samples
Figure BDA0003012133870000241
Sequence listing
<110> Shanghai cell therapy group Co., ltd
<120> RNA modified chimeric protein and application thereof
<130> 210138
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 635
<212> PRT
<213> Artificial Sequence
<400> 1
Met Asp Glu Ile Val Lys Asn Ile Arg Glu Gly Thr His Val Leu Leu
1 5 10 15
Pro Phe Tyr Glu Thr Leu Pro Glu Leu Asn Leu Ser Leu Gly Lys Ser
20 25 30
Pro Leu Pro Ser Leu Glu Tyr Gly Ala Asn Tyr Phe Leu Gln Ile Ser
35 40 45
Arg Val Asn Asp Leu Asn Arg Met Pro Thr Asp Met Leu Lys Leu Phe
50 55 60
Thr His Asp Ile Met Leu Pro Glu Ser Asp Leu Asp Lys Val Tyr Glu
65 70 75 80
Ile Leu Lys Ile Asn Ser Val Lys Tyr Tyr Gly Arg Ser Thr Lys Ala
85 90 95
Asp Ala Val Val Ala Asp Leu Ser Ala Arg Asn Lys Leu Phe Lys Arg
100 105 110
Glu Arg Asp Ala Ile Lys Ser Asn Asn His Leu Thr Glu Asn Asn Leu
115 120 125
Tyr Ile Ser Asp Tyr Lys Met Leu Thr Phe Asp Val Phe Arg Pro Leu
130 135 140
Phe Asp Phe Val Asn Glu Lys Tyr Cys Ile Ile Lys Leu Pro Thr Leu
145 150 155 160
Phe Gly Arg Gly Val Ile Asp Thr Met Arg Ile Tyr Cys Ser Leu Phe
165 170 175
Lys Asn Val Arg Leu Leu Lys Cys Val Ser Asp Ser Trp Leu Lys Asp
180 185 190
Ser Ala Ile Met Val Ala Ser Asp Val Cys Lys Lys Asn Leu Asp Leu
195 200 205
Phe Met Ser His Val Lys Ser Val Thr Lys Ser Ser Ser Trp Lys Asp
210 215 220
Val Asn Ser Val Gln Phe Ser Ile Leu Asn Asn Pro Val Asp Thr Glu
225 230 235 240
Phe Ile Asn Lys Phe Leu Glu Phe Ser Asn Arg Val Tyr Glu Ala Leu
245 250 255
Tyr Tyr Val His Ser Leu Leu Tyr Ser Ser Met Thr Ser Asp Ser Lys
260 265 270
Ser Ile Glu Asn Lys His Gln Arg Arg Leu Val Lys Leu Leu Leu Gly
275 280 285
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Met Asp
290 295 300
Val Val Ser Leu Asp Lys Pro Phe Met Tyr Phe Glu Glu Ile Asp Asn
305 310 315 320
Glu Leu Asp Tyr Glu Pro Glu Ser Ala Asn Glu Val Ala Lys Lys Leu
325 330 335
Pro Tyr Gln Gly Gln Leu Lys Leu Leu Leu Gly Glu Leu Phe Phe Leu
340 345 350
Ser Lys Leu Gln Arg His Gly Ile Leu Asp Gly Ala Thr Val Val Tyr
355 360 365
Ile Gly Ser Ala Pro Gly Thr His Ile Arg Tyr Leu Arg Asp His Phe
370 375 380
Tyr Asn Leu Gly Val Ile Ile Lys Trp Met Leu Ile Asp Gly Arg His
385 390 395 400
His Asp Pro Ile Leu Asn Gly Leu Arg Asp Val Thr Leu Val Thr Arg
405 410 415
Phe Val Asp Glu Glu Tyr Leu Arg Ser Ile Lys Lys Gln Leu His Pro
420 425 430
Ser Lys Ile Ile Leu Ile Ser Asp Val Arg Ser Lys Arg Gly Gly Asn
435 440 445
Glu Pro Ser Thr Ala Asp Leu Leu Ser Asn Tyr Ala Leu Gln Asn Val
450 455 460
Met Ile Ser Ile Leu Asn Pro Val Ala Ser Ser Leu Lys Trp Arg Cys
465 470 475 480
Pro Phe Pro Asp Gln Trp Ile Lys Asp Phe Tyr Ile Pro His Gly Asn
485 490 495
Lys Met Leu Gln Pro Phe Ala Pro Ser Tyr Ser Ala Glu Met Arg Leu
500 505 510
Leu Ser Ile Tyr Thr Gly Glu Asn Met Arg Leu Thr Arg Val Thr Lys
515 520 525
Ser Asp Ala Val Asn Tyr Glu Lys Lys Met Tyr Tyr Leu Asn Lys Ile
530 535 540
Val Arg Asn Lys Val Val Val Asn Phe Asp Tyr Pro Asn Gln Glu Tyr
545 550 555 560
Asp Tyr Phe His Met Tyr Phe Met Leu Arg Thr Val Tyr Cys Asn Lys
565 570 575
