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WO2025180432A1 - Enzyme pour coiffage d'arn et son système de réaction - Google Patents

Enzyme pour coiffage d'arn et son système de réaction

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Publication number
WO2025180432A1
WO2025180432A1 PCT/CN2025/079459 CN2025079459W WO2025180432A1 WO 2025180432 A1 WO2025180432 A1 WO 2025180432A1 CN 2025079459 W CN2025079459 W CN 2025079459W WO 2025180432 A1 WO2025180432 A1 WO 2025180432A1
Authority
WO
WIPO (PCT)
Prior art keywords
polynucleotide
ligase
polypeptide
capping
rna
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2025/079459
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English (en)
Chinese (zh)
Inventor
濮静逸
李尚阳
马超
吴良营
曹洋
程永升
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Pixel Biosciences Suzhou Co Ltd
Original Assignee
Pixel Biosciences Suzhou Co Ltd
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Filing date
Publication date
Application filed by Pixel Biosciences Suzhou Co Ltd filed Critical Pixel Biosciences Suzhou Co Ltd
Publication of WO2025180432A1 publication Critical patent/WO2025180432A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes

Definitions

  • the present application relates to the field of biomedicine, and specifically to a method for capping a polynucleotide.
  • RNA capping After mRNA is transcribed, a cap structure needs to be further added to the 5' end to increase stability and improve translation efficiency.
  • the most commonly used capping methods at present are enzymatic capping and co-transcriptional capping.
  • Traditional enzymatic capping requires purification after the initial transcript is synthesized, and then a capping reaction is performed. The capping process is cumbersome and requires three enzymatic reactions. There are many intermediate products in the capping reaction, which is difficult to purify and has poor consistency.
  • Co-transcriptional capping uses chemically synthesized cap analogs, which is relatively expensive. Capping is completed simultaneously during the transcription process, but the capping efficiency is low, and a large number of non-target products are obtained. Therefore, it is necessary to develop an efficient and specific RNA capping method.
  • RNA ligase e.g., T4 RNA ligase 1
  • a fusion enzyme containing a ligase e.g., a fusion enzyme of Sso7d and T4 RNA ligase 1
  • the use of RNA ligase for sequence ligation is highly efficient, and RNA fragments can be connected in a short time. Its capping efficiency is higher than that of the traditional three-step capping method, and the steps are simple, the purification difficulty is low, and the consistency is high.
  • the present application provides a method for capping a polynucleotide, comprising:
  • composition comprising at least: an uncapped polynucleotide, a capping enzyme, and a cap structure analog, wherein the capping enzyme comprises a polynucleotide ligase polypeptide;
  • the polynucleotide ligase polypeptide comprises a DNA ligase polypeptide.
  • the DNA ligase polypeptide is a prokaryotic DNA ligase, a prokaryotic DNA ligase variant, or a functional fragment thereof.
  • the DNA ligase polypeptide is a bacterial DNA ligase, a bacterial DNA ligase variant, or a functional fragment thereof.
  • the DNA ligase polypeptide is a viral DNA ligase, a viral DNA ligase variant, or a functional fragment thereof.
  • the DNA ligase polypeptide is T4 DNA ligase, a variant thereof, or a functional fragment thereof.
  • the polynucleotide ligase polypeptide comprises an RNA ligase polypeptide.
  • the RNA ligase polypeptide is a prokaryotic RNA ligase, a prokaryotic RNA ligase variant, or a functional fragment thereof.
  • the RNA ligase polypeptide is a bacterial RNA ligase, a bacterial RNA ligase variant, or a functional fragment thereof.
  • the RNA ligase polypeptide is a viral RNA ligase, a viral RNA ligase variant, or a functional fragment thereof.
  • the RNA ligase polypeptide is T4 RNA ligase, a variant thereof, or a functional fragment thereof.
  • the RNA ligase polypeptide is T4 RNA ligase.
  • the RNA ligase polypeptide is T4 RNA ligase 1.
  • the capping enzyme is a fusion polypeptide comprising a polynucleotide binding polypeptide fused to a polynucleotide ligase polypeptide.
  • the polynucleotide binding polypeptide comprises a DNA binding polypeptide.
  • the polynucleotide binding polypeptide comprises an RNA binding polypeptide.
  • the polynucleotide binding polypeptide is selected from one or more of a DNA double-strand binding domain, a DNA single-strand binding domain, an RNA/DNA composite chain binding domain, an RNA single-strand or RNA double-strand binding protein, wherein
  • the DNA double-strand binding domain includes Sso7d and/or NF-kappaB p50;
  • the RNA/DNA complex chain binding domain includes one or more of ScFV, RNaseH1 (D210N), and HBD of the monoclonal antibody S9.6;
  • RNA single-strand binding domains include: Sso7d, PKR, TRBP, PACT, Staufen, NFAR1, NFAR2, SPNR, RHA, NREBP, Kanadaptin, HYL1 Hyponastic leaves, ADAR1, ADAR2, ADAR3, TENR, RNaseIII, Dicer, and RDE-4.
  • the polynucleotide binding polypeptide includes one or more of Sso7d, NF-kappaB p50, ScFV of monoclonal antibody S9.6, RNaseH1 (D210N), HBD, PKR, TRBP, PACT, Staufen, NFAR1, NFAR2, SPNR, RHA, NREBP, Kanadaptin, HYL1 Hyponastic leaves, ADAR1, ADAR2, ADAR3, TENR, RNaseIII, Dicer, and RDE-4.
  • the polynucleotide binding polypeptide is Sso7d.
  • the C-terminus of the polynucleotide ligase polypeptide is linked to the N-terminus of the polynucleotide binding polypeptide.
  • the N-terminus of the polynucleotide ligase polypeptide is linked to the C-terminus of the polynucleotide binding polypeptide.
  • polynucleotide ligase polypeptide and the polynucleotide binding polypeptide are linked by a first linker.
  • the N-terminus of the polynucleotide ligase polypeptide is linked to the first linker, and the C-terminus of the polynucleotide binding polypeptide is linked to the first linker.
  • the C-terminus of the polynucleotide ligase polypeptide is linked to the first linker, and the N-terminus of the polynucleotide binding polypeptide is linked to the first linker.
  • the first linker is a G4S linker.
  • the polynucleotide ligase polypeptide comprises an amino acid sequence as shown in SEQ ID NO:9-12, 21-22.
  • the polynucleotide binding polypeptide comprises an amino acid sequence as shown in SEQ ID NO:13-17.
  • the capping enzyme comprises an amino acid sequence as shown in SEQ ID NO: 18-20, 24-29.
  • the uncapped polynucleotide is DNA or RNA.
  • the uncapped polynucleotide is chemically synthesized.
  • the uncapped polynucleotide is 1 to 150 nucleotides in length.
  • the base of the 5' terminal nucleotide of the uncapped polynucleotide is guanine.
  • the 5' terminal nucleotide of the uncapped polynucleotide is modified by phosphorylation.
  • sugars of the nucleotides of the uncapped polynucleotide are independently selected for each position from ribose and deoxyribose, and may comprise modifications comprising 2'-O-alkyl, 2'-O-methoxyethyl, 2'-O allyl, 2'-O alkylamine, 2'-fluororibose, and 2'-deoxyribose;
  • the bases of the nucleotides of the uncapped polynucleotide are independently selected for each position from adenine, uracil, guanine or cytosine, or an analog of adenine, uracil, guanine or cytosine,
  • nucleotide modified base can be selected from xanthine, allylaminouracil, allylaminothymidine, hypoxanthine, dioxyadenine, dioxycytosine, dioxyguanine, dioxyuracil, 6-chloropurine nucleoside, N6-methyladenine, methylpseudouracil, 2-thiocytosine, 2-thiouracil, 5-methyluracil, 4-thiothymidine, 4-thiouracil, 5,6-dihydro-5-methyluracil, 5,6-dihydrouracil, 5-[(3-indolyl)propionamide-N-allyl]uracil, 5-aminoallylcytosine, 5-aminoallyluracil, 5-bromouracil, 5-bromocytosine, 5-carboxycytosine, 5-carboxyuracil, 5-carboxyuracil, 5-fluorouracil, 5-formylcytosine, 5-formyl
  • the cap analog has the following general formula:
  • R3 is selected from guanine, adenine, cytosine, uracil, a guanine analog, an adenine analog, a cytosine analog, and a uracil analog;
  • R 4 is (N 1 p 1 ) x N 2 , wherein N 1 and N 2 are ribonucleosides, and N 1 and N 2 are the same or different;
  • each position is independently a phosphate group, a phosphorothioate, a phosphorodithioate, an alkylphosphonic acid, an arylphosphonic acid, or an N-phosphoramide bond;
  • X is an integer from 0 to 8, wherein if X ⁇ 2, the ribonucleosides N1 in (N1-p)x are identical to or different from each other;
  • the R 1 and R 2 groups are independently selected from O-alkyl, halogen, acetylamino (AcNH), hydrogen or hydroxy.
  • sugar in N1 and N2 is independently selected from ribose and deoxyribose for each position and may contain modifications including 2'-O-alkyl, 2'-O-methoxyethyl, 2'-O allyl, 2'-O alkylamine, 2'-fluororibose, and 2'-deoxyribose;
  • the bases in N1 and N2 are independently selected for each position from adenine, uracil, guanine or cytosine, or analogs of adenine, uracil, guanine or cytosine, and the nucleotide modified base may be selected from xanthine, allylaminouracil, allylaminothymidine, hypoxanthine, dioxyadenine, dioxycytosine, dioxyguanine, dioxyuracil, 6-chloropurine nucleoside, N6-methyladenine, methylpseudouracil, 2-thiocytosine, 2-thiouracil, 5-methyluracil, 4-thiothymidine, 4-thiouracil, 5,6-dihydro-5-methyl ...
  • Dihydrouracil 5-[(3-indolyl)propionamido-N-allyl]uracil, 5-aminoallylcytosine, 5-aminoallyluracil, 5-bromouracil, 5-bromocytosine, 5-carboxycytosine, 5-carboxyuracil, 5-fluorouracil, 5-formylcytosine, 5-formyluracil, 5-hydroxycytosine, 5-hydroxymethylcytosine, 5-hydroxymethyluracil, 5-hydroxyuracil, 5-iodocytosine, 5-iodouracil, 5-methoxycytosine, 5-methoxyuracil, 5-methylcytosine, 5-methyluracil , 5-propynylaminocytosine, 5-propynylaminouracil, 5-propynylcytosine, 5-propynyluracil, 6-azacytosine, 6-azauracil, 6-chloropurine, 6-thioguanine, 7-deazaadenine,
  • the cap structure analog is a dinucleotide cap analog or a trinucleotide cap analog.
