RNA modification chimeric proteins and uses thereof

Abstract

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

Description

Technical Field

[0001] This invention relates to the field of RNA synthesis, and more specifically to chimeric proteins used to modify RNA and their applications. Background Technology

[0002] Compared with DNA-based therapeutics, in vitro transcription (IVT) synthesis of mRNA has significant advantages in terms of biosafety. This is attributed to the fact that RNA is difficult to integrate into the genome, the short time window for RNA expression to translate proteins, and the fact that it does not require intracellular transcription steps, allowing for better control over the initiation time and expression level of the target protein.

[0003] Therapeutic mRNAs primarily mimic the natural structure of eukaryotic mRNAs. A key characteristic is the presence of a cap structure at the 5' end. This cap structure consists of the first nucleotide of the RNA linked to inverse 7-methylguanosine (m7G), connected via a triphosphate bridge to form a cap O (cap0). Figure 10 The structure is shown in Figure C. Cap 0 is sufficient to recruit translation initiation factors and prevent mRNA degradation. Then, 2′-hydroxymethylation of the proximal nucleotide of the first cap forms cap 1 (cap1, as shown in Figure C). Figure 10 (As shown in C). The main functions of the Cap0 cap structure include: regulating splicing; nuclear export; stabilizing the mRNA structure by protecting it from 5'-exonucleases; assisting the innate immune system in recognizing "self-RNA"; serving as an anchor for recruiting initiating factors; and initiating the 5' to 3' circularization of mRNA during protein synthesis and translation. The Cap1 structure is closely related to the recognition by the innate immune system; host immune proteins can recognize abnormal, uncapped RNA.

[0004] Because IVT does not directly produce biologically functional eukaryotic capped mRNA, but only 5'-triphosphorylated RNA, the generated RNA requires different strategies to achieve RNA capping modification. Currently, there are two main strategies for producing capped mRNA: one is capping via co-transcriptionalization with cap analogs, and the other is post-transcriptional capping via capping enzyme modification of RNA.

[0005] In capping via co-transcriptionalization using cap analogs, the cap analog is added directly to the in vitro transduction (IVT). Substrate-specific RNA polymerases (e.g., T3, T7, or SP6 RNA polymerases) can incorporate the cap analog at the 5' end of the RNA at transcription initiation, directly producing the corresponding 5'-capped mRNA. Limitations of co-transcriptional capping include: competition between GTP and the cap analog, resulting in only partially capped mRNA from the IVT, and difficulties in separating and determining the ratio of capped to uncapped mRNA. Another problem with using m7GpppG as a cap analog for co-transcriptionalization is that it causes RNA to extend in the "wrong" direction, specifically at the 3'-OH of m7G, resulting in mRNA with an incorrect cap orientation.

[0006] Posttranscriptional capping of RNA refers to the enzymatic capping reaction of the RNA product generated by IVT with specific capping enzymes and / or mRNA-modifying enzymes in a suitable reaction system. The enzymatic reaction of cap0 involves three consecutive steps: First, 5'-triphosphatase (TPase) hydrolyzes the γ-phosphate of RNA; next, guanylate transferase (GTase) uses GTP as a substrate to couple the β-phosphate terminus of the resulting 5'-diphosphate to GMP, forming 5'-5'-linked Gppp-RNA; finally, RNA (guanine-N7) methyltransferase (N7-MTase) uses S-adenosyl-L-methionine (AdoMet) as a substrate to catalyze the methylation of the cap structure at the N7 position. Finally, the m7G cap-specific 2′-O-methyltransferase (2-O-MTase) modifies the ribose on the first nucleotide to generate the cap 1 structure. (Example...) Figure 10 As shown in A.

[0007] To date, although various eukaryotic or viral capping enzymes and mRNA-modifying enzymes with capping modification functions have been reported in the literature, very few enzymes can actually be applied to the large-scale production of capped mRNA. There is still a need in this field for capping enzymes that combine high efficiency and low cost. Summary of the Invention

[0008] The purpose of this invention is to provide a protein or protein subunit for capping RNA and a method for modifying RNA using the same. The technical solution of this invention simplifies the production process of mRNA-related modifying enzymes and reduces the amount of raw materials required for mRNA production.

[0009] The first aspect of the present invention provides a chimeric protein subunit comprising interconnected (a) a D12 subunit of an RNA capping enzyme or a functional fragment thereof, or a variant thereof having at least 90% sequence identity and having D12 subunit activity, and (b) an RNA capping 2′-O-methyltransferase or a functional fragment thereof, or a variant thereof having at least 90% sequence identity and having RNA capping 2′-O-methyltransfer activity, and optionally (c) a linker between (a) and (b).

[0010] In one or more embodiments, the RNA capping enzyme is a vaccinia virus RNA capping enzyme.

[0011] In one or more embodiments, the RNA cap structure 2′-O-methyltransferase is a vaccinia virus 2′-O-methyltransferase.

[0012] In one or more embodiments, the carboxyl terminus of (a) is connected to the amino terminus of (b).

[0013] In one or more embodiments, (b) further comprises a His tag and / or an MBP tag located at its N-terminus or C-terminus. In one or more embodiments, the His tag is as shown in SEQ ID NO:8. In one or more embodiments, the MBP tag is as shown in SEQ ID NO:9.

[0014] 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, numbers 1-287.

[0015] In one or more embodiments, variants of the D12 subunit of the RNA capping enzyme or a functional segment thereof have mutations selected from one or more of the following: N42A, Y43A, L61A, K62A, F245A, L246A, K111A, R112A, N120A, N121A, N126A, N127A, F141A, R142A, K223A, D224A, H260A, S261A, E275A, N276A, R280A, R281A.

[0016] In one or more embodiments, the amino acid sequence of the RNA cap structure 2′-O-methyltransferase is shown in SEQ ID NO: 1, 303-635.

[0017] In one or more embodiments, the RNA cap structure 2′-O-methyltransferase or a variant of its functional fragment has the following characteristics:

[0018] (1) Having one or more mutations selected from the following: K41D, C178S, A201R, A201K, C272S; and / or

[0019] (2) One or more of the amino acids selected from R, K, H, Y, C, D or E are mutated to A.

[0020] In one or more embodiments, a variant of the RNA cap structure 2′-O-methyltransferase or a functional fragment thereof, as shown in SEQ ID NO:1, 303-635, and having a mutation selected from one or more of the following: K41D, C178S, A201R, A201K, C272S, or one or more amino acids selected from R, K, H, Y, C, D, E, mutated to A.

[0021] In one or more embodiments, the amino acid sequence of the linker is as shown in amino acids 288-302 of SEQ ID NO:1.

[0022] In one or more embodiments, the amino acid sequence of the chimeric protein subunit is shown in SEQ ID NO:1.

[0023] A second aspect of the present invention provides a chimeric protein comprising: (1) a chimeric protein subunit as described in any embodiment of the first aspect herein, and (2) an RNA capping enzyme D1 subunit or a functional fragment thereof, or a variant having at least 90% sequence identity with and corresponding activity thereof.

[0024] In one or more embodiments, the protein is a heterodimeric protein of (1) and (2).

[0025] In one or more embodiments, the RNA capping enzyme is a vaccinia virus RNA capping enzyme.

[0026] In one or more embodiments, the functional fragment of the RNA capping enzyme D1 subunit includes:

[0027] (1) The N7 methyltransferase (N7-MTase) domain or a functional fragment thereof;

[0028] (2) The N7-methyltransferase (N7-MTase) domain or a functional fragment thereof and the 5'-triphosphatase (TPase) domain or a functional fragment thereof;

[0029] (3) The N7-MTase domain or a functional fragment thereof and the guanylate transferase (GTase) domain or a functional fragment thereof; or

[0030] (4) The N7-methyltransferase (N7-MTase) domain or its functional fragment, the 5'-triphosphatase (TPase) domain or its functional fragment, and the guanylate transtransferase (GTase) domain or its functional fragment.

[0031] In one or more embodiments, the RNA capping enzyme D1 subunit is shown as SEQ ID NO:3.

[0032] In one or more embodiments, the functional fragment of the RNA capping enzyme D1 subunit comprises amino acids 498-844 or 540-844 of SEQ ID NO:3.

[0033] In one or more embodiments, the 5'-triphosphatase (TPase) domain is shown as amino acids 1-225 of SEQ ID NO:3.

[0034] In one or more embodiments, the guanylate transferase (GTase) domain is shown as amino acids 226-530 of SEQ ID NO:3.

[0035] In one or more embodiments, the N7 methyltransferase (N7-MTase) domain is shown as amino acids 531-844 of SEQ ID NO:3.

[0036] In one or more embodiments, variants of the N7-methyltransferase domain or its functional fragment have mutations selected from one or more of the following: D545A, R548A, N550D, Y555F, R560K, R794A, R808A, Y683S, Y684A, Y684F, D598A, G600A, G602A, I681A, S684A, F685A, T571A, L575A, L576A, M579A, F585A, L586A, D587A, D784A, N785A, R794A, F798A, M805A, E806A.