Thr Phe Pro Thr Thr Lys Ala Lys Val Leu Phe Leu Gln Gln Ser Ile
580 585 590
Phe Arg Phe Leu Asn Ile Pro Thr Thr Ser Thr Glu Lys Val Ser His
595 600 605
Glu Pro Ile Gln Arg Lys Ile Ser Ser Lys Asn Ser Met Ser Lys Asn
610 615 620
Arg Asn Ser Lys Arg Ser Val Arg Ser Asn Lys
625 630 635
<210> 2
<211> 1908
<212> DNA
<213> Artificial Sequence
<400> 2
atggacgaaa tcgttaaaaa catccgtgaa ggtacccacg ttctgctgcc gttctacgaa 60
accctgccgg aactgaacct gtctctgggt aaatctccgc tgccgtctct ggaatacggt 120
gctaactact tcctgcagat ctctcgtgtt aacgacctga accgtatgcc gaccgacatg 180
ctgaaactgt tcacccacga catcatgctg ccggaatctg acctggacaa agtttacgaa 240
atcctgaaaa tcaactctgt taaatactac ggtcgttcta ccaaagctga cgctgttgtt 300
gctgacctgt ctgctcgtaa caaactgttc aaacgtgaac gtgacgctat caaatctaac 360
aaccacctga ccgaaaacaa cctgtacatc tctgactaca aaatgctgac cttcgacgtt 420
ttccgtccgc tgttcgactt cgttaacgaa aaatactgca tcatcaaact gccgaccctg 480
ttcggtcgtg gtgttatcga caccatgcgt atctactgct ctctgttcaa aaacgttcgt 540
ctgctgaaat gcgtttctga ctcttggctg aaagactctg ctatcatggt tgcttctgac 600
gtttgcaaaa aaaacctgga cctgttcatg tctcacgtta aatctgttac caaatcttct 660
tcttggaaag acgttaactc tgttcagttc tctatcctga acaacccggt tgacaccgaa 720
ttcatcaaca aattcctgga attctctaac cgtgtttacg aagctctgta ctacgttcac 780
tctctgctgt actcttctat gacctctgac tctaaatcta tcgaaaacaa acaccagcgt 840
cgtctggtta aactgctgct gggtggtggt ggttctggtg gtggtggttc tggtggtggt 900
ggttctatgg acgttgtttc tctggacaaa ccgttcatgt acttcgaaga aatcgacaac 960
gaactggact acgaaccgga atctgctaac gaagttgcta aaaaactgcc gtaccagggt 1020
cagctgaaac tgctgctggg tgaactgttc ttcctgtcta aactgcagcg tcacggtatc 1080
ctggacggtg ctaccgttgt ttacatcggt tctgctccgg gtacccacat ccgttacctg 1140
cgtgaccact tctacaacct gggtgttatc atcaaatgga tgctgatcga cggtcgtcac 1200
cacgacccga tcctgaacgg tctgcgtgac gttaccctgg ttacccgttt cgttgacgaa 1260
gaatacctgc gttctatcaa aaaacagctg cacccgtcta aaatcatcct gatctctgac 1320
gttcgttcta aacgtggtgg taacgaaccg tctaccgctg acctgctgtc taactacgct 1380
ctgcagaacg ttatgatctc tatcctgaac ccggttgctt cttctctgaa atggcgttgc 1440
ccgttcccgg accagtggat caaagacttc tacatcccgc acggtaacaa aatgctgcag 1500
ccgttcgctc cgtcttactc tgctgaaatg cgtctgctgt ctatctacac cggtgaaaac 1560
atgcgtctga cccgtgttac caaatctgac gctgttaact acgaaaaaaa aatgtactac 1620
ctgaacaaaa tcgttcgtaa caaagttgtt gttaacttcg actacccgaa ccaggaatac 1680
gactacttcc acatgtactt catgctgcgt accgtttact gcaacaaaac cttcccgacc 1740
accaaagcta aagttctgtt cctgcagcag tctatcttcc gtttcctgaa catcccgacc 1800
acctctaccg aaaaagtttc tcacgaaccg atccagcgta aaatctcttc taaaaactct 1860
atgtctaaaa accgtaactc taaacgttct gttcgttcta acaaataa 1908
<210> 3
<211> 844
<212> PRT
<213> Artificial Sequence
<400> 3
Met Asp Ala Asn Val Val Ser Ser Ser Thr Ile Ala Thr Tyr Ile Asp
1 5 10 15
Ala Leu Ala Lys Asn Ala Ser Glu Leu Glu Gln Arg Ser Thr Ala Tyr
20 25 30
Glu Ile Asn Asn Glu Leu Glu Leu Val Phe Ile Lys Pro Pro Leu Ile
35 40 45
Thr Leu Thr Asn Val Val Asn Ile Ser Thr Ile Gln Glu Ser Phe Ile
50 55 60
Arg Phe Thr Val Thr Asn Lys Glu Gly Val Lys Ile Arg Thr Lys Ile
65 70 75 80