  • the cap structure analog has the following general formula:
  • X1 and X2 are bases, and X1 and X2 may be the same as or different from each other.
  • X 1 and/or X 2 are guanine or adenine.
  • the cap structure analog is selected from one of the following:
  • the reaction temperature of step S2 is 5°C to 80°C.
  • the reaction temperature of step S2 is 25°C to 60°C.
  • the composition of step S1 further comprises adenosine triphosphate.
  • the composition of step S1 further comprises a DNA and/or RNA buffer.
  • the composition of step S1 further comprises a DNase and/or RNase inhibitor.
  • the present application provides a capping enzyme for polynucleotide capping, wherein the capping enzyme is a fusion polypeptide comprising a polynucleotide binding polypeptide fused to a polynucleotide ligase polypeptide.
  • the polynucleotide ligase polypeptide comprises a DNA ligase polypeptide.
  • the DNA ligase polypeptide is a prokaryotic DNA ligase, a prokaryotic DNA ligase variant, or a functional fragment thereof.
  • the DNA ligase polypeptide is a bacterial DNA ligase, a bacterial DNA ligase variant, or a functional fragment thereof.
  • the DNA ligase polypeptide is a viral DNA ligase, a viral DNA ligase variant, or a functional fragment thereof.
  • the DNA ligase polypeptide is T4 DNA ligase, a variant thereof, or a functional fragment thereof.
  • the polynucleotide ligase polypeptide comprises an RNA ligase polypeptide.
  • the RNA ligase polypeptide is a prokaryotic RNA ligase, a prokaryotic RNA ligase variant, or a functional fragment thereof.
  • the RNA ligase polypeptide is a bacterial RNA ligase, a bacterial RNA ligase variant, or a functional fragment thereof.
  • the RNA ligase polypeptide is a viral RNA ligase, a viral RNA ligase variant, or a functional fragment thereof.
  • the RNA ligase polypeptide is T4 RNA ligase, a variant thereof, or a functional fragment thereof.
  • the RNA ligase polypeptide is T4 RNA ligase 1.
  • the capping enzyme is a fusion polypeptide comprising a polynucleotide binding polypeptide fused to a polynucleotide ligase polypeptide.
  • the polynucleotide binding polypeptide comprises a DNA binding polypeptide.
  • the polynucleotide binding polypeptide comprises an RNA binding polypeptide.
  • the polynucleotide binding polypeptide is selected from one or more of a DNA double-strand binding domain, a DNA single-strand binding domain, an RNA/DNA composite chain binding domain, an RNA single-strand or RNA double-strand binding protein, wherein
  • the DNA double-strand binding domain includes Sso7d and/or NF-kappaB p50;
  • the RNA/DNA complex chain binding domain includes one or more of ScFV, RNaseH1 (D210N), and HBD of the monoclonal antibody S9.6;
  • RNA single-strand binding domains include: Sso7d, PKR, TRBP, PACT, Staufen, NFAR1, NFAR2, SPNR, RHA, NREBP, Kanadaptin, HYL1 Hyponastic leaves, ADAR1, ADAR2, ADAR3, TENR, RNaseIII, Dicer, and RDE-4.
  • the polynucleotide binding polypeptide includes one or more of Sso7d, NF-kappaB p50, ScFV of monoclonal antibody S9.6, RNaseH1 (D210N), HBD, PKR, TRBP, PACT, Staufen, NFAR1, NFAR2, SPNR, RHA, NREBP, Kanadaptin, HYL1 Hyponastic leaves, ADAR1, ADAR2, ADAR3, TENR, RNaseIII, Dicer, and RDE-4.
  • the polynucleotide binding polypeptide is Sso7d.
  • the C-terminus of the polynucleotide ligase polypeptide is linked to the N-terminus of the polynucleotide binding polypeptide.
  • the N-terminus of the polynucleotide ligase polypeptide is linked to the C-terminus of the polynucleotide binding polypeptide.
  • polynucleotide ligase polypeptide and the polynucleotide binding polypeptide are linked by a first linker.
  • the first linker is a G4S linker.
  • the polynucleotide ligase polypeptide comprises an amino acid sequence as shown in SEQ ID NO:9-12, 21-22.
  • the polynucleotide binding polypeptide comprises an amino acid sequence as shown in SEQ ID NO:13-17.
  • the capping enzyme comprises an amino acid sequence as shown in SEQ ID NO: 18-20, 24-29.
  • the present application provides a kit comprising the aforementioned capping enzyme.
  • the present application provides a use of a capping enzyme in polynucleotide capping.
  • Figure 3 is a schematic diagram of the capping effect of wild-type RM378 RNA ligase 1;
  • FIG4 is the Urea-PAGE detection results of different enzyme capping temperature gradient tests
  • FIG7 is a Urea-PAGE assay result showing the concentration of cap analogs
  • FIG9 shows the Urea-PAGE detection results of different cap structure analogs and receptor RNA capping
  • Figure 11 shows the mass spectrometry results of the product of Oligo H1 linked to LzCap
  • Figure 13 shows the results of Urea PAGE detection of HiBiT mRNA ligation products
  • Figure 15 shows the Urea PAGE test results of HiBiT mRNA recovery products
  • genes or gene fragments e.g., probes, primers, EST or SAGE tags
  • exons introns
  • messenger RNA mRNA
  • transfer RNA tRNA
  • ribosomal RNA ribozymes
  • cDNA recombinant polynucleotides
  • branched polynucleotides plasmids
  • vectors isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, siRNA, shRNA, RNAi agents, and primers.
  • the term "uncapped polynucleotide” may refer to a given polynucleotide.
  • the uncapped polynucleotide may be a natural polynucleotide or an artificially designed non-natural polynucleotide.
  • the target polynucleotide may be a DNA molecule, an RNA molecule, or a combination of DNA and RNA molecules (e.g., a target polynucleotide that is partially DNA and partially RNA).
  • the uncapped polynucleotide may be a portion of the 5' untranslated region (5'UTR) of an mRNA.
  • ligase refers to an enzyme that can form a covalent bond between two polynucleotides. More specifically, a ligase can form a covalent bond (e.g., 5', 3'-phosphodiester bond) at the 5' end of a polynucleotide and the 3' end of another polynucleotide. These ligases can include DNA ligase or RNA ligase.
  • DNA ligase polypeptide or “DNA ligase” may refer to an enzyme that joins two DNA segments together, generally catalyzing the formation of a phosphodiester bond to join two DNA segments or one DNA segment and one RNA segment together.
  • DNA ligase polypeptides can join two double-stranded DNA segments, two single-stranded DNA segments, or one single-stranded DNA segment and one double-stranded DNA segment.
  • RNA ligase polypeptide or "RNA ligase” may refer to an enzyme that ligates two RNA segments together, generally catalyzing the formation of a phosphodiester bond to join two RNA segments or a DNA segment to an RNA segment.
  • RNA ligase polypeptides can ligate two double-stranded RNA segments, two single-stranded RNA segments, or one single-stranded RNA segment to a double-stranded RNA segment.
  • polynucleotide-binding polypeptide refers to a polypeptide capable of binding to a polynucleotide, including polypeptides that bind to single-stranded polynucleotides, polypeptides that bind to double-stranded polynucleotides, and polypeptides that bind to polynucleotides in other configurations.
  • a polynucleotide-binding polypeptide can be fused to a polynucleotide ligase polypeptide, for example, to the N-terminus or C-terminus of a polynucleotide ligase, without inactivating the polynucleotide-binding polypeptide or the ligase.
  • DNA-binding polypeptide refers to a polypeptide capable of binding to DNA, including polypeptides that bind to single-stranded DNA, polypeptides that bind to double-stranded DNA, and polypeptides that bind to DNA in other configurations.
  • a DNA-binding polypeptide can be fused to a DNA ligase polypeptide, for example, to the N-terminus or C-terminus of an RNA ligase polypeptide and/or a DNA ligase polypeptide, without inactivating the RNA ligase polypeptide and/or the DNA ligase polypeptide. It should be understood that RNA-binding polypeptides can also bind to polynucleotides other than RNA, such as DNA or known natural nucleotide analogs.
  • RNA-binding polypeptide refers to a polypeptide capable of binding to RNA, including polypeptides that bind to single-stranded RNA, polypeptides that bind to double-stranded RNA, and polypeptides that bind to other configurations of RNA.
  • the RNA-binding polypeptide can be fused to an RNA ligase polypeptide, for example, to the N-terminus or C-terminus of an RNA ligase polypeptide and/or a DNA ligase polypeptide, without inactivating the RNA ligase polypeptide and/or the DNA ligase polypeptide. It should be understood that the RNA-binding polypeptide can also bind to polynucleotides other than RNA, such as DNA or known natural nucleotide analogs.
  • fusion polypeptide refers to a polypeptide comprising two or more amino acid subsequences (e.g., two or more polypeptide domains) fused (e.g., via peptide chains through their respective amino and carboxyl residues) to form a single contiguous polypeptide. It should be understood that the two or more amino acid sequences can be fused directly or indirectly through their respective amino and carboxyl termini via a linker, spacer, or additional polypeptide.
  • a "fragment" of a polypeptide is a subsequence of the polypeptide that possesses the function required for enzymatic or binding activity and/or provides three-dimensional structure to the polypeptide.
  • domain refers to a unit of a protein or protein complex, including a polypeptide subsequence, a complete polypeptide sequence, or multiple polypeptide sequences, which unit has a defined function.
  • linker refers to an amino acid or nucleotide sequence that indirectly fuses two or more polypeptides or two or more nucleic acid sequences encoding two or more polypeptides.
  • the length of the linker or spacer is about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or about 100 amino acids or nucleotides.
  • the length of the linker or spacer is about 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or about 1000 amino acids or nucleotides.
  • 5' and 3' are conventional expressions used to describe the characteristics of nucleic acid sequences, and relate to the position of genetic elements and/or the direction of events such as RNA polymerase transcription or ribosomal translation that proceed in a 5' to 3' direction (5' to 3'). Synonymous terms are upstream (5') and downstream (3'). Typically, DNA sequences, gene maps, and RNA sequences can be drawn from left to right in a 5' to 3' direction, or the 5' to 3' direction can be represented by an arrow symbol, with the arrow pointing in the 3' direction.
  • 5' upstream
  • 3' downstream
  • 5' upstream
  • 3' downstream
  • the term “5' end” or “5' end” can be the first nucleotide of a polynucleotide along the 5' to 3' direction or a continuous stretch of nucleotides with this nucleotide as the endpoint
  • the "3' end” or “3' end” can be the last nucleotide of a polynucleotide along the 5' to 3' direction or a continuous stretch of nucleotides with this nucleotide as the endpoint.