[0037] The present invention also provides a nucleic acid molecule comprising a sequence selected from the following:

[0038] (1) The coding sequence of the chimeric protein subunit described in the first aspect of this paper.

[0039] (2) A variant that has at least 80% sequence identity with (1),

[0040] (3)(1) or (2) complementary sequences.

[0041] In one or more embodiments, the nucleic acid molecule has the sequence shown in SEQ ID NO:2, or a variant having at least 80% sequence identity with it, or a degenerate variant encoding the same amino acid sequence.

[0042] The present invention also provides a nucleic acid molecule comprising a sequence selected from the following

[0043] (1) The coding sequences of the chimeric protein subunits described in the first aspect of this document, and the coding sequences of the RNA capping enzyme D1 subunit or its functional fragments or variants.

[0044] (2) A variant that has at least 80% sequence identity with (1),

[0045] (3)(1) or (2) complementary sequences.

[0046] In one or more embodiments, the RNA capping enzyme is a vaccinia virus RNA capping enzyme.

[0047] In one or more embodiments, the functional fragment of the RNA capping enzyme D1 subunit includes:

[0048] (1) The N7 methyltransferase (N7-MTase) domain or a functional fragment thereof;

[0049] (2) The N7-methyltransferase (N7-MTase) domain or a functional fragment thereof and the 5'-triphosphatase (TPase) domain or a functional fragment thereof;

[0050] (3) The N7-MTase domain or a functional fragment thereof and the guanylate transferase (GTase) domain or a functional fragment thereof; or

[0051] (4) The N7-methyltransferase (N7-MTase) domain or its functional fragment, the 5'-triphosphatase (TPase) domain or its functional fragment, and the guanylate transtransferase (GTase) domain or its functional fragment.

[0052] In one or more embodiments, the RNA capping enzyme D1 subunit is shown as SEQ ID NO:3.

[0053] In one or more embodiments, the functional fragment of the RNA capping enzyme D1 subunit comprises amino acids 498-844 or 540-844 of SEQ ID NO:3.

[0054] In one or more embodiments, the 5'-triphosphatase (TPase) domain is shown as amino acids 1-225 of SEQ ID NO:3.

[0055] In one or more embodiments, the guanylate transferase (GTase) domain is shown as amino acids 226-530 of SEQ ID NO:3.

[0056] In one or more embodiments, the N7 methyltransferase (N7-MTase) domain is shown as amino acids 531-844 of SEQ ID NO:3.

[0057] In one or more embodiments, the coding sequence of the RNA capping enzyme D1 subunit or a functional fragment thereof is as shown in SEQ ID NO:4, or a variant having at least 80% sequence identity with it, or a degenerate variant encoding the same amino acid sequence.

[0058] In one or more embodiments, the coding sequence of the chimeric protein subunit is shown in SEQ ID NO:2.

[0059] Another aspect of the present invention provides a nucleic acid construct, wherein the nucleic acid construct is:

[0060] (1) Expressing the chimeric protein subunit described in the first aspect of this document, or the chimeric protein described in the second aspect.

[0061] (2) Includes the nucleic acid molecules described in this article.

[0062] In one or more embodiments, the nucleic acid construct contains an expression cassette of the chimeric protein subunit and an expression cassette of the RNA capping enzyme D1 subunit or a functional fragment thereof; or the nucleic acid construct is an expression cassette in which the coding sequences of the chimeric protein subunit and the coding sequences of the RNA capping enzyme D1 subunit or a functional fragment thereof are located.

[0063] In one or more embodiments, the nucleic acid construct is a cloning vector or an expression vector.

[0064] Another aspect of the present invention provides a host cell that contains, expresses, and / or secretes the chimeric protein subunits or chimeric proteins described herein.

[0065] In one or more embodiments, the host cell comprises the nucleic acid molecules and nucleic acid constructs described herein.

[0066] The present invention also provides a method for modifying target RNA into capped RNA or a method for preparing capped target RNA, comprising: contacting the target RNA with the chimeric protein described herein, or contacting the target RNA with a cell expressing the chimeric protein described herein, under conditions that allow RNA to be catalytically capped.

[0067] In one or more embodiments, the condition for allowing RNA capping is incubation at 37°C for at least 20 minutes.

[0068] In one or more embodiments, contacting the target RNA with the chimeric protein described herein includes expressing the target RNA in cells expressing the chimeric protein described herein, or mixing the target RNA with the chimeric protein described herein.

[0069] In one or more embodiments, the method includes the step of introducing a DNA sequence expressing the target RNA into the host cell.

[0070] In one or more embodiments, the method includes the steps of:

[0071] Optionally, (1) denature the target RNA, for example, by incubation at 50-70°C for 1-60 minutes, preferably at 65°C for 5-20 minutes.

[0072] Optionally (2) renatures the target RNA product of (1), for example, by incubating at 0°C for 2–10 minutes.

[0073] (3) Incubate the RNA with the chimeric protein described herein at 37°C for at least 20 minutes, for example, 30-90 minutes, to obtain capped RNA.

[0074] Optional (4) Purification of capped RNA.

[0075] In one or more embodiments, the incubated mixture further comprises one or more reagents selected from the following: GTP, SAM, and buffer.

[0076] In one or more embodiments, the target RNA is selected from 5'-triphosphorylated RNA, 5'-diphosphorylated RNA, and RNA having a 5'-Gppp cap structure.

[0077] In one or more embodiments, the method modifies the target RNA to a capped RNA having a 5'-m7Gppp structure, wherein the target RNA is selected from 5'-triphosphorylated RNA, 5'-diphosphoesterified RNA, and RNA having a 5'-Gppp cap structure, and the chimeric protein comprises an N7 methyltransferase domain or a functional fragment thereof, a 5'-triphosphatase domain or a functional fragment thereof, a guanylate transferase domain or a functional fragment thereof, and the chimeric protein subunit. Preferably, the chimeric protein comprises an RNA capping enzyme D1 subunit as shown in SEQ ID NO:3 and a chimeric protein subunit as shown in SEQ ID NO:1.

[0078] In one or more embodiments, the method modifies the target RNA to a capped RNA having a 5'-m7GpppNmp structure, wherein the target RNA is selected from 5'-triphosphorylated RNA, 5'-diphosphoesterified RNA, and RNA having a 5'-Gppp cap structure, and the chimeric protein comprises an N7 methyltransferase (N7-MTase) domain or a functional fragment thereof, a 5'-triphosphatase (TPase) domain or a functional fragment thereof, and a guanylate transferase (GTase) domain or a functional fragment thereof, and the chimeric protein subunit. Preferably, the chimeric protein comprises an RNA capping enzyme D1 subunit as shown in SEQ ID NO:3 and a chimeric protein subunit as shown in SEQ ID NO:1.

[0079] In one or more embodiments, the method is a method of capping target RNA, the target RNA being selected from 5'-bisphosphoesterified RNA and RNA having a 5'-Gppp structure, the chimeric protein comprising an N7 methyltransferase (N7-MTase) domain or a functional fragment thereof and a guanylate transferase (GTase) domain or a functional fragment thereof of the RNA capping enzyme D1 subunit, and the chimeric protein subunit. Preferably, the chimeric protein subunit is as shown in SEQ ID NO:1.

[0080] In one or more embodiments, the method is a method of capping target RNA, said target RNA being RNA having a 5'-Gppp structure, and said chimeric protein comprising an N7 methyltransferase (N7-MTase) domain or a functional fragment thereof of an RNA capping enzyme D1 subunit, and said chimeric protein subunit. Preferably, said chimeric protein subunit is as shown in SEQ ID NO:1.

[0081] In one or more embodiments, the 5'-triphosphatase (TPase) domain is shown as amino acids 1-225 of SEQ ID NO:3.

[0082] In one or more embodiments, the guanylate transferase (GTase) domain is shown as amino acids 226-530 of SEQ ID NO:3.

[0083] In one or more embodiments, the N7 methyltransferase (N7-MTase) domain is shown as amino acids 531-844 of SEQ ID NO:3.

[0084] The present invention also provides a method for methylating RNA with a 5'-m7Gppp structure, comprising: contacting the RNA with the chimeric protein subunit described herein, or contacting the RNA with a cell expressing the chimeric protein subunit described herein.

[0085] In one or more embodiments, contacting the RNA with the chimeric protein subunit described herein includes expressing the RNA in cells expressing the chimeric protein subunit described herein.

[0086] A third aspect of the present invention provides a fusion protein comprising:

[0087] (a) RNA cap 2′-O-methyltransferase or a functional fragment thereof, or a variant having at least 90% sequence identity with it and possessing RNA cap 2′-O-methyltransfer activity, and

[0088] (b) His tag and / or MBP tag located at the N or C end of (a).

[0089] In one or more embodiments, there is a joint between (a) and (b).