Pro Leu Ser Lys Val His Gly Leu Asp Val Lys Asn Val Gln Leu Val
85 90 95
Asp Ala Ile Asp Asn Ile Val Trp Glu Lys Lys Ser Leu Val Thr Glu
100 105 110
Asn Arg Leu His Lys Glu Cys Leu Leu Arg Leu Ser Thr Glu Glu Arg
115 120 125
His Ile Phe Leu Asp Tyr Lys Lys Tyr Gly Ser Ser Ile Arg Leu Glu
130 135 140
Leu Val Asn Leu Ile Gln Ala Lys Thr Lys Asn Phe Thr Ile Asp Phe
145 150 155 160
Lys Leu Lys Tyr Phe Leu Gly Ser Gly Ala Gln Ser Lys Ser Ser Leu
165 170 175
Leu His Ala Ile Asn His Pro Lys Ser Arg Pro Asn Thr Ser Leu Glu
180 185 190
Ile Glu Phe Thr Pro Arg Asp Asn Glu Thr Val Pro Tyr Asp Glu Leu
195 200 205
Ile Lys Glu Leu Thr Thr Leu Ser Arg His Ile Phe Met Ala Ser Pro
210 215 220
Glu Asn Val Ile Leu Ser Pro Pro Ile Asn Ala Pro Ile Lys Thr Phe
225 230 235 240
Met Leu Pro Lys Gln Asp Ile Val Gly Leu Asp Leu Glu Asn Leu Tyr
245 250 255
Ala Val Thr Lys Thr Asp Gly Ile Pro Ile Thr Ile Arg Val Thr Ser
260 265 270
Asn Gly Leu Tyr Cys Tyr Phe Thr His Leu Gly Tyr Ile Ile Arg Tyr
275 280 285
Pro Val Lys Arg Ile Ile Asp Ser Glu Val Val Val Phe Gly Glu Ala
290 295 300
Val Lys Asp Lys Asn Trp Thr Val Tyr Leu Ile Lys Leu Ile Glu Pro
305 310 315 320
Val Asn Ala Ile Asn Asp Arg Leu Glu Glu Ser Lys Tyr Val Glu Ser
325 330 335
Lys Leu Val Asp Ile Cys Asp Arg Ile Val Phe Lys Ser Lys Lys Tyr
340 345 350
Glu Gly Pro Phe Thr Thr Thr Ser Glu Val Val Asp Met Leu Ser Thr
355 360 365
Tyr Leu Pro Lys Gln Pro Glu Gly Val Ile Leu Phe Tyr Ser Lys Gly
370 375 380
Pro Lys Ser Asn Ile Asp Phe Lys Ile Lys Lys Glu Asn Thr Ile Asp
385 390 395 400
Gln Thr Ala Asn Val Val Phe Arg Tyr Met Ser Ser Glu Pro Ile Ile
405 410 415
Phe Gly Glu Ser Ser Ile Phe Val Glu Tyr Lys Lys Phe Ser Asn Asp
420 425 430
Lys Gly Phe Pro Lys Glu Tyr Gly Ser Gly Lys Ile Val Leu Tyr Asn
435 440 445
Gly Val Asn Tyr Leu Asn Asn Ile Tyr Cys Leu Glu Tyr Ile Asn Thr
450 455 460
His Asn Glu Val Gly Ile Lys Ser Val Val Val Pro Ile Lys Phe Ile
465 470 475 480
Ala Glu Phe Leu Val Asn Gly Glu Ile Leu Lys Pro Arg Ile Asp Lys
485 490 495
Thr Met Lys Tyr Ile Asn Ser Glu Asp Tyr Tyr Gly Asn Gln His Asn
500 505 510
Ile Ile Val Glu His Leu Arg Asp Gln Ser Ile Lys Ile Gly Asp Ile
515 520 525
Phe Asn Glu Asp Lys Leu Ser Asp Val Gly His Gln Tyr Ala Asn Asn
530 535 540
Asp Lys Phe Arg Leu Asn Pro Glu Val Ser Tyr Phe Thr Asn Lys Arg
545 550 555 560
Thr Arg Gly Pro Leu Gly Ile Leu Ser Asn Tyr Val Lys Thr Leu Leu
565 570 575
Ile Ser Met Tyr Cys Ser Lys Thr Phe Leu Asp Asp Ser Asn Lys Arg
580 585 590
Lys Val Leu Ala Ile Asp Phe Gly Asn Gly Ala Asp Leu Glu Lys Tyr
595 600 605
Phe Tyr Gly Glu Ile Ala Leu Leu Val Ala Thr Asp Pro Asp Ala Asp
610 615 620
Ala Ile Ala Arg Gly Asn Glu Arg Tyr Asn Lys Leu Asn Ser Gly Ile
625 630 635 640
Lys Thr Lys Tyr Tyr Lys Phe Asp Tyr Ile Gln Glu Thr Ile Arg Ser
645 650 655
Asp Thr Phe Val Ser Ser Val Arg Glu Val Phe Tyr Phe Gly Lys Phe
660 665 670
Asn Ile Ile Asp Trp Gln Phe Ala Ile His Tyr Ser Phe His Pro Arg
675 680 685
His Tyr Ala Thr Val Met Asn Asn Leu Ser Glu Leu Thr Ala Ser Gly
690 695 700
Gly Lys Val Leu Ile Thr Thr Met Asp Gly Asp Lys Leu Ser Lys Leu