  • C-terminus generally refers to the carboxyl terminus of a polypeptide.
  • N-terminus generally refers to the amino terminus of a polypeptide.
  • ligation refers to the formation of a covalent bond (e.g., a 5', 3'-phosphodiester bond) between two polynucleotides.
  • Ligation may also refer to the formation of a covalent bond (e.g., a 5', 3'-phosphodiester bond) between the 5' end of one polynucleotide and the 3' end of another polynucleotide.
  • capping may refer to the addition of a cap structure analog at the 5' and/or 3' end of a polynucleotide to generate a 5' and/or 3' capped polynucleotide.
  • cap analogue includes natural or artificial cap structures (cap), such as m7G and any compound of the following general formula:
  • Cap structure analogs include, for example, dinucleotide cap analogs of the formula m7G(5') p3 (5')G, in which a guanine nucleotide (G) is connected to a triphosphate bridge via its 5'OH.
  • the 3'-OH group is replaced by hydrogen or OCH3 (see US 7,074,596; Kore, Nucleotides, Nucleotides, and Nucleic Acids, 2006, 25:307-14; and Kore, Nucleotides, Nucleotides, and Nucleic Acids, 2006, 25:337-40).
  • Dinucleotide cap analogs include m7G(5') p3G , 3'-OMe-m7G(5') p3G (ARCA).
  • the term "cap structure analog” also includes trinucleotide cap analogs (defined below) and other longer molecules (e.g., cap analogs with four, five, or six or more nucleotides connected to a triphosphate bridge).
  • 'Cap structure analogs can include 5' capping nucleotides connected to the 5' end of the mRNA through a 5' to 5' triphosphate internucleotide bond.
  • the nucleotides connected to the mRNA through a 5' to 5' triphosphate internucleotide bond are referred to as "natural" cap structure analogs.
  • the natural cap structure analog is a 7-methylguanosine (m7G) nucleotide.
  • the 5' cap is a modified 5' cap that includes one or more modified nucleotides, such as a 5' capping nucleotide, or one or more modified internucleotide modifications, such as modifications to the 5' to 5' triphosphate internucleotide bond.
  • the 5' cap includes one or more nucleotides with a sugar modification (such as 2'-O-methylation).
  • RNA ligases DNA/RNA binding polypeptides
  • polypeptide sequences that are different from the specific identification sequence, in which one or more amino acid residues are deleted, replaced or added, or sequences that include specific identification sequence fragments.
  • Functional variants can be naturally occurring allelic variants or non-naturally occurring variants.
  • Functional variants can be from the same species or other species and can include homologues, collateral relatives and direct relatives.
  • Functional variants or functional fragments of a polypeptide have one or more biological activities of the native specific identification polypeptide, such as the ability to stimulate one or more biological effects of the native polypeptide.
  • functional fragments of DNA or RNA ligases are generally capable of catalyzing the formation of phosphodiester bonds.
  • mRNA messenger RNA
  • RNA ribonucleic acid
  • poly (A) tail a polyadenylic acid portion
  • a typical mature eukaryotic mRNA has the following structure: starting with the terminal nucleotide of the mRNA at the 5' end, followed by a 5' untranslated region (5'UTR) of nucleotides, then an open reading frame (ORF, which may be a protein coding sequence) starting with a start codon (which is an AUG triplet of nucleotide bases) and ending with a stop codon (which may be a UAA, UAG, or UGA triplet of nucleotide bases), and then a 3' untranslated region (3'UTR) of nucleotides, ending with a polyadenosine moiety.
  • ORF which may be a protein coding sequence
  • target polynucleotide may refer to a given polynucleotide.
  • the target polynucleotide may be a natural polynucleotide or an artificially designed non-natural polynucleotide.
  • the target polynucleotide may be a DNA molecule, an RNA molecule, or a combination of DNA and RNA molecules (e.g., a target polynucleotide comprising a portion of DNA and a portion of RNA).
  • polynucleotide to be linked refers to a portion of a target polynucleotide.
  • the polynucleotide to be linked can be a continuous or discontinuous sequence of the target polynucleotide.
  • the polynucleotide to be linked can include the 5' end or the 3' end of the target polynucleotide.
  • the polynucleotide to be linked can be a non-Poly A region of the target polynucleotide.
  • first polynucleotide and “second polynucleotide” may be part of a target polynucleotide.
  • the polynucleotide to be connected, the first polynucleotide, and the second polynucleotide are arranged in a 5' to 3' direction.
  • the 3' end of the polynucleotide to be connected may form a covalent bond with the 5' end of the first polynucleotide
  • the 3' end of the first polynucleotide may form a covalent bond with the 5' end of the second polynucleotide.
  • the first polynucleotide may include a PolyA region and a non-PolyA region. In some embodiments, the first polynucleotide may only include a PolyA region and a non-PolyA region. In some embodiments, the second polynucleotide is entirely a PolyA region.
  • poly(A) portion refers to a nucleic acid sequence that is attached to the 3' end of a nucleic acid (e.g., RNA) and is composed primarily of adenosine nucleotides (either adenosine or deoxyadenosine).
  • a nucleic acid e.g., RNA
  • the poly(A) portion can be composed of 10%-100%, 25%-100%, 30%-100%, 40%-100%, 50%-100%, 60%-100%, 70%-100%, 80%-100%, 90%-100%, 95%-100%, 96%-100%, 97%-100%, 98%-100%, or 99%-100% adenosine nucleotides.
  • the polyadenosine portion (PolyA region) may be composed entirely of adenosine nucleotides (which may be adenosine or deoxyadenosine).
  • the adenosine nucleotides included in the polyadenosine portion may be standard adenosine nucleotides or modified (non-standard) adenosine nucleotides.
  • non-Poly A region can be a sequence other than the Poly A region in the target polynucleotide.
  • a typical mature eukaryotic mRNA has the following structure: starting from the mRNA terminal nucleotide at the 5' end, followed by a 5' untranslated region (5'UTR) of nucleotides, then an open reading frame (which can be a protein coding sequence), and then a 3' untranslated region (3'UTR) of nucleotides, ending with a polyadenosine moiety.
  • the non-Poly A region of the mRNA can include at least a portion of the 5'UTR, at least a portion of the 3'UTR, at least a portion of the open reading frame, or any combination thereof.
  • the term "first functional region” can be a segment of a target polynucleic acid that has a certain function.
  • the function can be related to gene translation, expression, or regulation.
  • the first functional region in DNA can include a coding region or a non-coding region.
  • the first functional region can include a 5'UTR, a 3'UTR, or an open reading frame.
  • splint oligonucleotide generally refers to an oligonucleotide having a first sequence of nucleotides that are regionally complementary or reversely complementary to a first oligonucleotide adjacent to an end, and a second sequence of nucleotides that are regionally complementary or reversely complementary to a second oligonucleotide adjacent to an end.
  • the splint oligonucleotide is capable of binding to the first oligonucleotide and the second oligonucleotide to produce a complex, thereby bringing the ends of the first oligonucleotide and the second oligonucleotide into close spatial proximity.
  • the splint oligonucleotide increases the chance of connection between the first oligonucleotide and the second oligonucleotide.
  • the splint oligonucleotide can increase the chance of connection between the polynucleotide to be connected and the first polynucleotide.
  • the splint oligonucleotide can increase the chance of connection between the polynucleotide to be connected and the second complex.
  • polynucleotide ligase polypeptides include DNA ligase polypeptides.
  • the DNA ligase polypeptide can be a prokaryotic DNA ligase, a prokaryotic DNA ligase variant, or a functional fragment thereof.
  • the DNA ligase polypeptide can be a bacterial DNA ligase, a bacterial DNA ligase variant, or a functional fragment thereof.
  • the DNA ligase polypeptide can be a viral DNA ligase, a viral DNA ligase variant, or a functional fragment thereof.
  • the DNA ligase polypeptide can be T4 DNA ligase, a variant thereof or a functional fragment thereof.
  • the polynucleotide ligase polypeptide may include an RNA ligase polypeptide.
  • the RNA ligase polypeptide may be a prokaryotic RNA ligase, a prokaryotic RNA ligase variant, or a functional fragment thereof.
  • the RNA ligase polypeptide may be a bacterial RNA ligase, a bacterial RNA ligase variant, or a functional fragment thereof.
  • the RNA ligase polypeptide may be a viral RNA ligase, a viral RNA ligase variant, or a functional fragment thereof.
  • RNA ligase polypeptide is T4 RNA ligase, its variant or its functional fragment.
  • the RNA ligase polypeptide is T4 RNA ligase.
  • the RNA ligase polypeptide is T4 RNA ligase 1 or a functional fragment of T4 RNA ligase 1.
  • the RNA ligase polypeptide is T4 RNA ligase 2 or a functional fragment of T4 RNA ligase 2.
  • the capping enzyme can be a fusion polypeptide comprising a polynucleotide binding polypeptide fused to a polynucleotide ligase polypeptide, wherein the polynucleotide binding polypeptide comprises a DNA binding polypeptide and/or an RNA binding polypeptide.
  • the polynucleotide binding polypeptide is selected from one or more of a DNA double-strand binding domain, a DNA single-strand binding domain, an RNA/DNA composite chain binding domain, an RNA single-strand or RNA double-strand binding protein.
  • the double-stranded DNA binding domain may include Sso7d and/or NF-kappaB p50; the RNA/DNA complex chain binding domain may include one or more of ScFV, RNaseH1 (D210N), and HBD of monoclonal antibody S9.6; the single-stranded RNA binding domain may include one or more of Sso7d, PKR, TRBP, PACT, Staufen, NFAR1, NFAR2, SPNR, RHA, NREBP, Kanadaptin, HYL1 Hyponastic leaves, ADAR1, ADAR2, ADAR3, TENR, RNaseIII, Dicer, and RDE-4.
  • the polynucleotide binding polypeptide includes one or more of Sso7d, NF-kappaB p50, ScFV of monoclonal antibody S9.6, RNaseH1 (D210N), HBD, PKR, TRBP, PACT, Staufen, NFAR1, NFAR2, SPNR, RHA, NREBP, Kanadaptin, HYL1 Hyponastic leaves, ADAR1, ADAR2, ADAR3, TENR, RNaseIII, Dicer, and RDE-4.
  • polynucleotide binding polypeptide is Sso7d.
  • the capping enzyme is a fusion polypeptide, wherein the C-terminus of the polynucleotide ligase polypeptide is linked to the N-terminus of the polynucleotide binding polypeptide, or the N-terminus of the polynucleotide ligase polypeptide is linked to the C-terminus of the polynucleotide binding polypeptide.
  • the polynucleotide ligase polypeptide and the polynucleotide binding polypeptide are connected by a first joint.