[0090] In one or more embodiments, the RNA cap structure 2′-O-methyltransferase is a vaccinia virus 2′-O-methyltransferase.

[0091] In one or more embodiments, the amino acid sequence of the RNA cap structure 2′-O-methyltransferase is shown in SEQ ID NO: 1, 303-635.

[0092] In one or more embodiments, the RNA cap structure 2′-O-methyltransferase or a variant of its functional fragment has the following characteristics:

[0093] (1) Having one or more mutations selected from the following: K41D, C178S, A201R, A201K, C272S; and / or

[0094] (2) One or more of the amino acids selected from R, K, H, Y, C, D or E are mutated to A.

[0095] In one or more embodiments, a variant of the RNA cap structure 2′-O-methyltransferase or a functional fragment thereof, as shown in SEQ ID NO:1, 303-635, and having a mutation selected from one or more of the following: K41D, C178S, A201R, A201K, C272S, or one or more amino acids selected from R, K, H, Y, C, D, E, mutated to A.

[0096] In one or more embodiments, the His label is as shown in SEQ ID NO:8.

[0097] In one or more embodiments, the MBP label is as shown in SEQ ID NO:9.

[0098] The present invention also provides nucleic acid sequences encoding the fusion protein described in the third aspect herein, nucleic acid constructs comprising said nucleic acid sequences, or host cells. Attached Figure Description

[0099] Figure 1 Image of vectors for 2-O-MTase, MBP-(2-O-MTase), vaccinia capped enzyme D1:D12, and chimeric enzyme D1:D12-((2-O-MTase)).

[0100] Figure 2 The N-terminus of 2-O-methyltransferase is added with MBP and / or His tags. The bacterial cell lysate is identified by SDS-PAGE.

[0101] Figure 3 SDS-PAGE identification of vaccinia virus capping enzyme and chimeric enzyme cell lysate.

[0102] Figure 4 SDS-PAGE analysis of wild-type vaccinia virus capping enzyme, 2-O-methyltransferase, and chimeric enzyme proteins.

[0103] Figure 5 HPLC-MS was used to analyze the activity of 30 units of vaccinia virus capping enzyme VVCE-modified triphosphate RNA.

[0104] Figure 6 HPLC-MS was used to analyze the activity of 30 units of 2-O-methyltransferase modified cap0 RNA.

[0105] Figure 7 HPLC-MS was used to analyze the activity of 30 units of triphosphate RNA synergistically modified by vaccinia virus capping enzyme and 2-O-methyltransferase.

[0106] Figure 8 HPLC-MS was used to analyze the activity of 30 units of chimeric enzyme-modified triphosphate RNA.

[0107] Figure 9 The cellular biological functions of eGFP mRNA prepared by modification with wild-type enzymes and chimeric enzymes were compared. Specifically, 1) eGFP mRNA was prepared by synergistic modification with vaccinia virus capping enzyme and vaccinia virus 2-O-methyltransferase; 2) eGFP mRNA was prepared by modification with chimeric enzyme alone; and 3) unmodified control group consisted of uncapped eGFP mRNA.

[0108] Figure 10 A, Schematic diagram of RNA capping modification enzymatic reaction; B, Schematic diagram of exemplary embodiment of the present invention; C, Schematic diagram of cap0 and cap1 cap structures. Detailed Implementation

[0109] The inventors have proposed a novel RNA modification chimeric enzyme that combines the activities of capped RNA triphosphatase, guanylate transferase, and guanine methyltransferase with the methyltransferase activity of mRNA cap structure 2′-O-methyltransferase, which simplifies the process of mRNA production.

[0110] In their research on the prokaryotic expression of mRNA-capped 2′-O-methyltransferase, the inventors discovered that the expression of the native 2′-O-methyltransferase protein alone was relatively unstable, often resulting in little or no yield of the target protein during fermentation. However, by fusing an MBP lysing tag to the N-terminus of the 2′-O-methyltransferase, the fusion protein could be expressed stably and in high yields during fermentation. Furthermore, the inventors found that when expressing vaccinia virus capped enzymes using a dual-promoter vector, the expression levels of the D1 and D12 protein subunits could not reach the optimal 1:1 ratio, hindering the formation of more D1:D12 complexes. The expression level of the D12 protein subunit was significantly higher than that of the D1 protein subunit, which greatly reduced the expression yield of the complete capped enzyme D1:D12 complex structure.

[0111] In light of the above findings, the applicant linked 2′-O-methyltransferase to the C-terminus of the D12 protein subunit ( Figure 10 (B) This design serves two purposes: firstly, to balance and reduce D12 expression, and secondly, to stabilize 2′-O-methyltransferase. Surprisingly, the chimeric enzyme designed above completely retains the native activities of 5′-triphosphatase, guanylate transferase, N7-methyltransferase, and 2′-O-methyltransferase.

[0112] Therefore, a first aspect of the present invention provides a chimeric protein subunit comprising interconnected (a) a D12 subunit of an RNA capping enzyme or a functional fragment thereof, or a variant having at least 90% sequence identity with the D12 subunit and having D12 subunit activity, and (b) a 2′-O-methyltransferase of an RNA cap structure or a functional fragment thereof, or a variant having at least 90% sequence identity with the RNA cap structure and having 2′-O-methyltransfer activity, and optionally (c) a linker between (a) and (b). The present invention also provides a chimeric protein comprising said chimeric protein subunit and an RNA capping enzyme D1 subunit or a functional fragment thereof. The chimeric protein is a heterodimer.

[0113] "Chimeric enzymes" are non-natural enzymes that do not exist in nature. Chimeric enzymes can contain catalytic domains derived from different sources (e.g., from different enzymes) or from the same source (e.g., from the same enzyme) but arranged in a different manner than those found in nature. Chimeric enzymes can be one (i.e., a single subunit) or multiple (i.e., multi-subunit) catalytic domains or proteins linked covalently or non-covalently. "Catalytic domain" refers to a protein domain that is essential and sufficient (especially in terms of its three-dimensional structure) for ensuring enzyme function. "Oligomerases" are multi-subunit enzymes composed of at least two polypeptide chains linked covalently or non-covalently. "Oligomerases" include homooligomerases and heterooligomerases. Homooligomerases are multi-subunit enzymes composed of only one type of monomer (subunit), while heterooligomerases are composed of different types of monomers (subunits), such as heterodimers.

[0114] 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 (844aa) and D12 (287aa) (D1:D12).

[0115] The chimeric protein described herein may comprise an RNA capping enzyme D1 subunit or a variant thereof. The D1 subunit possesses three catalytic domains, thus encompassing three enzymatic activities capable of performing all three steps in m7GpppRNA synthesis. These three catalytic domains are bound together in a 97kDa D1 protein, with the catalytic domains of RNA 5'-triphosphatase (TPase) and guanylate transferase (GTase) located at the N-terminal portion, and the catalytic domain of N7-methyltransferase (N7-MTase) located at the C-terminal portion of the D1 protein. Exemplarily, the vaccinia virus RNA capping enzyme D1 subunit is shown in SEQ ID NO:3, the 5'-triphosphatase domain as shown in amino acids 1-225 of SEQ ID NO:3, the guanylate transferase domain as shown in amino acids 226-530 of SEQ ID NO:3, and the N7-methyltransferase domain as shown in amino acids 531-844 of SEQ ID NO:3.

[0116] The chimeric proteins described herein may also include truncated RNA capping enzyme D1 subunits or variants thereof, preferably containing an N7-MTase domain. Truncated vaccinia capping enzymes containing an N7-MTase domain and possessing biological enzymatic activity are known in the art, for example, as described by Shuhuman ZS et al. (Shuman ZS, RNA, 2008; Higman MA et al., Journal of Biological Chemistry, 1994), including but not limited to amino acid fragments of D1 protein 498-844 or 540-844 amino acid fragments of D1 protein. Therefore, the chimeric protein of this article also includes a functional fragment of the RNA capping enzyme D1 subunit, comprising: (1) an N7 methyltransferase domain or a functional fragment thereof; (2) an N7 methyltransferase domain or a functional fragment thereof and a 5'-triphosphatase domain or a functional fragment thereof; (3) an N7 methyltransferase domain or a functional fragment thereof and a guanylate transferase domain or a functional fragment thereof; or (4) an N7 methyltransferase domain or a functional fragment thereof, a 5'-triphosphatase domain or a functional fragment thereof and a guanylate transferase domain or a functional fragment thereof. In one or more embodiments, the functional fragment of the RNA capping enzyme D1 subunit comprises amino acids 498-844 or 540-844 of SEQ ID NO:3.

[0117] The D12 subunit (33 kDa) itself does not possess methyltransferase catalytic activity, but it can be activated to enhance the methyltransferase activity of the D1 protein. The amino acid sequence of the D12 subunit of the vaccinia virus RNA capping enzyme is shown in SEQ ID NO:1, lines 1-287.