705 710 715 720
Thr Asp Lys Lys Thr Phe Ile Ile His Lys Asn Leu Pro Ser Ser Glu
725 730 735
Asn Tyr Met Ser Val Glu Lys Ile Ala Asp Asp Arg Ile Val Val Tyr
740 745 750
Asn Pro Ser Thr Met Ser Thr Pro Met Thr Glu Tyr Ile Ile Lys Lys
755 760 765
Asn Asp Ile Val Arg Val Phe Asn Glu Tyr Gly Phe Val Leu Val Asp
770 775 780
Asn Val Asp Phe Ala Thr Ile Ile Glu Arg Ser Lys Lys Phe Ile Asn
785 790 795 800
Gly Ala Ser Thr Met Glu Asp Arg Pro Ser Thr Arg Asn Phe Phe Glu
805 810 815
Leu Asn Arg Gly Ala Ile Lys Cys Glu Gly Leu Asp Val Glu Asp Leu
820 825 830
Leu Ser Tyr Tyr Val Val Tyr Val Phe Ser Lys Arg
835 840
<210> 4
<211> 2535
<212> DNA
<213> Artificial Sequence
<400> 4
atggacgcta acgttgtttc ttcttctacc atcgctacct acatcgacgc tctggctaaa 60
aacgcttctg aactggaaca gcgttctacc gcttacgaaa tcaacaacga actggaactg 120
gttttcatca aaccgccgct gatcaccctg accaacgttg ttaacatctc taccatccag 180
gaatctttca tccgtttcac cgttaccaac aaagaaggtg ttaaaatccg taccaaaatc 240
ccgctgtcta aagttcacgg tctggacgtt aaaaacgttc agctggttga cgctatcgac 300
aacatcgttt gggaaaaaaa atctctggtt accgaaaacc gtctgcacaa agaatgcctg 360
ctgcgtctgt ctaccgaaga acgtcacatc ttcctggact acaaaaaata cggttcttct 420
atccgtctgg aactggttaa cctgatccag gctaaaacca aaaacttcac catcgacttc 480
aaactgaaat acttcctggg ttctggtgct cagtctaaat cttctctgct gcacgctatc 540
aaccacccga aatctcgtcc gaacacctct ctggaaatcg aattcacccc gcgtgacaac 600
gaaaccgttc cgtacgacga actgatcaaa gaactgacca ccctgtctcg tcacatcttc 660
atggcttctc cggaaaacgt tatcctgtct ccgccgatca acgctccgat caaaaccttc 720
atgctgccga aacaggacat cgttggtctg gacctggaaa acctgtacgc tgttaccaaa 780
accgacggta tcccgatcac catccgtgtt acctctaacg gtctgtactg ctacttcacc 840
cacctgggtt acatcatccg ttacccggtt aaacgtatca tcgactctga agttgttgtt 900
ttcggtgaag ctgttaaaga caaaaactgg accgtttacc tgatcaaact gatcgaaccg 960
gttaacgcta tcaacgaccg tctggaagaa tctaaatacg ttgaatctaa actggttgac 1020
atctgcgacc gtatcgtttt caaatctaaa aaatacgaag gtccgttcac caccacctct 1080
gaagttgttg acatgctgtc tacctacctg ccgaaacagc cggaaggtgt tatcctgttc 1140
tactctaaag gtccgaaatc taacatcgac ttcaaaatca aaaaagaaaa caccatcgac 1200
cagaccgcta acgttgtttt ccgttacatg tcttctgaac cgatcatctt cggtgaatct 1260
tctatcttcg ttgaatacaa aaaattctct aacgacaaag gtttcccgaa agaatacggt 1320
tctggtaaaa tcgttctgta caacggtgtt aactacctga acaacatcta ctgcctggaa 1380
tacatcaaca cccacaacga agttggtatc aaatctgttg ttgttccgat caaattcatc 1440
gctgaattcc tggttaacgg tgaaatcctg aaaccgcgta tcgacaaaac catgaaatac 1500
atcaactctg aagactacta cggtaaccag cacaacatca tcgttgaaca cctgcgtgac 1560
cagtctatca aaatcggtga catcttcaac gaagacaaac tgtctgacgt tggtcaccag 1620
tacgctaaca acgacaaatt ccgtctgaac ccggaagttt cttacttcac caacaaacgt 1680
acccgtggtc cgctgggtat cctgtctaac tacgttaaaa ccctgctgat ctctatgtac 1740
tgctctaaaa ccttcctgga cgactctaac aaacgtaaag ttctggctat cgacttcggt 1800
aacggtgctg acctggaaaa atacttctac ggtgaaatcg ctctgctggt tgctaccgac 1860
ccggacgctg acgctatcgc tcgtggtaac gaacgttaca acaaactgaa ctctggtatc 1920
aaaaccaaat actacaaatt cgactacatc caggaaacca tccgttctga caccttcgtt 1980