  • the first joint indirectly fuses two or more polypeptides or two or more nucleic acid sequences encoding two or more polypeptides or nucleotide sequences.
  • the length of the first joint is about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or about 100 amino acids or nucleotides.
  • the length of the connector or spacer is about 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or about 1000 amino acids or nucleotides.
  • the first linker has a length of about 1 to about 1000 amino acids or nucleotides, about 10 to about 1000, about 50 to about 1000, about 100 to about 1000, about 200 to about 1000, about 300 to about 1000, about 400 to about 1000, about 500 to about 1000, about 600 to about 1000, about 700 to about 1000, about 800 to about 1000, or about 900 to about 1000 amino acids or nucleotides.
  • the N-terminus of the polynucleotide ligase polypeptide is connected to the first linker, and the C-terminus of the polynucleotide binding polypeptide is connected to the first linker; or the C-terminus of the polynucleotide ligase polypeptide is connected to the first linker, and the N-terminus of the polynucleotide binding polypeptide is connected to the first linker.
  • the first linker can include a G4S linker (e.g., GGGGS (SEQ ID NO: 5)).
  • the first linker can be a repeat of multiple G4S linkers, such as the sequence shown in SEQ ID 23.
  • the N-terminus or C-terminus of the capping enzyme may have a tag for protein purification, for example, the protein purification tag includes one or more of 6xHis, GST, DYKDDDDK (SEQ ID NO: 45) (FLAG), c-myc, or HA.
  • the protein purification tag includes one or more of 6xHis, GST, DYKDDDDK (SEQ ID NO: 45) (FLAG), c-myc, or HA.
  • polynucleotide ligase polypeptide includes an amino acid sequence as shown in any one of SEQ ID NO: 9-12, 21-22.
  • polynucleotide ligase polypeptide is encoded by a nucleotide sequence as shown in any one of SEQ ID NO:33-35.
  • polynucleotide ligase polypeptide is encoded by the nucleotide sequence shown in SEQ ID NO: 47.
  • polynucleotide binding polypeptide includes the amino acid sequence shown in any one of SEQ ID NO: 13-17.
  • the polynucleotide binding polypeptide may include an amino acid sequence as shown in SEQ ID NO:46.
  • the polynucleotide binding polypeptide includes a nucleotide sequence encoded by any one of SEQ ID NO:30-32.
  • the capping enzyme includes an amino acid sequence as shown in any one of SEQ ID NO: 18-20, 24-29.
  • the capping enzyme can be an amino acid sequence as shown in SEQ ID NO:48.
  • the capping enzyme is encoded by a nucleotide sequence as shown in any one of SEQ ID NO:36-44.
  • the capping enzyme can be encoded by a nucleotide sequence as shown in SEQ ID NO:49.
  • polynucleotide ligase polypeptide includes the amino acid sequence shown in any one of SEQ ID NO: 9-12, 21-22.
  • polynucleotide binding polypeptide is the amino acid sequence shown in any one of SEQ ID NO:13-17.
  • the capping enzyme is the amino acid sequence shown in any one of SEQ ID NO: 18-20, 24-29.
  • the present application also includes conservative substitutions of one or more amino acids in the polypeptide sequence without significantly altering its biological activity. Skilled artisans will know methods for making phenotypically silent amino acid substitutions (e.g., see Bowie et al., 1990, Science 247, 1306). Similarly, the present invention also includes functional variants produced by substitutions (including non-conservative substitutions) of one or more amino acids.
  • variant refers to polypeptides, including naturally occurring, recombinant, and synthetic polypeptides, that are at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 5%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence of the invention. Identity is found over a comparison window of at least 20 amino acid positions, preferably at least 50 amino acid positions, at least 100 amino acid positions, or over the entire length of the polypeptide of the invention.
  • cap structure analogue which may have the following general formula:
  • R 3 is selected from guanine, adenine, cytosine, uracil, a guanine analog, an adenine analog, a cytosine analog, a uracil analog;
  • R 4 is (N 1 p) x N 2 , wherein N 1 and N 2 are ribonucleosides, and N 1 and N 2 are the same or different;
  • p 1 is independently a phosphate group, a thiophosphate, a dithiophosphate, an alkylphosphonic acid, an arylphosphonic acid or an N-phosphoramide bond at each position;
  • X is an integer from 0 to 8, wherein if X ⁇ 2, the ribonucleosides N1 in (N1-p) x are the same or different from each other;
  • R 1 and R 2 groups are independently selected from O-alkyl, halogen, acetylamino (AcNH), hydrogen or hydroxyl.
  • the sugars in N1 and N2 are independently selected from ribose and deoxyribose for each position, and may contain modifications including 2'-O-alkyl, 2'-O-methoxyethyl, 2'-O allyl, 2'-O alkylamine, 2'-fluororibose and 2'-deoxyribose, for example, the bases in N1 and N2 are independently selected from adenine, uracil, guanine or cytosine for each position, or analogs of adenine, uracil, guanine or cytidine, and the nucleotide modified bases may be selected from xanthine, allylaminouracil, allylaminothymidine, hypoxanthine, dioxyadenine, dioxycytosine, dioxyguanine, dioxyuracil, 6-chloropurine nucleoside, N6 -methyladenine, methylpseu
  • the cap structure analogue may be a dinucleotide cap analogue or a trinucleotide cap analogue.
  • cap structure analogue may have the following general formula:
  • R 5 and R 6 groups are independently selected from O-alkyl (O-methyl), halogen, label, hydrogen or hydroxyl;
  • the R 2 group is selected from -CH 2 NHAc, O-alkyl (O-methyl), halogen, tag, hydrogen or hydroxyl;
  • X1 and X2 are bases, and X1 and X2 may be the same as or different from each other.
  • X 1 and/or X 2 are guanine or adenine.
  • cap structure analog is selected from one of the following:
  • the present application provides a method for capping a polynucleotide, comprising:
  • composition comprising at least: an uncapped polynucleotide, the aforementioned capping enzyme, and the aforementioned cap structure analog, wherein the capping enzyme comprises a polynucleotide ligase polypeptide;
  • an uncapped polynucleotide is DNA or RNA.
  • uncapped polynucleotides are chemically synthesized.
  • the length of the uncapped polynucleotide is 1 to 2000 nucleotides.
  • the length of the uncapped polynucleotide is 1 to 2000 nucleotides.
  • the length of the uncapped polynucleotide can be 1 to 2000 nucleotides, 1 to 1500 nucleotides, 1 to 1000 nucleotides, 1 to 500 nucleotides, or 1 to 100 nucleotides.
  • the number of uncapped polynucleotides can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83 3, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,
  • the number of nucleotides in an uncapped polynucleotide can range from 1-150, 1-140, 1-130, 1-120, 1-110, 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, 1-20, 1-10, 10-150, 10-140, 10-130, 10-120, 10-110, 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, 10-20, 20-150, 20-140, 20-130, 20-120, 20-110, 20-100, 20-90, 20-80 , 20-70, 20-60, 20-50, 20-40, 20-30, 30-150, 30-140, 30-130, 30-120, 30-110, 30-100, 30-90, 30-80, 30-70, 30-60, 30-50, 30-40, 40-150, 40-140, 40-130, 40-120, 40-110, 40-100, 40-90, 40-80, 40-70, 40-60, 40-50, 50-150, 50-140, 50-130, 50-120, 50-
  • the 5' end of the uncapped polynucleotide can be a specific nucleotide, or a nucleotide containing a specific modification, or a nucleotide containing a specific sequence.
  • the base of the 5' end nucleotide of the uncapped polynucleotide is guanine.
  • the base of the 5' end nucleotide of the uncapped polynucleotide is adenine.
  • the base of the 5' end nucleotide of the uncapped polynucleotide is cytosine.
  • the base of the 5' end nucleotide of the uncapped polynucleotide is guanine.
  • the connection method disclosed in the present application can have different efficiencies for different bases of the 5' end nucleotide. Those skilled in the art can select the base of the 5' end nucleotide with higher connection efficiency according to the method disclosed in the present application.
  • the 5' terminal nucleotide of the uncapped polynucleotide is modified by phosphorylation.
  • the sugars of the nucleotides of the uncapped polynucleotide are independently selected from ribose and deoxyribose for each position and may contain modifications including 2'-O-alkyl, 2'-O-methoxyethyl, 2'-O allyl, 2'-O alkylamine, 2'-fluororibose and 2'-deoxyribose.
  • the bases of the nucleotides of the uncapped polynucleotide are independently selected from adenine, uracil, guanine or cytosine for each position, or analogs of adenine, uracil, guanine or cytosine, and the nucleotide modifications may be selected from xanthine, allylaminouracil, allylaminothymidine, hypoxanthine, dioxyadenine, dioxycytosine, dioxyguanine, dioxyuracil, 6-chloropurine nucleoside, N6-methyladenine, methylpseudouracil, 2-thiocytosine, , 2-thiouracil, 5-methyluracil, 4-thiothymidine, 4-thiouracil, 5,6-dihydro-5-methyluracil, 5,6-dihydrouracil, 5-[(3-indolyl)propionamido-N-allyl]urac
  • the reaction temperature of step S2 is 5° C. to 80° C.
  • it can be 5° C. to 80° C., 5° C. to 70° C., 5° C. to 60° C., 5° C. to 50° C., 5° C. to 40° C., 5° C. to 30° C., 10° C. to 80° C., 10° C. to 70° C., 10° C. to 60° C., 10° C. to 50° C., 10° C. to 40° C., 10° C. to 30° C., 20° C. to 80° C., 20° C. to 70° C., 20° C. to 60° C., 20° C. to 50° C., 20° C. to 40° C., or 20° C. to 30° C.
  • reaction temperature of step S2 is 25°C to 60°C.
  • the composition of step S1 further includes adenosine triphosphate.
  • the composition of step S1 further includes a DNA and/or RNA buffer.
  • the composition of step S1 further includes a DNase and/or RNase inhibitor.
  • the present application further provides a kit comprising the capping enzyme as described above.
  • the present application further provides use of the aforementioned capping enzyme in polynucleotide capping.
  • mRNA 100 may include a 5'UTR 101, an ORF 102, a 3'UTR 103, and a PolyA region 104.
  • Traditional chemical synthesis or in vitro transcription methods can obtain 5'UTR 101, ORF 102, and 3'UTR 103, but cannot obtain a complete and stable-length PolyA region 104.
  • One of the main problems addressed by the present application is how to obtain a complete and stable-length PolyA region 104.
  • the target polynucleotide 100 (taking mRNA as an example) is divided into a polynucleotide to be connected 201, a first polynucleotide 202, and a second polynucleotide 203.
  • the polynucleotide to be connected 201, the first polynucleotide 202, and the second polynucleotide 203 can constitute the complete target polynucleotide 100.