[0118] In some embodiments, the RNA cap 2′-O-methyltransferase described herein is a vaccinia virus 2′-O-methyltransferase (2-O-MTase). This 39 kDa 2′-O-methyltransferase, also known as the VP39 protein, enables cap-specific mRNA (nucleoside 2′-O-)-methyl transfer, converting the cap-0 structure to the cap-1 structure. The amino acid sequence of the vaccinia virus RNA cap 2′-O-methyltransferase is shown in SEQ ID NO:1, 303-635.

[0119] In some aspects, the present invention also provides a 2′-O-methyltransferase protein fused with an MBP tag for improving protein expression stability, having bioenzymatic activity equivalent to or better than that of natural vaccinia virus 2′-O-methyltransferase. The fusion protein comprises (a) an RNA-capped 2′-O-methyltransferase or a functional fragment thereof, or a variant having at least 90% sequence identity with the RNA-capped 2′-O-methyltransferase and possessing RNA-capped 2′-O-methyltransfer activity, and (b) a His tag and / or an MBP tag located at the N-terminus or C-terminus of (a), and optionally a linker between (a) and (b). The RNA-capped 2′-O-methyltransferase is as previously described. The His tag described herein is a short peptide containing one or more consecutive histidine residues. The MBP tag described herein has its conventional meaning in the art.

[0120] In this invention, polypeptides or proteins (e.g., vaccinia virus capping enzymes or their subunits or domains, RNA cap 2′-O-methyltransferases) also include mutants that have at least 70% sequence identity and retain the activity of the polypeptide or protein (e.g., 5′-triphosphorylation activity, guanosine transfer activity, N7 methyl transfer activity, 2-O-methyl transfer activity). The mutants include amino acid sequences that have at least 70%, at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 97% sequence identity with a reference sequence and retain the biological activity of the reference sequence. Sequence identity between two aligned sequences can be calculated using, for example, NCBI's BLASTp. The mutants also include amino acid sequences that have one or more mutations (insertions, deletions, or substitutions) in the said amino acid sequence while still retaining the biological activity of the reference sequence. The number of mutations typically refers to 1-50, for example 1-20, 1-10, 1-8, 1-5, or 1-3. Substitutions are preferably conserved substitutions. For scFv, mutations can occur within the CDR region (including mutations within the CDR region described above) or within the FR region, as long as the biological activity of the reference sequence is preserved after the mutation. For example, in the art, conserved substitutions with amino acids of similar or comparable properties typically do not alter the function (e.g., enzyme activity) of the protein or peptide. "Amino acids with similar or comparable properties" includes, for example, families of amino acid residues having similar side chains. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, replacing one or more sites in the polypeptide of the present invention with another amino acid residue from the same side chain class will not substantially affect its activity.

[0121] For example, it is known in the art that variants of the D12 subunit of RNA capped enzymes can have mutations selected from one or more of the following while retaining 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, and R281A. As another example, Schnierle BS et al. disclosed in their paper a series of effective mutant amino acid sequences of mRNA capped 2′-O-methyltransferases, in which the mutants still retained high enzymatic catalytic activity. These mutants include, but are not limited to, the C178S, C272S, K41D, A201R, and A210K mutants of 2′-O-methyltransferase; the charged amino acids R, K, H, Y, C, D, and E of the 2′-O-methyltransferase are randomly replaced with A. Zheng S, Colin PY, and Mao X have disclosed a series of effective mutant amino acid sequences of the N7-methyltransferase domain of vaccinia virus capping enzyme in their respective literatures, and the mutants also retain high 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, and E806A. (Schnierle BS et al., J BiolChem. 1994; Zheng S et al., RNA. 2008; Mao X et al., Biochemistry. 1996; Kyrieleis OJ et al., Structure. 2014; Colin PY. Sci Rep. 2020; Nayanendu Saha et al., Virology, 2001; Nayanendu Saha et al., J. VIROL, 2003) The above references are included in this paper in full by citation.

[0122] In some embodiments, the polypeptide or protein described herein further includes a signal peptide that guides it to subcellular structures. The signal peptide may be located at the N-terminus or C-terminus of the polypeptide. The subcellular structures include, but are not limited to, the Golgi apparatus or endoplasmic reticulum, the proteasome, the cell membrane, or the lysosome.

[0123] In this document, a linker is a polypeptide fragment that connects different proteins or polypeptides, with the purpose of maintaining the spatial conformation of the linked proteins or polypeptides to preserve their function or activity. Exemplary linkers include those containing G and / or S. Typically, a linker contains one or more repeating motifs. Preferably, the motifs are adjacent in the linker sequence, with no inserted amino acid residues between the repeats. The linker sequence may consist of 1, 2, 3, 4, or 5 repeating motifs. The length of the linker can be 3-25 amino acid residues, for example, 3-15, 5-15, or 10-20 amino acid residues. In some embodiments, the linker sequence is a polyglycine linker sequence. The number of glycine residues in the linker sequence is not particularly limited, typically 2-20, for example, 2-15, 2-10, or 2-8. In addition to glycine and serine, the linker may also contain other known amino acid residues, such as alanine (A), leucine (L), threonine (T), glutamic acid (E), phenylalanine (F), arginine (R), glutamine (Q), etc. In some embodiments, different proteins or polypeptides of the present 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 shown in amino acids 288-302 of SEQ ID NO:1.

[0124] Within the scope of this invention, those skilled in the art are familiar with polypeptide linkers that can be used herein without affecting the folding of the enzyme catalytic domain and can achieve the chimeric enzyme effect described in this invention, including but not limited to GGGGIAPSMVGGGGS (Turner, Ritter et al. 1997), SPNGASNSGSAPDTSSAPGSQ (Hennecke, Krebber et al. 1998), EGKSSGSGSESKSTE (Bird, Hardman et al. 1988), EGKSSGSGSESKEF (Newton, Xue et al. 1996), GGGSGGGSGGGTGGGSGGG (Robinson and Sauer). 1998), GSTSGSGKSSEGKG (Bedzyk, Weidner et al. 1990), YPRSIYIRRRHPSPSLTT (Tang, Jiang et al. 1996), STSGSGKPGSGEGS (Ting, Kain et al. 2001), SSADDAKKDAAKKDDAK KDDAKKDA (Pantoliano, Bird et al. 1991), GSADDAXXDAAXKDDAKKDDAKKDGS (Gregoire, Lin et al. 1996), LSADDAKKDAAKKDDAKKDDAKKDL (Pavlinkova, Beresford et al. 199 9) AEAAAAKEAAAKEAAAKA (Wickham, Carrion et al. 1995), GSTGSSGKPGSGEGSTGAGGAGSTSGSSGKPSGEG (Ting, Kain et al. 2001), LSLEVAEEIARLEAEV (Ting, Kain et al. 2001), GTPTPTPTPTGEF (Gustavsson, Lehtio et al. 2001), GSTGSSGKPGSGEGSTKG (Whitlow, Bell et al. 1993) and GSHGSSGKP (Ting, Kain et al. 2001), or the generic connectors described in US20130042334. The above references are incorporated herein by reference in their entirety.

[0125] This invention includes polynucleotides encoding the chimeric protein subunits or chimeric proteins described herein. The polynucleotides of this invention can be in DNA or RNA form. DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. DNA can be single-stranded or double-stranded. DNA can be a coding strand or a non-coding strand. This invention also includes degenerate variants of polynucleotides encoding polypeptides or proteins, i.e., polynucleotides encoding the same amino acid sequence but with different nucleotide sequences.

[0126] The polynucleotides described herein include sequences that have undergone codon optimization, provided that the amino acid sequence encoded by the polynucleotide remains unchanged. Codon-optimized sequences may exhibit more suitable expression for a specific species. Methods for codon optimization of polynucleotide sequences are well known in the art.

[0127] The polynucleotides of this invention can be the coding sequences of the chimeric protein subunit and the coding sequences of the RNA capping enzyme D1 subunit or its functional fragments, or the expression cassettes of the chimeric protein subunit and the expression cassettes of the RNA capping enzyme D1 subunit or its functional fragments. In this document, the coding sequence refers to the portion of a nucleic acid sequence that directly identifies its protein product (e.g., a polypeptide of the chimeric protein subunit, the RNA capping enzyme D1 subunit, or its functional fragments). The boundary of the coding sequence is typically defined 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. The coding sequence may include, but is not limited to, DNA, cDNA, and recombinant nucleic acid sequences. In this document, the expression cassette refers to the complete element required to express the gene of interest, including the promoter, gene coding sequence, and PolyA tailing signal sequence. The polynucleotide described herein can be two independent nucleic acid molecules, each containing a coding sequence for a chimeric protein subunit and a coding sequence for an RNA capping enzyme D1 subunit or a functional fragment thereof, such as an expression cassette for the chimeric protein subunit and an expression cassette for the RNA capping enzyme D1 subunit or a functional fragment thereof, respectively; or, the coding sequences for the chimeric protein subunit and the RNA capping enzyme D1 subunit or a functional fragment thereof can be linked into a single nucleic acid molecule via a linker, such as the coding sequences for the chimeric protein subunit and the RNA capping enzyme D1 subunit or a functional fragment thereof being in the same expression cassette, or the two expression cassettes being linked into the same nucleic acid molecule via a suitable linker. In some embodiments, the polynucleotide of the present invention is a nucleic acid molecule in which the coding sequences for the chimeric protein subunit and the RNA capping enzyme D1 subunit or a functional fragment thereof are in the same expression cassette, and contains a promoter, coding sequences for the chimeric protein subunit and the RNA capping enzyme D1 subunit or a functional fragment thereof, and a PolyA tailing signal. 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.