tcttctgttc gtgaagtttt ctacttcggt aaattcaaca tcatcgactg gcagttcgct 2040
atccactact ctttccaccc gcgtcactac gctaccgtta tgaacaacct gtctgaactg 2100
accgcttctg gtggtaaagt tctgatcacc accatggacg gtgacaaact gtctaaactg 2160
accgacaaaa aaaccttcat catccacaaa aacctgccgt cttctgaaaa ctacatgtct 2220
gttgaaaaaa tcgctgacga ccgtatcgtt gtttacaacc cgtctaccat gtctaccccg 2280
atgaccgaat acatcatcaa aaaaaacgac atcgttcgtg ttttcaacga atacggtttc 2340
gttctggttg acaacgttga cttcgctacc atcatcgaac gttctaaaaa attcatcaac 2400
ggtgcttcta ccatggaaga ccgtccgtct acccgtaact tcttcgaact gaaccgtggt 2460
gctatcaaat gcgaaggtct ggacgttgaa gacctgctgt cttactacgt tgtttacgtt 2520
ttctctaaac gttaa 2535
<210> 5
<211> 50
<212> DNA
<213> Artificial Sequence
<400> 5
gataatacga ctcactatag ggaaggagag gaaggaaagg gaagaaagaa 50
<210> 6
<211> 50
<212> DNA
<213> Artificial Sequence
<400> 6
ttctttcttc cctttccttc ctctccttcc ctatagtgag tcgtattatc 50
<210> 7
<211> 1173
<212> DNA
<213> Artificial Sequence
<400> 7
tctagataat acgactcact atagggagaa ttcgccacca tggtgagcaa gggcgaggag 60
ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag 120
ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc 180
atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac 240
ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc 300
gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac 360
aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag 420
ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac 480
agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag 540
atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc 600
cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc 660
ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc 720
gccgggatca ctctcggcat ggacgagctg tacaagtaag gatcctgcac tagtgctgtc 780
gacgctcgct ttcttgctgt ccaatttcta ttaaaggttc ctttgttccc taagtccaac 840
tactaaactg ggggatatta tgaagggcct tgagcatctg gattctgcct aataaaaaac 900
atttattttc attgcgctcg ctttcttgct gtccaatttc tattaaaggt tcctttgttc 960
cctaagtcca actactaaac tgggggatat tatgaagggc cttgagcatc tggattctgc 1020
ctaataaaaa acatttattt tcattgcaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1080
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1140
aaaaaaaaaa aaaaaaaaaa aaaaaaaggt acc 1173
<210> 8
<211> 6
<212> PRT
<213> Artificial Sequence
<400> 8
His His His His His His
1 5
<210> 9
<211> 370
<212> PRT
<213> Artificial Sequence
<400> 9
Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp Lys Gly
1 5 10 15
Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp Thr Gly
20 25 30
Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys Phe Pro
35 40 45
Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp Ala His
50 55 60
Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile Thr
65 70 75 80
Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp Asp Ala
85 90 95
Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val Glu Ala
100 105 110
Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys Thr
115 120 125
Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys Gly Lys