  • the first polynucleotide 202 can contain a non-PolyA region 2021 and a PolyA region 2022.
  • the non-PolyA region 2021 can be a portion of the target polynucleotide sequence 103.
  • the non-PolyA region 2021 can be connected to a portion of the polynucleotide to be connected 201 to form a complete 3'UTR 103.
  • the second polynucleotide 203 can be entirely a PolyA region.
  • the PolyA region 2022 can constitute the complete PolyA region 104 of the target polynucleotide 100 with the second polynucleotide 203.
  • the first polynucleotide 202 and the second polynucleotide 203 can be chemically synthesized, and their lengths are stable and controllable. By dividing the target polynucleotide into multiple fragments, synthesizing them separately, and then connecting them with T4 RNA ligase, stable and controllable PolyA mRNA can be obtained.
  • a splint oligonucleotide 300 can be designed.
  • the splint oligonucleotide 300 can be divided into a first region 301 and a second region 302.
  • the first region 301 corresponds to at least a portion of the polynucleotide 201 to be ligated (complementary or reverse complementary)
  • the first region 302 corresponds to at least a portion of the first polynucleotide 202 (complementary or reverse complementary).
  • the second region 302 can include a second subregion 3021 and a first subregion 3022.
  • the second subregion 3021 can correspond to the non-polyA region 2021 of the first polynucleotide 202 (complementary or reverse complementary), and the first subregion 3022 can correspond to the polyA region 2022 of the first polynucleotide 202 (complementary or reverse complementary).
  • the splint oligonucleotide can be a DNA sequence or an RNA sequence.
  • the first region 301 is reverse complementary to at least a portion of the polynucleotide to be connected 201
  • the first region 302 is reverse complementary to at least a portion of the first polynucleotide 202.
  • the second region 302 may include a second subregion 3021 and a first subregion 3022, wherein the second subregion 3021 may be reverse complementary to the non-polyA region 2021 of the first polynucleotide 202, and the first subregion 3022 may be reverse complementary to the polyA region 2022 of the first polynucleotide 202.
  • the above is a specific embodiment of the component design of the present application.
  • the polynucleotide to be connected, the first polynucleotide and the second polynucleotide described in the present application are not limited to the above embodiment.
  • one approach of the present application is to divide the target polynucleotide into multiple fragments, obtain these fragments by chemical synthesis or in vitro transcription, at least two of which contain Poly A regions, and then synthesize these fragments into a complete target polynucleotide using a ligase.
  • the target polynucleotide obtained by the above method has at least the following advantages:
  • a PolyA tail of controllable length can be obtained in the target polynucleotide
  • the PolyA tail can be modified in a controllable manner.
  • the present application provides a method for preparing a target polynucleotide, which may include the following steps:
  • Step S1 providing polynucleotides to be connected, providing a first polynucleotide, and providing a second polynucleotide, wherein the first polynucleotide and/or the second polynucleotide includes a Poly A region;
  • Step S2 covalently linking the polynucleotide to be linked, the first polynucleotide, and the second polynucleotide to obtain the target polynucleotide.
  • the first polynucleotide, and/or the second polynucleotide, and/or the polynucleotide to be joined, and/or the target polynucleotide are ribonucleic acid (RNA) or deoxyribonucleic acid (DNA).
  • RNA ribonucleic acid
  • DNA deoxyribonucleic acid
  • the target polynucleotide e.g., mRNA
  • the first polynucleotide, the second polynucleotide, and the polynucleotide to be joined are all RNA.
  • the target polynucleotide is DNA
  • the first polynucleotide, the second polynucleotide, and the polynucleotide to be joined are all deoxyribonucleic acid (DNA).
  • the first polynucleotide, and/or the second polynucleotide, and/or the polynucleotide to be ligated, and/or the target polynucleotide is a combination of ribonucleic acid (RNA) and deoxyribonucleic acid (DNA).
  • RNA ribonucleic acid
  • DNA deoxyribonucleic acid
  • the first polynucleotide, and/or the second polynucleotide, and/or the polynucleotide to be joined are chemically synthesized or in vitro transcribed.
  • the first polynucleotide and the second polynucleotide are chemically synthesized, and the polynucleotide to be joined is in vitro transcribed.
  • the polynucleotide to be joined, the first polynucleotide, and the second polynucleotide are all chemically synthesized.
  • the advantages of chemical synthesis for obtaining the first polynucleotide, and/or the second polynucleotide, and/or the polynucleotide to be linked are: (1) chemical synthesis is more controllable, and the length of the obtained polynucleotides can be controlled, especially the first polynucleotide and/or the second polynucleotide, so that the length of the obtained poly A is easily controlled; (2) modification is convenient, and modified nucleotides can be added as required; (3) compared to traditional enzymatic polymerization methods, less biological raw materials are used, making it easier to meet GMP requirements.
  • the synthesis of polynucleotides can be carried out using the method described in Example 5.
  • the first polynucleotide and/or the second polynucleotide may include a polyA region.
  • the first polynucleotide may include a polyA region and a non-polyA region
  • the second polynucleotide may be polyA.
  • the first polynucleotide and the second polynucleotide may both be poly A.
  • the first polynucleotide and the second polynucleotide each include a poly A region and a non-poly A region.
  • the non-poly A region may include 5-100 nucleotides.
  • the length of the first polynucleotide and/or the second polynucleotide may be 1 to 150 nucleotides.
  • the number of nucleotides in the first polynucleotide and/or the second polynucleotide may be 1 to 150 nucleotides.
  • the first polynucleotide and the second polynucleotide may be of the same length or different lengths, for example, the first polynucleotide is longer than the second polynucleotide, or the first polynucleotide is shorter than the second polynucleotide.
  • the polynucleotides to be linked may not contain a polyA region.
  • the target polynucleotide may include a first functional region.
  • the first functional region in mRNA may include one or more of a 3'UTR, an ORF, and a 5'UTR.
  • the first functional region in DNA may include a coding region or a non-coding region.
  • the polynucleotide to be linked is covalently linked to the non-polyA region of the first polynucleotide and/or the second polynucleotide to form the first functional region.
  • the first polynucleotide includes a non-polyA region and a polyA region
  • the second polynucleotide includes a polyA region
  • the non-polyA region includes a portion of the first functional region (e.g., a portion of the 3'UTR)
  • the polynucleotide to be linked includes the remaining portion of the first functional region (e.g., another portion of the 3'UTR).
  • the first polynucleotide including the non-polyA region and the polynucleotide to be linked are linked to obtain the first functional region.
  • the method for preparing a target polynucleotide further comprises covalently linking the polynucleotide to be linked, the first polynucleotide, and the second polynucleotide in the presence of a ligase to obtain the target polynucleotide.
  • the ligase may comprise a DNA ligase or an RNA ligase.
  • the ligase may comprise one or more of T4 DNA ligase and T4 RNA ligase.
  • the T4 RNA ligase may comprise one or more of T4 RNA ligase 1 and T4 RNA ligase 2.
  • the ligase may comprise a single-stranded ligase or a double-stranded ligase.
  • the single-stranded ligase may comprise one or more of T4 RNA ligase 1, RM378 RNA ligase, and TS2126 RNA ligase
  • the double-stranded ligase may comprise one or more of T4 DNA ligase, T3 DNA ligase, and T4 RNA ligase 2.
  • the ligase can further include one or more of RM378 RNA ligase, TS2126 RNA ligase, E.
  • coli RNA ligase Mth RNA ligase, RTCB RNA ligase, T3 DNA ligase, T7 DNA ligase, Taq DNA ligase, marine archaea Thermococcus sp DNA ligase, Chlorella virus DNA ligase, and RtcB DNA ligase.
  • the method for preparing a target polynucleotide further comprises covalently linking the polynucleotide to be linked, the first polynucleotide, and the second polynucleotide in the presence of a splint oligonucleotide to obtain the target polynucleotide.
  • the method for preparing a target polynucleotide further comprises covalently linking the polynucleotide to be linked, the first polynucleotide, and the second polynucleotide in the presence of a splint oligonucleotide to obtain the target polynucleotide.
  • the method for preparing a target polynucleotide further comprises covalently ligating the polynucleotide to be ligated, the first polynucleotide, and the second polynucleotide in the presence of a splint oligonucleotide and a double-stranded ligase to obtain the target polynucleotide, wherein the double-stranded ligase may be T4 RNA ligase 2.
  • the splint oligonucleotide comprises a first region and a second region, wherein the first region corresponds to the polynucleotide to be connected, and the second region corresponds to the first polynucleotide and/or the second polynucleotide.
  • the first region may be complementary or reverse complementary to the '3' end sequence of the polynucleotide to be connected, and the second region may be complementary or reverse complementary to the '5' end sequence of the first polynucleotide and/or the second polynucleotide.
  • the second region may be complementary or reverse complementary to at least the non-PolyA region of the first polynucleotide.
  • the second region may only be complementary or reverse complementary to at least part of the non-PolyA region of the first polynucleotide, the second region may also be complementary or reverse complementary to at least part of the non-PolyA region and at least part of the polyA region of the first polynucleotide, the second region may also be complementary or reverse complementary to all non-PolyA regions and at least part of the polyA region of the first polynucleotide, or the second region may be complementary or reverse complementary to all non-PolyA regions and all polyA regions of the first polynucleotide.
  • step S2 of the method for preparing a target polynucleotide further comprises:
  • Step S.2.1.1 Covalently linking the first polynucleotide to the polynucleotide to be linked to obtain a first complex
  • Step S.2.1.2 Covalently link the second polynucleotide to the first complex to obtain the target polynucleotide.
  • the first complex may refer to the product of covalently linking the polynucleotide to be linked and the first polynucleotide.
  • step S.2.1.1 may further include covalently linking the 3' end of the polynucleotide to be linked and the 5' end of the first polynucleotide to obtain the first complex.
  • step S.2.1.1 the 3' and 5' end groups of the polynucleotides to be linked can be hydroxyl groups, the 3' end group of the first polynucleotide can be a hydroxyl group, and the 5' end group can be a phosphate group.
  • step S.2.1.2 the 3' and 5' end groups of the second polynucleotide can be phosphate groups.
  • the linking polynucleotide and/or the first polynucleotide and/or the second polynucleotide may be phosphorylated or hydroxylated.
  • obtaining the first complex may further include performing a first modification on the 3' end of the polynucleotide to be linked and/or performing a second modification on the 5' end of the polynucleotide to be linked, and then covalently linking the polynucleotide to be linked to obtain the first complex.
  • obtaining the first complex may further include performing a first modification on the 3' end of the first polynucleotide and/or performing a second modification on the 5' end of the first polynucleotide, and then covalently linking the polynucleotide to be linked to obtain the first complex.