[0128] In some embodiments, the coding sequence or expression cassette is integrated into the genome of the cell. Thus, in these embodiments, the genome of the cell described herein contains a stably integrated expression cassette encoding the chimeric protein subunit and the RNA capping enzyme D1 subunit or a functional fragment thereof.

[0129] This invention also relates to nucleic acid constructs containing the polynucleotides described herein, and one or more regulatory sequences operatively linked to these sequences. The polynucleotides described herein can be manipulated in various ways to ensure the expression of the chimeric protein subunits or chimeric proteins. The nucleic acid constructs can be manipulated prior to insertion into a vector, depending on the expression vector or requirements. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.

[0130] The regulatory sequence can be a suitable promoter sequence. The promoter sequence is typically operatively linked to the coding sequence of the protein to be expressed. The promoter can be any nucleotide sequence exhibiting transcriptional activity in the selected host cell, including mutant, truncated, and heterozygous promoters, and can be obtained from a gene encoding an extracellular or intracellular polypeptide homologous or heterologous to that of the host cell. The regulatory sequence can also be a suitable transcription terminator sequence, a sequence recognized by the host cell to terminate transcription. The terminator sequence is operatively linked to the 3' end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the selected host cell can be used in this invention. The regulatory sequence can also be a suitable leader sequence, the untranslated region of mRNA important for translation in the host cell. The leader sequence is operatively linked to the 5' end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the selected host cell can be used in this invention.

[0131] In some embodiments, the nucleic acid construct is a vector. The vector can be a cloning vector, an expression vector, or a homologous recombination vector. The polynucleotides of the present invention can be cloned into many types of vectors, such as plasmids, phage particles, phage derivatives, animal viruses, and granules. Cloning vectors can be used to provide coding sequences for therapeutic proteins and peptides of the present invention, such as a nucleic acid molecule containing coding sequences for both therapeutic proteins and peptides. Expression vectors can be provided to cells in the form of viral vectors. Expression of the polynucleotides of the present invention is typically achieved by operably linking the polynucleotides of the present invention to a promoter and incorporating the construct into an expression vector. This vector is suitable for replication and integration into eukaryotic cells. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters that can be used to regulate the expression of the desired nucleic acid sequence. Homologous recombination vectors are used to integrate the expression cassettes described herein into the host genome.

[0132] Typically, a suitable vector contains a replication origin that functions in at least one organism, a promoter sequence, a convenient restriction enzyme site, and one or more optional markers. For example, in some embodiments, the present invention uses a lentiviral vector containing a replication origin site, a 3'LTR, a 5'LTR, the polynucleotide described herein, and optional markers.

[0133] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strongly constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operatively linked thereto. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences known in the art and suitable for prokaryotic or eukaryotic cells may also be used.

[0134] To evaluate the expression of therapeutic proteins, peptides, or portions thereof, expression vectors introduced into cells may also contain one or both of optional marker genes or reporter genes to facilitate the identification and selection of expressing cells from cell populations seeking transfection or infection via viral vectors. Alternatively, optional markers may be carried on a separate DNA segment and used in co-transfection procedures. Both the optional marker and the reporter gene may have suitable regulatory sequences flanking them to enable expression in host cells. Useful optional markers include, for example, antibiotic resistance genes. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase, or green fluorescent protein genes. Suitable expression systems are well-known and can be prepared using known techniques or are commercially available.

[0135] The polynucleotides described herein can generally be obtained using PCR amplification. Specifically, primers can be designed based on the nucleotide sequences disclosed herein, especially the open reading frame sequences, and the relevant sequences can be amplified using commercially available cDNA libraries or cDNA libraries prepared according to conventional methods known to those skilled in the art. When the sequences are long, two or more PCR amplifications are often required, and then the fragments amplified from each amplification are spliced ​​together in the correct order. Alternatively, the nucleic acid molecules described herein can be synthesized directly.

[0136] Methods for introducing genes into cells and expressing genes into cells are known in the art. Vectors can be readily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method in the art. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0137] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipid transfection, particle bombardment, microinjection, electroporation, and others. Biological methods for introducing polynucleotides of interest into host cells include the use of DNA vectors, RNA vectors, or viral vectors, such as lentiviral vectors. Chemical methods for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, and beads; and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes.

[0138] In this document, host cells contain, express, and / or secrete the protein subunits or proteins described herein. When referring to a cell containing, expressing, or 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; and "secreting" means that the cell secretes the expressed molecule extracellularly. Host cells include cells ultimately used to secrete chimeric proteins or their subunits, cells that produce RNA, and various cells used in the production of these cells, such as *E. coli* cells, for purposes such as providing the coding sequence of the proteins of this invention or providing the vectors described herein. After a host cell expresses the protein subunits or proteins described herein, the protein subunits or proteins can be purified using conventional protein purification methods in the art (e.g., chromatography, including affinity chromatography, ion exchange chromatography, etc.).

[0139] The chimeric protein subunits or chimeric proteins of the present invention can be used to perform chemical modifications in one or more steps of the RNA capping process. Therefore, the present invention provides a method for modifying target RNA into capped RNA or a method for preparing capped target RNA, comprising: contacting the target RNA with the chimeric protein described herein, or contacting the target RNA with a cell expressing the chimeric protein described herein.

[0140] In this article, "target RNA" refers to RNA of interest obtained in vivo or in vitro. Typically, target RNA is uncapped or partially capped RNA. Target RNA can originate from any source, such as in vitro transcription (IVT), chemical synthesis, or extraction from the body. In vitro transcription refers to the enzymatic synthesis of RNA using a DNA template with T7, T3, or SP6 promoters and the corresponding DNA-dependent RNA polymerase in a suitable buffer system. The buffer system includes, but is not limited to, NTPs and Mg²⁺. 2+ The RNA contains components such as ribonuclease inhibitors (RI) and inorganic pyrophosphatase (iPPase). Compared with solid-phase RNA synthesis, in vitro transcription synthesis can produce large quantities of long-chain RNA with high quality, making it suitable for industrial production of mRNA drugs. In one or more embodiments, the target RNA described herein is an in vitro transcribed capless single-stranded RNA (ssRNA) that possesses all the characteristics of mRNA except for the cap structure, including but not limited to the 5'-UTR, 3'-UTR, and protein or polypeptide translation coding regions.

[0141] In vitro transcription methods for RNA substrates are well known in the art. Exemplary polymerase (e.g., T7, T3, SP6) transcription methods involve mixing and incubating a transpolymerase, rNTPs, and a DNA transcription template (containing a promoter recognized by the polymerase). The reaction system also contains one or more reagents selected from: MgCl2, buffer, iPPase, inhibitors, nuclease-free water, etc. The DNA used as the transcription template can be obtained by conventional methods in the art, such as synthesis, hybridization, PCR, etc., and the reagents required in these methods are also well known in the art.

[0142] To avoid being limited by theory, the target RNAs described herein include 5'-triphosphorylated RNA, 5'-diphosphoesterified RNA, and RNA with a 5'-Gppp structure. In this paper, "capped RNA" includes capped RNA with a 5'-m7Gppp structure (cap0) or capped RNA with a 5'-m7GpppNmp structure (cap1). In each of these structures, "5'" indicates that the group is located at the 5' end of the RNA, "m" indicates methylation, the number "7" indicates the methylation position, "G" is guanosine monophosphate, "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 processes for purifying and denaturing / renaturing RNA are known in the art, such as incubating the RNA sample at 50-70°C for 1-60 minutes (e.g., 65°C for 5-20 minutes) followed by incubation at 0°C for 2-10 minutes. However, in the method of this invention, RNA capping can be achieved even without purification and denaturation / renaturation.

[0143] To enable the target RNA to contact the chimeric protein described herein, the target RNA can be expressed in cells expressing the chimeric protein described herein (e.g., by introducing a DNA sequence expressing the target RNA into the host cell described herein). Suitable methods known in the art can be used to express the target RNA in cells, such as introducing DNA encoding the RNA into the cell, where the DNA is transcribed to form the target RNA, which is then capped by the chimeric protein or a chimeric protein subunit expressed in the cell.