130 135 140
Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp Pro Leu
145 150 155 160
Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly Lys Tyr
165 170 175
Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly Leu
180 185 190
Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala Asp Thr
195 200 205
Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala Met
210 215 220
Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys Val
225 230 235 240
Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro Ser Lys
245 250 255
Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser Pro Asn
260 265 270
Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr Asp Glu
275 280 285
Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val Ala Leu
290 295 300
Lys Ser Tyr Glu Glu Glu Leu Val Lys Asp Pro Arg Ile Ala Ala Thr
305 310 315 320
Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro Gln Met
325 330 335
Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala Ala Ser
340 345 350
Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr Gly Ala
355 360 365
Ser Thr
370

Claims (12)

1. A chimeric protein subunit comprising, fused to (a) the D12 subunit of an RNA capping enzyme or a functional fragment thereof, or a variant thereof having at least 90% sequence identity thereto and having D12 subunit activity, and (b) an RNA cap structure 2 '-O-methyltransferase or a functional fragment thereof, or a variant thereof having at least 90% sequence identity thereto and having RNA cap structure 2' -O-methyltransferase activity, and optionally (c) a linker between (a) and (b),
preferably, the first and second electrodes are formed of a metal,
the RNA capping enzyme is a vaccinia virus RNA capping enzyme,
the RNA cap structure 2 '-O-methyltransferase is vaccinia virus 2' -O-methyltransferase,
(a) The carboxy terminus of (b) is linked to the amino terminus of (b).
2. The chimeric protein subunit of claim 1,
the amino acid sequence of the D12 subunit of the RNA capping enzyme is shown in SEQ ID NO 1, 1-287, and/or
The amino acid sequence of the RNA cap structure 2' -O-methyltransferase is shown as SEQ ID NO. 1, 303-635, and/or
A variant of the D12 subunit of the RNA capping enzyme or a functional fragment thereof has a mutation selected from one or more of: N42A, Y43A, L61A, K62A, F245A, L246A, K111A, R112A, N120A, N121A, N126A, N127A, F141A, R142A, K223A, D224A, H260A, S261A, E275A, N276A, R280A, R281A, and/or
A variant of RNA cap structure 2' -O-methyltransferase or a functional fragment thereof has the following characteristics: (1) a mutation selected from one or more of: K41D, C178S, a201R, a201K, C272S; and/or (2) one or more amino acids selected from R, K, H, Y, C, D or E are mutated to A, and/or
(b) Further has a His tag and/or MBP tag at its N-or C-terminus,
the amino acid sequence of the linker is shown as the 288 th-302 th amino acid of SEQ ID NO. 1,
preferably, the amino acid sequence of the subunit of the chimeric protein is shown in SEQ ID NO 1.