  • obtaining the target polynucleotide may further include performing a first modification on the 3' end of the first complex and/or performing a second modification on the 5' end of the first complex, and then covalently linking the polynucleotide to be linked to obtain the target polynucleotide.
  • obtaining the target polynucleotide may further include performing a first modification on the 3' end of the second polynucleotide and/or performing a second modification on the 5' end of the second polynucleotide, and then covalently linking the polynucleotide to the first complex, and then obtaining the first complex.
  • obtaining the target polynucleotide further includes performing a first modification on the 3' end of the first complex.
  • obtaining the target polynucleotide further includes performing a second modification on the 5' end of the first complex.
  • obtaining the target polynucleotide further includes performing a first modification on the 3' end of the second polynucleotide.
  • obtaining the target polynucleotide further includes performing a second modification on the 5' end of the second polynucleotide.
  • the first modification and/or the second modification can be a hydroxylation modification or a phosphorylation modification.
  • the phosphorylation modification can be performed using T4PNK enzyme.
  • step S.2.1.1 of obtaining the first complex may further include covalently linking the first polynucleotide to the polynucleotide to be linked in the presence of a splint oligonucleotide to obtain the first complex.
  • step S.2.1.1 it can further include obtaining a first complex in the presence of a ligase, wherein the ligase can be a double-stranded ligase, for example, the double-stranded ligase can be T4 RNA ligase 2.
  • the ligase can be a double-stranded ligase, for example, the double-stranded ligase can be T4 RNA ligase 2.
  • step S.2.1.2 it can further include obtaining the target polynucleotide in the presence of a ligase, wherein the ligase can be a single-stranded ligase, for example, the single-stranded ligase can be T4 RNA ligase 1.
  • the ligase can be a single-stranded ligase, for example, the single-stranded ligase can be T4 RNA ligase 1.
  • the method of preparing a target polynucleotide further comprises:
  • Step S.2.2.1 connecting the first polynucleotide to the second polynucleotide to obtain a second complex
  • Step S.2.2.2 Connect the polynucleotide to be connected with the second complex to obtain the target polynucleotide.
  • the second complex may refer to a product obtained by covalently linking the first polynucleotide and the second polynucleotide.
  • the 3' end of the first polynucleotide and the 5' end of the second polynucleotide may be covalently linked to obtain the second complex.
  • step S.2.2.1 the 3' and 5' end groups of the polynucleotides to be linked can be hydroxyl groups, and the 3' and 5' end groups of the first polynucleotide can be hydroxyl groups.
  • step S.2.2.2 the 3' and 5' end groups of the second polynucleotide can be phosphate groups.
  • phosphorylation modification or hydroxylation modification can be performed on the ligating polynucleotide and/or the first polynucleotide and/or the second polynucleotide.
  • obtaining the second complex further includes performing a first modification on the 3' end of the first polynucleotide and/or performing a second modification on the 5' end of the first polynucleotide, and then covalently linking it with the second polynucleotide to obtain the second complex.
  • obtaining the second complex further includes performing a first modification on the 3' end of the second polynucleotide and/or performing a second modification on the 5' end of the second polynucleotide, and then covalently linking it with the first polynucleotide to obtain the second complex.
  • obtaining the target polynucleotide further includes performing a first modification on the 3' end of the polynucleotide to be linked and/or performing a second modification on the 5' end of the polynucleotide to be linked, and then covalently linking it with the second complex to obtain the target polynucleotide.
  • obtaining the target polynucleotide further includes performing a first modification on the 3' end of the second complex and/or performing a second modification on the 5' end of the second complex, and then covalently linking with the nucleotide to be linked to obtain the target polynucleotide.
  • obtaining the second complex further comprises performing a first modification on the 3' end of the first polynucleotide.
  • obtaining the second complex further comprises performing a second modification on the 5' end of the first polynucleotide.
  • obtaining the second complex further comprises performing a first modification on the 3' end of the second polynucleotide.
  • obtaining the second complex further comprises performing a second modification on the 5' end of the second polynucleotide.
  • the first modification and/or the second modification may be a hydroxylation modification or a phosphorylation modification.
  • the phosphorylation modification may be performed using T4PNK enzyme.
  • obtaining the target polynucleotide further includes performing a first modification on the 3' end of the polynucleotide to be connected.
  • obtaining the target polynucleotide further includes performing a second modification on the 5' end of the polynucleotide to be connected.
  • obtaining the target polynucleotide further includes performing a first modification on the 3' end of the second complex.
  • obtaining the target polynucleotide further includes performing a second modification on the 5' end of the second complex.
  • the first modification and/or the second modification can be a hydroxylation modification or a phosphorylation modification.
  • the phosphorylation modification can be performed using T4PNK enzyme.
  • obtaining the target polynucleotide may further include covalently linking the second complex to the polynucleotide to be linked in the presence of a splint oligonucleotide to obtain the first complex.
  • step S.2.2.1. it can further include obtaining a second complex in the presence of a ligase, wherein the ligase can be a single-strand ligase, for example, the double-strand ligase can be T4 RNA ligase 1.
  • the ligase can be a single-strand ligase, for example, the double-strand ligase can be T4 RNA ligase 1.
  • step S.2.2.2. it can further include obtaining the target polynucleotide in the presence of a ligase, wherein the ligase can be a double-stranded ligase, for example, the double-stranded ligase can be T4 RNA ligase 2.
  • the ligase can be a double-stranded ligase, for example, the double-stranded ligase can be T4 RNA ligase 2.
  • one approach of the present application is to divide the target polynucleotide into multiple fragments, obtain these fragments separately by chemical synthesis or in vitro transcription, at least two of which contain the sequence to be joined and the first nucleotide, and then synthesize these fragments into a complete target polynucleotide using a ligase.
  • the target polynucleotide obtained in this manner has at least the following advantages:
  • a PolyA tail of controllable length can be obtained in the target polynucleotide
  • the PolyA tail can be modified in a controllable manner.
  • the present application provides a method for preparing a target polynucleotide, which may include the following steps:
  • A1 Providing a first polynucleotide, providing a polynucleotide to be connected, wherein the first polynucleotide comprises a non-PolyA region and a PolyA region;
  • step A1 further comprises providing a second polynucleotide
  • step A2 further comprises covalently linking the polynucleotide to be linked, the first polynucleotide, and the second polynucleotide to obtain the target polynucleotide.
  • the second polynucleotide is polyA.
  • the first polynucleotide, and/or the second polynucleotide, and/or the polynucleotide to be joined, and/or the target polynucleotide are ribonucleic acid (RNA) or deoxyribonucleic acid (DNA).
  • RNA ribonucleic acid
  • DNA deoxyribonucleic acid
  • the target polynucleotide e.g., mRNA
  • the first polynucleotide, the second polynucleotide, and the polynucleotide to be joined are all RNA.
  • the target polynucleotide is DNA
  • the first polynucleotide, the second polynucleotide, and the polynucleotide to be joined are all deoxyribonucleic acid (DNA).
  • the first polynucleotide, and/or the second polynucleotide, and/or the polynucleotide to be linked, and/or the target polynucleotide is a combination of ribonucleic acid (RNA) and deoxyribonucleic acid (DNA).
  • RNA ribonucleic acid
  • DNA deoxyribonucleic acid
  • the first polynucleotide and the second polynucleotide are RNA
  • the polynucleotide to be linked is DNA
  • the target polynucleotide obtained according to the method of the present application is a combination of ribonucleic acid (RNA) and deoxyribonucleic acid (DNA).
  • the first polynucleotide includes a polyA region and a non-polyA region, wherein the non-polyA region is DNA and the polyA region is RNA.
  • the first polynucleotide obtained in this way is a combination of DNA and RNA, and the target polynucleotide further obtained is a combination of ribonucleic acid (RNA) and deoxyribonucleic acid (DNA).
  • the first polynucleotide, and/or the second polynucleotide, and/or the polynucleotide to be joined are chemically synthesized or in vitro transcribed.
  • the first polynucleotide and the second polynucleotide are chemically synthesized, and the polynucleotide to be joined is in vitro transcribed.
  • the polynucleotide to be joined, the first polynucleotide, and the second polynucleotide are all chemically synthesized.
  • the advantages of chemical synthesis for obtaining the first polynucleotide, and/or the second polynucleotide, and/or the polynucleotide to be linked are: (1) chemical synthesis is more controllable, and the length of the obtained polynucleotides can be controlled, especially the first polynucleotide and/or the second polynucleotide, so that the length of the obtained poly A is easily controlled; (2) modification is convenient, and modified nucleotides can be added as required; (3) compared to traditional enzymatic polymerization and in vitro transcription methods, less biological raw materials are used, making it easier to meet GMP requirements. Chemical synthesis of polynucleotides can be carried out using the method described in Example 5.
  • the first polynucleotide and/or the second polynucleotide may include a polyA region.
  • the first polynucleotide may include a polyA region and a non-polyA region
  • the second polynucleotide may be polyA.
  • the first polynucleotide and the second polynucleotide may both be poly A.
  • the first polynucleotide and the second polynucleotide each include a poly A region and a non-poly A region.
  • the first polynucleotide and the second polynucleotide may be of the same length or different lengths, for example, the first polynucleotide is longer than the second polynucleotide, or the first polynucleotide is shorter than the second polynucleotide.
  • the polynucleotides to be linked may not contain a polyA region.
  • the target polynucleotide may include a first functional region.
  • the first functional region in mRNA may include one or more of a 3'UTR, an ORF, and a 5'UTR.
  • the first functional region in DNA may include a coding region or a non-coding region.
  • the polynucleotide to be linked is covalently linked to the non-polyA region of the first polynucleotide and/or the second polynucleotide to form the first functional region.
  • the first polynucleotide includes a non-polyA region and a polyA region
  • the second polynucleotide includes a polyA region
  • the non-polyA region includes a portion of the first functional region (e.g., a portion of the 3'UTR)
  • the polynucleotide to be linked includes the remaining portion of the first functional region (e.g., another portion of the 3'UTR).
  • the first polynucleotide including the non-polyA region and the polynucleotide to be linked are linked to obtain the first functional region.
  • the method for preparing a target polynucleotide further comprises covalently linking the polynucleotide to be linked, the first polynucleotide, and the second polynucleotide in the presence of a ligase to obtain the target polynucleotide.
  • the ligase may comprise a DNA ligase or an RNA ligase.
  • the ligase may comprise one or more of T4 DNA ligase and T4 RNA ligase.
  • the T4 RNA ligase may comprise one or more of T4 RNA ligase 1 and T4 RNA ligase 2.
  • the ligase may comprise a single-stranded ligase or a double-stranded ligase.