[0144] Alternatively, the target RNA (e.g., RNA transcribed in vitro) can be mixed in solution with the chimeric protein described herein to achieve contact. Prior to mixing, the RNA may be purified and / or renatured. Procedures and conditions for RNA capping in solution are known in the art, such as mixing the target RNA in nuclease-free water with capping buffer, GTP, SAM, and the chimeric protein described herein, and incubating. Incubation steps include, for example, incubation at 37°C for 30–90 minutes. The modified capped RNA product can be purified, for example, by magnetic bead purification.

[0145] In the capping method described herein, the molar ratio of the target RNA to the chimeric protein is less than 865:1, preferably 86.5-173:1. The inventors have discovered that different molar ratios yield different capped RNAs. 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 into a capped RNA (cap0) with a 5'-m7Gppp structure. If the molar ratio of the target RNA to the chimeric protein is less than 86.5-173:1, the method modifies the target RNA into a capped RNA (cap1) with a 5'-m7GpppNmp structure.

[0146] Furthermore, the domains contained in the chimeric protein may differ depending on the target RNA. If the target RNA is 5'-triphosphorylated RNA, the chimeric protein needs to have the activities of 5'-triphosphatase, guanylate transferase, N7-methyltransferase, and 2-O-methyltransferase. That is, the chimeric protein contains the N7-methyltransferase (N7-MTase) domain or a functional fragment thereof, the 5'-triphosphatase (TPase) domain or a functional fragment thereof, and the guanylate transferase (GTase) domain or a functional fragment thereof, as well as the chimeric protein subunit.

[0147] If the target RNA is 5'-bisphosphoesterified RNA, the chimeric protein needs to have guanylate transferase, N7 methyltransferase, and 2-O-methyltransferase activities. That is, the chimeric protein contains the N7 methyltransferase (N7-MTase) domain or its functional fragment and the guanylate transferase (GTase) domain or its functional fragment of the RNA capping enzyme D1 subunit, and the chimeric protein subunit.

[0148] If the target RNA is RNA with a 5'-Gppp structure, the chimeric protein needs to have N7 methyltransferase (N7-MTase) and 2-O-methyltransferase activities, that is, the chimeric protein contains the N7 methyltransferase (N7-MTase) domain or a functional fragment thereof of the RNA capping enzyme D1 subunit and the chimeric protein subunit.

[0149] In another aspect, the present invention may also modify a capped RNA (cap0) having a 5'-m7Gppp structure to a capped RNA (cap1) having a 5'-m7GpppNmp structure, comprising the steps of: contacting the RNA with the chimeric protein subunit described herein (e.g., expressing the RNA in cells expressing the chimeric protein subunit described herein), or contacting the RNA with cells expressing the chimeric protein subunit described herein.

[0150] Advantages of this invention:

[0151] 1. Chimeric enzymes that possess the biological activities of both capped enzymes and 2′-O-methyltransferases can reduce production processes and lower production costs;

[0152] 2. Cap0 capping and cap1 capping are continuous reactions. The chimeric enzyme covalently couples the two enzymes together, which is more conducive to continuous contact with RNA substrate and improves modification efficiency.

[0153] Unless otherwise defined, 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 pertains. While the invention may be practiced or tested using any methods and materials similar to or equivalent to those described herein, preferred methods and materials are described hereafter. All publications and patents specifically mentioned herein are incorporated herein by reference in their entirety for all purposes, including description and disclosure of chemical substances, devices, statistical analyses, and methods reported in said publications that may be used in connection with the invention. All references cited in this specification should be regarded as indications of the level of skill in the art. Nothing herein should be construed as an admission that the invention does not precede these disclosures by virtue of prior invention.

[0154] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Science Press, 2002, or according to the manufacturer's recommendations.

[0155] Example

[0156] Example 1: Expression and purification of natural vaccinia virus capping enzyme, 2-O-methyltransferase, and chimeric enzyme.

[0157] To overexpress vaccinia virus capping enzymes D1:D12, 2-O-methyltransferase 2-O-MTase, and D1:D12-(2-O-MTase) chimeric enzymes in *E. coli*, we optimized DNA sequences based on the corresponding amino acid sequences of these proteins and cloned them into commercial prokaryotic expression vectors pET28a (Novagen), pMAL-c5X (NEB), and pRSF-Duet1 (Novagen). The amino acid and DNA sequences of the enzymes are shown in SEQ ID NO:1-4, and the prokaryotic expression vector diagram is shown below. Figure 1 .

[0158] To ensure high-level enzyme expression and allow for direct and reproducible purification of vaccinia virus capped enzymes or chimeric enzyme complexes, a single His tag was added to the N-terminus of the D1 enzyme to facilitate the purification of the intact D1:D12 or D1:D12-(2-O-MTase) protein complex. To make the 2-O-methyltransferase more stable and easier to purify, a His tag and / or an MBP tag were added to the N-terminus of the 2-O-methyltransferase. Following the instructions of the commercial expression vectors, plasmids were transformed into *E. coli* BL21(DE3) expression bacteria. Single colonies were picked and cultured at 37°C to the logarithmic growth phase, followed by induction with 1 mM IPTG at 25°C for 16 hours. After collecting the induced bacteria, all enzyme proteins were purified using two standard purification steps: Ni affinity chromatography and Capto SP ImpRes or Capto Q ImpRes (Cytiva) ion exchange chromatography to obtain the final enzyme protein. The bacterial cell lysates before and after induction, as well as the finally purified enzyme protein, were subjected to SDS-PAGE (polyacrylamide gel electrophoresis). Figure 2 SDS-PAGE analysis of bacterial lysate with His and / or MBP tags added to the N-terminus of 2-O-methyltransferase. Figure 3 SDS-PAGE images of vaccinia virus cell lysates containing capping enzymes and chimeric enzymes. Figure 4 SDS-PAGE images of purified MBP-(2-O-MTase), vaccinia virus capped enzyme D1:D12, and chimeric enzyme D1:D12-((2-O-MTase)) are shown.

[0159] like Figure 2 As shown, when 2-O-methyltransferase lacks an MBP tag, protein expression is unstable and yield is low. Therefore, the 2-O-methyltransferases used in this invention are all MBP-tagged 2-O-methyltransferases, i.e., MBP-(2-O-MTase). Figure 3 As shown, when expressing the capping enzyme of natural vaccinia virus, the expression level of the D12 protein subunit is much higher than that of the D1 protein subunit, which is not conducive to the formation of a stable 1:1 D1:D12 complex. When 2-O-MTase is fused with D12, the expression levels of the fusion protein subunit and D1 are closer, which is conducive to the formation of the protein complex D1:D12-(2-O-MTase). Figure 4As shown, all three expression schemes yielded proteins or protein subunits of the target size after purification. The vaccinia virus capping enzyme contained a 33kD D12 protein subunit and a 100kD D1 protein subunit; the chimeric enzyme contained a 73kD D12-(2-O-MTase) fusion protein subunit and a 100kD D1 protein subunit, which met the expected protein size; the MBP-(2-O-MTase) fusion protein was 83kD, which is equal to the sum of the 44kD his-MBP protein tag and the 39kD 2-O-MTase protein, also meeting the expected protein size.

[0160] Example 2: In vitro transcription and enzymatic modification of RNA substrates to synthesize cap0 mRNA and cap1 mRNA

[0161] Primers containing the T7 promoter sequence were designed and synthesized as follows: RNA30-T7F: 5'-gataatacgactcactataGGGAAGGAGAGGAAGGAAAGGGAAGAAAGAA-3' (SEQ ID NO: 5); RNA30-R: 5'-TTCTTTCTTCCCTTTCCTTCCTCTCCTTCCCtatagtgagtcgtattatc-3' (SEQ ID NO: 6). The primers were mixed according to the proportions in Table 1 and subjected to an annealing reaction. The annealing program was: pre-denaturation at 95℃ for 10 min, followed by a gradient temperature decrease to 20℃ over 2 hours, and holding at 20℃ for one hour. After purification with magnetic beads, the resulting 30nt RNA (RNA30) transcribed DNA template was obtained.

[0162] Table 1. Primer annealing reaction system

[0163] reagents volume 10× Annealing Buffer 5μL RNA30-T7F (100μM) 20μL RNA30-R (100μM) 20μL Nuclease-free water Up to 50 μL

[0164] Transcription of RNA: Prepare a suitable T7 RNA polymerase reaction system at room temperature and add the reaction components in the order shown in the table. The reaction system can be scaled up or down proportionally. Incubate at 37°C for 6-16 hours. In this example, the transcribed RNA is 30 nt RNA. After 2 hours, the reaction is complete. Add 2 U DNase 1 and digest at 37°C for 15 minutes. Then, purify the RNA using magnetic beads for the next step of RNA capping modification enzymatic reaction.

[0165] Table 2. RNA transcription and synthesis system

[0166]

[0167] Capping modified RNA: Transfer 10 μg of purified RNA to a 1.5 ml centrifuge tube and dilute to 14 μl with nuclease-free water; heat at 65°C for 10 minutes, then remove the centrifuge tube and place it on ice for 5 minutes; add the following components in order according to Table 3-6, depending on the specific RNA modification objective, and incubate at 37°C for 30-90 minutes. This step is suitable for capping reactions with 10 μg of RNA, and the reaction substrate volume can be scaled up proportionally as needed. After the reaction, the final modified RNA product can be obtained by magnetic bead purification.