3. A chimeric protein comprising: (1) The chimeric protein subunit of claim 1 or 2, and (2) an RNA capping enzyme D1 subunit or a functional fragment thereof, or a variant thereof having at least 90% sequence identity thereto and corresponding activity,
preferably, the first and second liquid crystal display panels are,
the chimeric protein is a heterodimeric protein, and/or
The RNA capping enzyme is a vaccinia virus RNA capping enzyme, and/or
Functional fragments of the D1 subunit of RNA capping enzyme comprise:
(1) An N7 methyltransferase domain or a functional fragment thereof;
(2) An N7 methyltransferase domain or a functional fragment thereof and a5' -triphosphatase domain or a functional fragment thereof;
(3) An N7 methyltransferase domain or functional fragment thereof and a guanosine transferase domain or functional fragment thereof; or
(4) An N7 methyltransferase domain or a functional fragment thereof, a5' -triphosphatase domain or a functional fragment thereof, and a guanosine transferase domain or a functional fragment thereof.
4. The chimeric protein of claim 3,
the 5' -triphosphatase domain is shown as amino acids 1-225 of SEQ ID NO. 3, and/or
The guanosine transferase domain is shown as SEQ ID NO:3 amino acids 226-530, and/or
The structure domain of the N7 methyltransferase is shown as amino acids 531-844 of SEQ ID NO. 3, and/or
The subunit of the RNA capping enzyme D1 is shown as SEQ ID NO 3, and/or
The functional fragment of the D1 subunit of the RNA capping enzyme comprises amino acids 498-844 or 540-844 of SEQ ID NO. 3, and/or
A variant of the N7-methyltransferase domain or a functional fragment thereof has a mutation selected from one or more of: 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. A nucleic acid molecule comprising a sequence selected from:
(1) The coding sequence of a subunit of the chimeric protein of claim 1 or 2, or the coding sequence of the chimeric protein of claim 3 or 4,
(2) A variant having at least 80% sequence identity to (1),
(3) The complementary sequence of (1) or (2),
preferably, the first and second liquid crystal display panels are,
the coding sequence of the chimeric protein subunit is shown as SEQ ID NO. 2, or a variant having at least 80% sequence identity with the same, or a degenerate variant encoding the same amino acid sequence with the same, and/or
The coding sequence of the chimeric protein, the coding sequence of the RNA capping enzyme D1 subunit or a functional fragment thereof is shown as SEQ ID NO. 4, or a variant having at least 80% sequence identity therewith, or a degenerate variant encoding the same amino acid sequence therewith.
6. A nucleic acid construct, said nucleic acid construct:
(1) Expressing the chimeric protein subunit of claim 1 or 2, or the chimeric protein of claim 3 or 4,
(2) Comprising the nucleic acid molecule of claim 5,
preferably, the first and second liquid crystal display panels are,
the nucleic acid construct comprises an expression cassette for the chimeric protein subunit and an expression cassette for the RNA capping enzyme D1 subunit or a functional fragment thereof; or the nucleic acid construct is an expression cassette, wherein the coding sequence for the chimeric protein subunit and the coding sequence for the D1 subunit of RNA capping enzyme or a functional fragment thereof are in the expression cassette, and/or
The nucleic acid construct is a cloning vector or an expression vector.
7. A host cell comprising, expressing and/or secreting a chimeric protein subunit of claim 1 or 2, or a chimeric protein of claim 3 or 4,
preferably, the host cell comprises the nucleic acid molecule of claim 5 and/or the nucleic acid construct of claim 6.
8. A method of modifying a target RNA to a capped RNA or a method of preparing a capped target RNA, comprising: contacting a target RNA with the chimeric protein subunit of claim 1 or 2 or the chimeric protein of claim 3 or 4, or contacting a target RNA with a cell comprising, expressing and/or secreting the chimeric protein subunit of claim 1 or 2 or the chimeric protein of claim 3 or 4, under conditions that allow for RNA capping,
preferably, the first and second electrodes are formed of a metal,
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 mixing the target RNA with the chimeric protein subunit or chimeric protein, and/or
The method comprises the step of introducing into the cell a DNA sequence expressing an RNA of interest.