  • the single-stranded ligase may comprise one or more of T4 RNA ligase 1, RM378 RNA ligase, and TS2126 RNA ligase
  • the double-stranded ligase may comprise one or more of T4 DNA ligase, T3 DNA ligase, and T4 RNA ligase 2.
  • the ligase can further include one or more of RM378 RNA ligase, TS2126 RNA ligase, E.
  • coli RNA ligase Mth RNA ligase, RTCB RNA ligase, T3 DNA ligase, T7 DNA ligase, Taq DNA ligase, marine archaea Thermococcus sp DNA ligase, Chlorella virus DNA ligase, and RtcB DNA ligase.
  • the method for preparing a target polynucleotide further comprises covalently linking the polynucleotide to be linked, the first polynucleotide, and the second polynucleotide in the presence of a splint oligonucleotide to obtain the target polynucleotide.
  • the method for preparing a target polynucleotide further comprises covalently linking the polynucleotide to be linked, the first polynucleotide, and the second polynucleotide in the presence of a splint oligonucleotide to obtain the target polynucleotide.
  • the splint oligonucleotide comprises a first region and a second region, wherein the first region corresponds to the polynucleotide to be connected, and the second region corresponds to the first polynucleotide and/or the second polynucleotide.
  • the first region may be complementary or reverse complementary to the '3' end sequence of the polynucleotide to be connected, and the second region may be complementary or reverse complementary to the '5' end sequence of the first polynucleotide and/or the second polynucleotide.
  • the second region may be complementary or reverse complementary to at least the non-PolyA region of the first polynucleotide.
  • the second region may only be complementary or reverse complementary to at least part of the non-PolyA region of the first polynucleotide, the second region may also be complementary or reverse complementary to at least part of the non-PolyA region and at least part of the polyA region of the first polynucleotide, the second region may also be complementary or reverse complementary to all non-PolyA regions and at least part of the polyA region of the first polynucleotide, or the second region may be complementary or reverse complementary to all non-PolyA regions and all polyA regions of the first polynucleotide.
  • the second region of the splint oligonucleotide may include a first subregion and a second subregion, wherein the first subregion corresponds to a polyA region and the second subregion corresponds to a non-polyA region.
  • the first subregion is complementary or reverse complementary to at least a portion of the polyA region of the first polynucleotide
  • the second subregion is complementary or reverse complementary to at least a portion of the non-polyA region of the first polynucleotide.
  • the first subregion is complementary or reverse complementary to at least a portion of the polyA region of the first polynucleotide, and the second subregion is complementary or reverse complementary to the entire non-polyA region of the first polynucleotide.
  • step A2 of the method for preparing a target polynucleotide further comprises:
  • Step A.2.1.1 Covalently linking the first polynucleotide to the polynucleotide to be linked to obtain a first complex
  • Step A.2.1.2 Covalently link the second polynucleotide to the first complex to obtain the target polynucleotide.
  • the first complex may refer to the product of covalently linking the polynucleotide to be linked and the first polynucleotide.
  • step A.2.1.1 may further include covalently linking the 3' end of the polynucleotide to be linked and the 5' end of the first polynucleotide to obtain the first complex.
  • step A.2.1.1 the 3' and 5' end groups of the polynucleotides to be linked can be hydroxyl groups, the 3' end group of the first polynucleotide can be hydroxyl groups, and the 5' end group can be a phosphate group.
  • step A.2.1.2 the 3' and 5' end groups of the second polynucleotide can be phosphate groups.
  • the linking polynucleotide and/or the first polynucleotide and/or the second polynucleotide may be phosphorylated or hydroxylated.
  • obtaining the first complex may further include performing a first modification on the 3' end of the polynucleotide to be linked and/or performing a second modification on the 5' end of the polynucleotide to be linked, and then covalently linking the polynucleotide to be linked to obtain the first complex.
  • obtaining the first complex may further include performing a first modification on the 3' end of the first polynucleotide and/or performing a second modification on the 5' end of the first polynucleotide, and then covalently linking the polynucleotide to be linked to obtain the first complex.
  • obtaining the target polynucleotide may further include performing a first modification on the 3' end of the first complex and/or performing a second modification on the 5' end of the first complex, and then covalently linking the polynucleotide to be linked to obtain the target polynucleotide.
  • obtaining the target polynucleotide may further include performing a first modification on the 3' end of the second polynucleotide and/or performing a second modification on the 5' end of the second polynucleotide, and then covalently linking the polynucleotide to the first complex, and then obtaining the first complex.
  • obtaining the first complex further comprises performing a first modification on the 3' end of the polynucleotide to be connected.
  • obtaining the first complex further comprises performing a second modification on the 5' end of the polynucleotide to be connected.
  • obtaining the first complex further comprises performing a first modification on the 3' end of the first polynucleotide.
  • obtaining the first complex further comprises performing a second modification on the 5' end of the first polynucleotide.
  • the first modification and/or the second modification may be a hydroxylation modification or a phosphorylation modification.
  • the phosphorylation modification may be performed using T4PNK enzyme.
  • obtaining the target polynucleotide further includes performing a first modification on the 3' end of the first complex.
  • obtaining the target polynucleotide further includes performing a second modification on the 5' end of the first complex.
  • obtaining the target polynucleotide further includes performing a first modification on the 3' end of the second polynucleotide.
  • obtaining the target polynucleotide further includes performing a second modification on the 5' end of the second polynucleotide.
  • the first modification and/or the second modification can be a hydroxylation modification or a phosphorylation modification.
  • the phosphorylation modification can be performed using T4PNK enzyme.
  • step A.2.1.1 of obtaining the first complex may further include covalently linking the first polynucleotide to the polynucleotide to be linked in the presence of a splint oligonucleotide to obtain the first complex.
  • step A.2.1.1 it can further include obtaining a first complex in the presence of a ligase, wherein the ligase can be a double-stranded ligase, for example, the double-stranded ligase can be T4 RNA ligase 2.
  • the ligase can be a double-stranded ligase, for example, the double-stranded ligase can be T4 RNA ligase 2.
  • step A.2.1.2 it can further include obtaining the target polynucleotide in the presence of a ligase, wherein the ligase can be a single-stranded ligase, for example, the single-stranded ligase can be T4 RNA ligase 1.
  • the ligase can be a single-stranded ligase, for example, the single-stranded ligase can be T4 RNA ligase 1.
  • step A2 of the method for preparing a target polynucleotide further comprises:
  • Step A.2.2.1 connecting the first polynucleotide to the second polynucleotide to obtain a second complex
  • Step A.2.2.2 Connect the polynucleotide to be connected with the second complex to obtain the target polynucleotide.
  • the second complex may refer to a product obtained by covalently linking the first polynucleotide and the second polynucleotide.
  • the 3' end of the first polynucleotide and the 5' end of the second polynucleotide may be covalently linked to obtain the second complex.
  • the ligating polynucleotide and/or the first polynucleotide and/or the second polynucleotide may be phosphorylated or hydroxylated.
  • phosphorylation modification or hydroxylation modification can be performed on the ligating polynucleotide and/or the first polynucleotide and/or the second polynucleotide.
  • obtaining the second complex further includes performing a first modification on the 3' end of the first polynucleotide and/or performing a second modification on the 5' end of the first polynucleotide, and then covalently linking it with the second polynucleotide to obtain the second complex.
  • obtaining the target polynucleotide further includes performing a first modification on the 3' end of the second complex and/or performing a second modification on the 5' end of the second complex, and then covalently linking with the nucleotide to be linked to obtain the target polynucleotide.
  • obtaining the second complex further includes performing a first modification on the 3' end of the first polynucleotide.
  • obtaining the second complex further includes performing a second modification on the 5' end of the first polynucleotide.
  • obtaining the second complex further includes performing a first modification on the 3' end of the second polynucleotide.
  • obtaining the second complex further includes performing a second modification on the 5' end of the second polynucleotide.
  • the first modification and/or the second modification may be a hydroxylation modification or a phosphorylation modification.
  • the phosphorylation modification may be performed using the T4PNK enzyme.
  • obtaining the target polynucleotide further includes performing a first modification on the 3' end of the polynucleotide to be connected.
  • obtaining the target polynucleotide further includes performing a second modification on the 5' end of the polynucleotide to be connected.
  • obtaining the target polynucleotide further includes performing a first modification on the 3' end of the second complex.
  • obtaining the target polynucleotide further includes performing a second modification on the 5' end of the second complex.
  • the first modification and/or the second modification can be a hydroxylation modification or a phosphorylation modification.
  • the phosphorylation modification can be performed using T4PNK enzyme.
  • step A.2.2.1. it can further include obtaining a second complex in the presence of a ligase, wherein the ligase can be a single-stranded ligase, for example, the single-stranded ligase can be T4 RNA ligase 1.
  • the ligase can be a single-stranded ligase, for example, the single-stranded ligase can be T4 RNA ligase 1.
  • step A.2.2.2. it can further include obtaining the target polynucleotide in the presence of a ligase, wherein the ligase can be a double-stranded ligase, for example, the double-stranded ligase can be T4 RNA ligase 2.
  • the ligase can be a double-stranded ligase, for example, the double-stranded ligase can be T4 RNA ligase 2.
  • the first polynucleotide and/or the second polynucleotide comprises one or more modified nucleotides.
  • first modified nucleotide is present in the first polynucleotide and a second modified nucleotide is present in the second polynucleotide, wherein the distance of the first modified nucleotide relative to the 3' end of the first polynucleotide on the first polynucleotide is equal to the distance of the second modified nucleotide relative to the 3' end of the second polynucleotide on the second polynucleotide.
  • the modified nucleotides may include base-modified nucleotides, sugar-modified nucleotides, or phosphate-modified nucleotides, or a combination thereof.
  • RNA ligase polypeptide is a prokaryotic RNA ligase, a prokaryotic RNA ligase variant, or a functional fragment thereof.
  • RNA ligase polypeptide is a bacterial RNA ligase, a bacterial RNA ligase variant, or a functional fragment thereof.
  • RNA ligase polypeptide is a viral RNA ligase, a viral RNA ligase variant, or a functional fragment thereof.
  • RNA ligase polypeptide is T4 RNA ligase, a variant thereof, or a functional fragment thereof.
  • RNA ligase polypeptide is T4 RNA ligase.
  • RNA ligase polypeptide is T4 RNA ligase 1.
  • capping enzyme is a fusion polypeptide comprising a polynucleotide binding polypeptide fused to a polynucleotide ligase polypeptide.