[0168] Table 3. Capped RNA synthesis systems for capless RNA enzymatic modification and capping.

[0169]

[0170] Table 4. Capped RNA synthesis system for cap0 RNA enzymatic modification.

[0171]

[0172]

[0173] Table 5. Capped RNA synthesis system (natural enzymes) for enzymatic modification of capless RNA.

[0174] Components volume denatured capless RNA 10μg 10* Capped Buffer 2.0μl GTP (10mM) 1.0μl SAM (32mM) 1.0μl Vaccinia virus capping enzyme D1:D12 Xμg (e.g.) Figure 7 (As shown) MBP-(2-O-MTase) Yμg (e.g.) Figure 7 (As shown) Nuclease-free water Up to 20 μl

[0175] Table 6. Capped RNA synthesization system (chimeric enzyme) for enzymatic modification of capless RNA.

[0176] Components volume denatured capless RNA 10μg 10* Capped Buffer 2.0μl GTP (10mM) 1.0μl SAM (32mM) 1.0μl Chimeric enzyme D1: D12-(2-O-MTase) Xμg (e.g.) Figure 8 (As shown) Nuclease-free water Up to 20 μl

[0177] Example 3: HPLC-MS method for detecting the efficiency of different RNA-modifying enzymes in catalyzing RNA 5' capping.

[0178] As shown in Table 7, the expected molecular weights of the intermediate or final products of transcription or enzymatic modification in Example 2 can be calculated. These samples were then analyzed by HPLC-MS (liquid chromatography-mass spectrometry). By comparing the detected approximate molecular weights, the proportion of target products produced after the enzymatic catalytic reaction of these RNA substrates can be inferred, thus leading to the conclusion of the different enzymatic activities of these RNA-modifying enzymes. The results are as follows... Figure 5-8 As shown.

[0179] Table 7. Expected molecular weight of RNA30 with different cap structures

[0180]

[0181]

[0182] *Note: The actual molecular weight detected by mass spectrometry may sometimes deviate from the predicted molecular weight by approximately 5 Da, but this does not affect the identification of the target product.

[0183] Figure 5 The results showed that, under conditions where the vaccinia virus capping enzyme (approximately 133 kDa) was greater than 0.1 μg, it could modify uncapped triphosphate RNA30 with a expected molecular weight of 10623 Da into cap0 RNA30 with a expected molecular weight of 10903 Da. When the enzyme dosage was less than 0.05 μg, the modification of uncapped triphosphate RNA30 was incomplete, with only a portion of the substrate being converted to cap0 RNA30 and a small amount being converted to G-cap RNA30. In other words, complete capping of cap0 can be achieved when the substrate RNA:capping enzyme molar ratio is less than 1330. (Note: Approximately 10 kDa for RNA is used for rapid calculation.)

[0184] Figure 6 The results showed that, under conditions where the 2-O-methyltransferase (approximately 81 kDa) was greater than 0.5 μg, it could modify cap0 RNA30 with a expected molecular weight of 10903 Da to cap1 RNA30 with a expected molecular weight of 10918 Da. When the enzyme dosage was less than 0.25 μg, the modification of cap0 RNA30 was incomplete, with only a portion of the substrate being converted to cap1 RNA30. In other words, when the molar ratio of substrate RNA to 2-O-methyltransferase was less than 16:2, complete modification of cap0 to the cap1 cap could be achieved.

[0185] Figure 7 This study demonstrates the activity of vaccinia virus capping enzyme and 2-O-methyltransferase in co-modifying RNA30 triphosphate units using HPLC-MS. Based on the detection results of capping enzyme-modified RNA30 triphosphate into cap0 RNA30, 0.2 μg of vaccinia virus capping enzyme was uniformly added to each group in this experiment to ensure complete conversion of RNA30 triphosphate into cap0 RNA30, while 2-O-methyltransferase was added in gradients of 0.25 μg, 0.5 μg, 1 μg, and 2 μg. Figure 7The results showed that when 0.25 μg of 2-O-methyltransferase was added, only half of the substrate was converted to cap1 RNA30; when 0.5 μg of 2-O-methyltransferase was added, a small amount of substrate was still not converted to cap1 RNA30; and only when the amount of 2-O-methyltransferase was greater than 1 μg could co-modified RNA30 be completely converted to cap1 RNA. This indicates that one-step RNA modification to cap1 requires more 2-O-methyltransferase protein than stepwise RNA modification to cap1. In the co-capping system, when the substrate RNA:capping enzyme molar ratio was less than 665:1, complete capping of cap0 could be achieved; when the RNA:2-O-methyltransferase molar ratio was between 81 and 162:1, complete modification of cap0 to the cap1 cap could be achieved. This indicates that the amount of enzyme used in the co-capping reaction system is similar to that in the stepwise capping reaction system.

[0186] Figure 8 The results showed that the chimeric enzyme (173kDa, 133kDa + 39kDa) at 0.1-0.2 μg could completely convert capless triphosphate RNA30 with a expected molecular weight of 10623 Da into cap0 RNA30 with a expected molecular weight of 10903 Da; at 0.1 μg, approximately 90% of the RNA30 was completely capped. This indicates that the chimeric enzyme completely retains the activities of the catalytic domains of 5'-triphosphatase, guanylate transferase, and N7-guanine methyltransferase in the wild-type vaccinia virus capping enzyme, and the dosage used is comparable to that of the wild-type enzyme alone. When the chimeric enzyme dosage reached 2 μg, it could further completely convert the already converted 10903 Da cap0 RNA30 into cap1 RNA30 with a expected molecular weight of 10918 Da. This shows that the chimeric enzyme not only completely retains the activity of the 2-O-methyltransferase catalytic domain, but can also catalyze the acquisition of cap1 RNA30 more efficiently and completely. In the chimeric enzyme capping system, when the molar ratio of substrate RNA to chimeric enzyme is less than 865:1, complete capping of cap0 can be achieved; when the molar ratio of RNA to chimeric enzyme is between 86.5 and 173:1, complete modification of cap0 to cap1 can be achieved. These indicators are comparable to the molar ratios of 665:1 and 81-162:1 for mixed enzyme systems, indicating that at the same molar concentration, the chimeric enzyme can achieve the same function as the mixed enzyme. This proves that the chimeric enzyme does not alter the catalytic activity of each functional domain, which is beneficial for simplifying enzyme production and use (compared to using two enzymes, only one enzyme needs to be produced, saving step costs, and only one enzyme needs to be added during the reaction).

[0187] Example 4: Comparison of cell biological functions of eGFP mRNA prepared by modification with wild-type enzyme and chimeric enzyme respectively.

[0188] The template DNA (SEQ ID NO:7) for eGFP mRNA transcription was synthesized and subcloned into a commercial vector, such as pUC57. The plasmid was transformed into Top10 *E. coli*, extracted, and linearized with restriction endonucleases. The linear template DNA was recovered using magnetic beads for RNA transcription synthesis experiments. The transcription synthesis system and steps for eGFP mRNA are described in Table 2 of Example 2. The reaction system and steps for enzymatic modification to prepare cap1 eGFP mRNA after transcription synthesis are described in Tables 5 and 6 of Example 2. Completely capped mRNA samples were used for transfection experiments. Cap1 eGFP mRNA prepared by modifying RNA with natural vaccinia virus capping enzyme combined with 2-O-methyltransferase was designated as sample 1; cap1 eGFP mRNA prepared by chimeric enzyme modification was designated as sample 2; and unmodified capless eGFP mRNA was designated as sample 3. These samples were transfected into Chinese hamster ovary (CHO) cells according to the following cell transfection procedure to verify the cell biological function of the modified cap1 eGFP mRNA. Transfection procedure: (1) One day before transfection, CHO cells were digested, plated in 24-well plates at a density of 1x10⁵ / well. (2) For transfection, two 1.5ml EP tubes were used, each containing 50ul of opti-MEM culture medium. 2.25ul of PEI transfection reagent was added to one tube, and 1.5ug of mRNA was added to the other. After standing for 5 minutes, the PEI solution was added to the mRNA solution, mixed thoroughly, and then stood for 20 minutes. (3) The prepared PEI-mRNA solution was added to CHO cells containing serum-free culture medium and placed in a 37°C incubator. After 4 hours of culture, the cells were replaced with complete culture medium and cultured for another 4 hours. (4) After 24 hours of culture, fluorescence imaging and flow cytometry were performed to detect the EGFP positivity rate. All three samples were replicated in parallel.

[0189] The results are as follows Figure 9 As shown in Table 8, the green fluorescence positivity rate and fluorescence intensity of eGFP mRNA samples 1 and 2 transfected into CHO cells were at the same level, with no significant difference. This indicates that using chimeric enzyme modification alone can achieve the same effect as modifying RNA with a combination of natural vaccinia virus capping enzyme and 2-O-methyltransferase.