9. The method of claim 8, wherein the method comprises the steps of:
optionally (1) denaturing the target RNA,
optionally (2) renaturing the target RNA,
(3) Incubating RNA with a chimeric protein subunit of claim 1 or 2 or a chimeric protein of claim 3 or 4 under conditions that allow for RNA capping to obtain capped RNA,
optionally (4) purifying the capped RNA,
preferably, the first and second liquid crystal display panels are,
the conditions that allow RNA capping are incubation at 37 ℃ for at least 20 minutes, and/or
The conditions that allow RNA capping further comprise the presence of one or more agents selected from the group consisting of: GTP, SAM, buffer, and/or
The target RNA is selected from the group consisting of 5' -triphosphorylated RNA, 5' -diphosphonated RNA, and RNA having a5' -Gppp cap structure.
10. The method of claim 8 or 9,
the method modifies a target RNA selected from the group consisting of 5'-m7 Gppp-structured capped RNA, 5' -triphosphorylated RNA, 5 '-diphosphonated RNA, and RNA having a5' -Gppp cap structure into a capped RNA having a 5'-m7Gppp structure, the chimeric protein comprises an N7 methyltransferase domain of a D1 subunit of an RNA capping enzyme or a functional fragment thereof, a5' -triphosphatase domain or a functional fragment thereof, a guanosine transferase domain or a functional fragment thereof, and the chimeric protein subunit; preferably, the chimeric protein comprises an RNA capping enzyme D1 subunit shown as SEQ ID NO. 3 and a chimeric protein subunit shown as SEQ ID NO. 1,
the method modifies a target RNA selected from the group consisting of 5'-m7 gppnp-structured capped RNA, 5' -triphosphorylated RNA, 5 '-diphosphonated RNA, and RNA having a5' -Gppp cap structure into a capped RNA having a 5'-m7 gppnp structure, the chimeric protein comprising an N7 methyltransferase (N7-MTase) domain of RNA-capping enzyme D1 subunit or a functional fragment thereof, a5' -triphosphatase (TPase) domain or a functional fragment thereof, and a guanosine transferase (GTase) domain or a functional fragment thereof, and the chimeric protein subunit; preferably, the chimeric protein comprises an RNA capping enzyme D1 subunit shown as SEQ ID NO. 3 and a chimeric protein subunit shown as SEQ ID NO. 1,
the method is a method of capping a target RNA selected from the group consisting of 5 '-diphosphonated RNA and RNA having a5' -Gppp structure, the chimeric protein comprising an N7 methyltransferase (N7-MTase) domain of the D1 subunit of RNA capping enzyme or a functional fragment thereof and a guanosine transferase (GTase) domain or a functional fragment thereof, and the chimeric protein subunit; preferably, the subunit of the chimeric protein is represented by SEQ ID NO 1, or
The method is a method of capping a target RNA that is an RNA having a5' -Gppp structure, the chimeric protein comprises an N7 methyltransferase (N7-MTase) domain of the D1 subunit of the RNA capping enzyme, or a functional fragment thereof, and the chimeric protein subunit; preferably, the subunit of the chimeric protein is shown as SEQ ID NO: 1.
11. A fusion protein comprising
(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-methyltransferase activity, and
(b) A His-tag and/or MBP-tag at (a) the N-or C-terminus,
preferably, the first and second electrodes are formed of a metal,
(a) And (b) there is a joint between them,
the RNA cap structure 2 '-O-methyltransferase is vaccinia virus 2' -O-methyltransferase,
the amino acid sequence of the RNA cap structure 2' -O-methyltransferase is shown as SEQ ID NO. 1, 303-635,
the variant of said RNA cap structure 2' -O-methyltransferase or a functional fragment thereof has the following characteristics: (1) a mutation selected from one or more of: K41D, C178S, a201R, a201K, C272S; and/or (2) one or more amino acids selected from R, K, H, Y, C, D or E are mutated to A, and/or
His tag is shown as SEQ ID NO:8, and/or
The MBP tag is shown as SEQ ID NO 9.
12. A nucleic acid sequence encoding the fusion protein of claim 11, a nucleic acid construct or a host cell comprising the nucleic acid sequence.
CN202110379093.3A 2021-04-08 2021-04-08 RNA modified chimeric protein and application thereof Pending CN115197327A (en)

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