[0190] Table 8. EGFP positivity and fluorescence intensity of cells transfected with different mRNA samples.

[0191] sequence list <110> Shanghai Cell Therapy Group Co., Ltd. <120> RNA-modified chimeric proteins and their applications <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 Glue Tyr Cys Ile Ile Lys 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 Will Arg With Lys Cys Will Be Asp Ser Trp With Lys Asp 180 185 190 Ser Ala Ile Met Val Ser Asp Val Cys 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 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 Met Thr Ser Asp Ser Lys 260 265 270 Glu Asn Lys Is Gln 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 caccccgtcta 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 Light Leu Val Asp Ile Cys Asp Arg Ile Val Phe Light Ser Light Light 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 ttctctacc atcgctacct acatcgacgc tctggctaaa 60 aacgctctg aactggaaca gcgttctacc gcttacgaa tcacaacca actggactg 120 gttttcatca aaccgccgct gatcaccctg accaacgttg ttaacctc taccatccag 180 gatctttca tccgtttcac cgttaccaac aaagaaggtg ttaaaatccg taccaaatc 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 tccttttgttc 960 cctaagtcca actactaaac tgggggatat tatgaagggc cttgagcatc tggattctgc 1020 ctaataaaaa acatttattt tcattgcaaa aaaaaaaaaa aaaaaaaaa aaaaaaaaaa 1080 1140 aaaaaaaaaa aaaaaaaaaa aaaaaaggt acc 1173 <210> 8 <211> 6 <212> PRT <213> Artificial Sequence <400> 8 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

1. A chimeric protein subunit consisting of: a fusion of (a) a D12 subunit of RNA capping enzyme, and (b) a RNA cap structure 2'-O-methyltransferase, and optionally (c) a linker between (a) and (b), wherein, the amino acid sequence of the D12 subunit of RNA capping enzyme is set forth in SEQ ID NO: 1, positions 1-287, the amino acid sequence of the RNA cap structure 2'-O-methyltransferase is set forth in SEQ ID NO: 1, positions 303-635.

2. The chimeric protein subunit of claim 1, wherein, the carboxy terminus of (a) is linked to the amino terminus of (b).

3. The chimeric protein subunit of claim 1, wherein, the amino acid sequence of the linker is set forth in SEQ ID NO: 1, positions 288-302.

4. A chimeric protein subunit consisting of: a fusion of (a) a D12 subunit of RNA capping enzyme, (b) a RNA cap structure 2'-O-methyltransferase, and optionally (c) a linker between (a) and (b), and (d) a His tag and a MBP tag at the N-terminus or C-terminus, wherein the amino acid sequence of the D12 subunit of RNA capping enzyme is set forth in SEQ ID NO: 1, positions 1-287, and the amino acid sequence of the RNA cap structure 2'-O-methyltransferase is set forth in SEQ ID NO: 1, positions 303-635.

5. The chimeric protein subunit of claim 4, wherein, the amino acid sequence of the linker is set forth in SEQ ID NO: 1, positions 288-302.

6. The chimeric protein subunit of claim 1, wherein, the amino acid sequence of the chimeric protein subunit is set forth in SEQ ID NO:

1.

7. A chimeric protein consisting of: (1) the chimeric protein subunit of any one of claims 1-6, and (2) a D1 subunit of RNA capping enzyme, wherein, the D1 subunit of RNA capping enzyme is set forth in SEQ ID NO:

3.

8. The chimeric protein of claim 7, wherein, the chimeric protein is a heterodimeric protein.

9. A nucleic acid molecule selected from the group consisting of a coding sequence of the chimeric protein subunit of any one of claims 1-6, or a coding sequence of the chimeric protein of claim 7 or 8.

10. The nucleic acid molecule of claim 9, wherein, the coding sequence of the chimeric protein subunit is set forth in SEQ ID NO: 2, and / or the coding sequence of the chimeric protein, the coding sequence of the D1 subunit of RNA capping enzyme is set forth in SEQ ID NO:

4.

11. A nucleic acid construct, the nucleic acid construct: (1) expressing the chimeric protein subunit of any one of claims 1-6, or the chimeric protein of claim 7 or 8, (2) comprising the nucleic acid molecule of claim 9 or 10.

12. The nucleic acid construct of claim 11, wherein, the nucleic acid construct contains an expression cassette of the chimeric protein subunit and an expression cassette of the D1 subunit of RNA capping enzyme; or the nucleic acid construct is one expression cassette, wherein the coding sequence of the chimeric protein subunit and the coding sequence of the D1 subunit of RNA capping enzyme are within the expression cassette, and / or the nucleic acid construct is a cloning vector or an expression vector.

13. A host cell, the host cell comprising, expressing and / or secreting the chimeric protein subunit of any one of claims 1-6, or the chimeric protein of claim 7 or 8.

14. The host cell of claim 13, comprising the nucleic acid molecule of claim 9 or 10 and / or the nucleic acid construct of claim 11 or 12.

15. A method of modifying a target RNA to a capped RNA or a method of making a capped target RNA, comprising: contacting a target RNA with the chimeric protein subunit of any one of claims 1 to 6 or the chimeric protein of claim 7 or 8, or contacting a target RNA with a cell comprising, expressing and / or secreting the chimeric protein subunit of any one of claims 1 to 6 or the chimeric protein of claim 7 or 8, under conditions allowing capping of RNA.

16. The method of claim 15, wherein contacting a target RNA with the chimeric protein subunit or the chimeric protein comprises expressing the target RNA in a cell expressing the chimeric protein subunit or the chimeric protein, or mixing the target RNA with the chimeric protein subunit or the chimeric protein, and / or the method comprises the step of introducing a DNA sequence expressing the target RNA into the cell.

17. The method of claim 15, wherein, the method comprises the steps of: optionally (1) denaturing the target RNA, optionally (2) renaturing the target RNA, (3) incubating the RNA with the chimeric protein subunit of any one of claims 1 to 6 or the chimeric protein of claim 7 or 8, under conditions allowing capping of RNA, to obtain a capped RNA, optionally (4) purifying the capped RNA.

18. The method of claim 17, wherein, the conditions allowing capping of RNA are incubation at 37°C for at least 20 minutes, and / or the conditions allowing capping of RNA further comprise the presence of one or more reagents selected from the group consisting of GTP, SAM, a buffer, and / or the target RNA is selected from the group consisting of 5'-triphosphorylated RNA, 5'-diphosphorylated RNA and RNA having a 5'-Gppp cap structure.

19. The method of any one of claims 15 to 18, wherein the method modifies a target RNA selected from the group consisting of 5'-triphosphorylated RNA, 5'-diphosphorylated RNA and RNA having a 5'-Gppp cap structure, to a capped RNA having a 5'-m7Gppp structure, the chimeric protein comprises an N7-methyltransferase domain of a RNA capping enzyme D1 subunit, a 5'-triphosphatase domain, a guanylyltransferase domain, and the chimeric protein subunit.

20. The method of claim 19, wherein, the chimeric protein comprises a RNA capping enzyme D1 subunit as set forth in SEQ ID NO: 3 and a chimeric protein subunit as set forth in SEQ ID NO:

1.

21. The method of any one of claims 15-18, wherein, the method modifies a target RNA selected from the group consisting of 5'-triphosphorylated RNA, 5'-diphosphorylated RNA and RNA having a 5'-Gppp cap structure, to a capped RNA having a 5'-m7GpppNmp structure, the chimeric protein comprises an N7-methyltransferase (N7-MTase) domain, a 5'-triphosphatase (TPase) domain and a guanylyltransferase (GTase) domain of a RNA capping enzyme D1 subunit, and the chimeric protein subunit.

22. The method of claim 21, wherein, The chimeric protein comprises an RNA capping enzyme D1 subunit as set forth in SEQ ID NO: 3 and a chimeric protein subunit as set forth in SEQ ID NO:

1.

23. The method of any one of claims 15-18, wherein, The method is a method of capping a target RNA selected from the group consisting of 5'-diphosphorylated RNA and RNA having a 5'-Gppp structure, the chimeric protein comprises an N7-methyltransferase (N7-MTase) domain and a guanylyltransferase (GTase) domain of an RNA capping enzyme D1 subunit, and the chimeric protein subunit.

24. The method of claim 23, wherein, The chimeric protein subunit is as set forth in SEQ ID NO:

1.

25. The method of any one of claims 15-18, wherein, The method is a method of capping a target RNA selected from the group consisting of 5'-diphosphorylated RNA and RNA having a 5'-Gppp structure, the chimeric protein comprises an N7-methyltransferase (N7-MTase) domain and a guanylyltransferase (GTase) domain of an RNA capping enzyme D1 subunit, and the chimeric protein subunit.

26. The method of claim 25, wherein, The chimeric protein subunit is as set forth in SEQ ID NO: 1.

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