  • the DNA double-strand binding domain includes Sso7d and/or NF-kappaB p50;
  • the RNA/DNA complex chain binding domain includes one or more of ScFV, RNaseH1 (D210N), and HBD of the monoclonal antibody S9.6;
  • RNA single-strand binding domains include: Sso7d, PKR, TRBP, PACT, Staufen, NFAR1, NFAR2, SPNR, RHA, NREBP, Kanadaptin, HYL1 Hyponastic leaves, ADAR1, ADAR2, ADAR3, TENR, RNaseIII, Dicer, and RDE-4.
  • polynucleotide binding polypeptide comprises one or more of Sso7d, NF-kappaB p50, ScFV of monoclonal antibody S9.6, RNaseH1 (D210N), HBD, PKR, TRBP, PACT, Staufen, NFAR1, NFAR2, SPNR, RHA, NREBP, Kanadaptin, HYL1Hyponastic leaves, ADAR1, ADAR2, ADAR3, TENR, RNaseIII, Dicer, and RDE-4.
  • the bases of the nucleotides of the uncapped polynucleotide are independently selected for each position from adenine, uracil, guanine or cytosine, or an analog of adenine, uracil, guanine or cytosine,
  • nucleotide modified base can be selected from xanthine, allylaminouracil, allylaminothymidine, hypoxanthine, dioxyadenine, dioxycytosine, dioxyguanine, dioxyuracil, 6-chloropurine nucleoside, N6-methyladenine, methylpseudouracil, 2-thiocytosine, 2-thiouracil, 5-methyluracil, 4-thiothymidine, 4-thiouracil, 5,6-dihydro-5-methyluracil, 5,6-dihydrouracil, 5-[(3-indolyl)propionamide-N-allyl]uracil, 5-aminoallylcytosine, 5-aminoallyluracil, 5-bromouracil, 5-bromocytosine, 5-carboxycytosine, 5-carboxyuracil, 5-carboxyuracil, 5-fluorouracil, 5-formylcytosine, 5-formyl
  • R3 is selected from guanine, adenine, cytosine, uracil, a guanine analog, an adenine analog, a cytosine analog, and a uracil analog;
  • R 4 is (N 1 p) x N 2 , wherein N 1 and N 2 are ribonucleosides, and N 1 and N 2 are the same or different;
  • each position is independently a phosphate group, a phosphorothioate, a phosphorodithioate, an alkylphosphonic acid, an arylphosphonic acid, or an N-phosphoramide bond;
  • X is an integer from 0 to 8, wherein if X ⁇ 2, the ribonucleosides N1 in ( N1p )x are identical to or different from each other;
  • the R 1 and R 2 groups are independently selected from -O-alkyl, halogen, acetylamino (AcNH), hydrogen or hydroxy.
  • sugars in N1 and N2 are independently selected from ribose and deoxyribose for each position and may contain modifications including 2'-O-alkyl, 2'-O-methoxyethyl, 2'-O allyl, 2'-O alkylamine, 2'-fluororibose, and 2'-deoxyribose;
  • the bases in N1 and N2 are independently selected for each position from adenine, uracil, guanine or cytosine, or analogs of adenine, uracil, guanine or cytosine, and the nucleotide modified base may be selected from xanthine, allylaminouracil, allylaminothymidine, hypoxanthine, dioxyadenine, dioxycytosine, dioxyguanine, dioxyuracil, 6-chloropurine nucleoside, N6-methyladenine, methylpseudouracil, 2-thiocytosine, 2-thiouracil, 5-methyluracil, 4-thiothymidine, 4-thiouracil, 5,6-dihydro-5-methyl ...
  • Dihydrouracil 5-[(3-indolyl)propionamido-N-allyl]uracil, 5-aminoallylcytosine, 5-aminoallyluracil, 5-bromouracil, 5-bromocytosine, 5-carboxycytosine, 5-carboxyuracil, 5-fluorouracil, 5-formylcytosine, 5-formyluracil, 5-hydroxycytosine, 5-hydroxymethylcytosine, 5-hydroxymethyluracil, 5-hydroxyuracil, 5-iodocytosine, 5-iodouracil, 5-methoxycytosine, 5-methoxyuracil, 5-methylcytosine, 5-methyluracil , 5-propynylaminocytosine, 5-propynylaminouracil, 5-propynylcytosine, 5-propynyluracil, 6-azacytosine, 6-azauracil, 6-chloropurine, 6-thioguanine, 7-deazaadenine,
  • R 5 and R 6 groups are independently selected from O-alkyl (O-methyl), halogen, label, hydrogen or hydroxyl;
  • X1 and X2 are bases, and X1 and X2 may be the same as or different from each other.
  • step S2 The method according to embodiment 41, wherein the reaction temperature of step S2 is 25°C to 60°C.
  • step S1 further comprises adenosine triphosphate.
  • step S1 further comprises a DNA/RNA buffer.
  • step S1 further comprises a DNase and/or RNase inhibitor.
  • a capping enzyme for capping a polynucleotide wherein the capping enzyme is a fusion polypeptide comprising a polynucleotide binding polypeptide fused to a polynucleotide ligase polypeptide.
  • the capping enzyme of embodiment 47, wherein the DNA ligase polypeptide is a viral DNA ligase, a viral DNA ligase variant, or a functional fragment thereof.
  • RNA ligase polypeptide is a prokaryotic RNA ligase, a prokaryotic RNA ligase variant, or a functional fragment thereof.
  • RNA ligase polypeptide is a bacterial RNA ligase, a bacterial RNA ligase variant, or a functional fragment thereof.
  • RNA ligase polypeptide is a viral RNA ligase, a viral RNA ligase variant, or a functional fragment thereof.
  • RNA ligase polypeptide is T4 RNA ligase, a variant thereof, or a functional fragment thereof.
  • RNA ligase polypeptide is T4 RNA ligase.
  • RNA ligase polypeptide is T4 RNA ligase 1.
  • polynucleotide binding polypeptide is selected from one or more of a DNA double-strand binding domain, a DNA single-strand binding domain, an RNA/DNA composite strand binding domain, an RNA single-strand or RNA double-strand binding protein, wherein
  • DNA duplex binding domains include Sso7d and/or NF-kappaB p50;
  • the RNA/DNA complex chain binding domain includes one or more of ScFV, RNaseH1 (D210N), and HBD of the monoclonal antibody S9.6;
  • RNA single-strand binding domains include: Sso7d, PKR, TRBP, PACT, Staufen, NFAR1, NFAR2, SPNR, RHA, NREBP, Kanadaptin, HYL1 Hyponastic leaves, ADAR1, ADAR2, ADAR3, TENR, RNaseIII, Dicer, and RDE-4.
  • kits comprising the capping enzyme of embodiments 46-71.
  • RNA ligase for efficient sequence ligation, allowing RNA fragments to be joined quickly. Therefore, we selected RNA oligonucleotide sequences, added a phosphate group to the 5' end, and chemically synthesized them. Then, we enzymatically ligated them using RNA ligase 1 to add a cap structure.
  • a schematic diagram of the capping ligation is shown in Figure 1.
  • RNA ligase 1 derived from T4 phage and RM378 phage for capping test.
  • T4RL1 T4 RNA ligase 1
  • a 6 ⁇ His tag was added to the N-terminus of the above protein to construct a fusion protein.
  • a flexible amino acid sequence G4S linker (GGGGSGGGGSGGGGS, SEQ ID NO: 23) was added after the His tag for protein fusion expression.
  • RNA ligases 1 wild-type T4 RNA ligase 1, T4 RNA ligase 1 purchased from NEB, RM378 RNA ligase 1, and Sso7d-T4RL1 to compare their performance. Because different enzymes have different optimal reaction temperatures, a temperature gradient was established to screen for the optimal capping temperature for each enzyme.
  • RNA and cap analogs were tested whether wild-type T4 RNA ligase 1 could ligate RNA and cap analogs.
  • the oligonucleotide (oligo) sequences used are shown in Table 1.
  • a phosphate group was added to the 5' end of Oligo 1, allowing the ligase to form a ligation with the -OH group at the 3' end of the cap structure.
  • the addition of a phosphate group to the 3' end of Oligo 1 prevented self-ligation of the oligonucleotide.
  • the cap analog, LzCap@AG(3'Acm) was selected. Its molecular structure is shown below. We attempted to ligate it to the 5' end of the oligo using an enzymatic reaction.
  • the ligation system is shown in Table 2. All components were mixed in a centrifuge tube and evenly divided into several tubes. Incubate at different temperatures (25°C, 37°C, 50°C, and 60°C) for 1 hour. The reaction was terminated with 2 ⁇ RNA loading buffer and denatured at 60°C for 5 minutes. Analysis was performed by Urea-PAGE gel electrophoresis. The final concentration of wild-type T4 RNA ligase 1 was 6 ⁇ M.
  • T4 RNA ligase 1s purchased from NEB and Sso7d-T4RL1
  • the experimental method was the same as in Example 3, except that wild-type T4 RNA ligase 1 was replaced with T4 RNA ligase 1 purchased from NEB (NEB, M0204S) and Sso7d-T4 RNA ligase 1.
  • NEB-T4RL1 was purchased commercially at a concentration of 10,000 units/ml. According to the instructions, 10 units were added to a 10 ⁇ l reaction system.
  • the Urea-PAGE detection results are shown in Figure 4.
  • the grayscale analysis of the image was performed using Image J software, and the connection efficiency was calculated as Production grayscale value/(Oligo 1 grayscale value + Production grayscale value). The results are shown in Table 3.
  • the optimal temperature for capping of Sso7d-T4RL1 was 25°C, and the highest ligation efficiency was 32.4%, which was most suitable for capping reaction.
  • the ligation system is shown in Table 5. All components were mixed in a centrifuge tube and evenly divided into several tubes. The mixture was incubated at different temperatures (25°C, 37°C, 45°C, 50°C, and 60°C) for 1 hour. The reaction was terminated with 2 ⁇ RNA loading buffer and denatured at 60°C for 5 minutes. The samples were analyzed by Urea-PAGE gel electrophoresis.
  • the Urea-PAGE detection results are shown in Figure 6.
  • the grayscale analysis of the image was performed using Image J software, and the connection efficiency was calculated as Production grayscale value/(Oligo 2 grayscale value + Production grayscale value). The results are shown in Table 6.
  • Ligation efficiency analysis revealed that when ligating conventional oligonucleotides, Sso7d-T4RL1 had an optimal temperature of 37°C, with a maximum ligation efficiency of 87.6%. Activity gradually decreased with increasing temperature. NEB-T4RL1 had an optimal temperature of 43°C, with a maximum ligation efficiency of 86.7%. However, activity decreased rapidly with increasing temperature, reaching only 40% of Sso7d-T4RL1 at 60°C. T4RL1 also had an optimal temperature of 43°C, with a maximum ligation efficiency of only 34.6%. RM378RL1 had an optimal temperature of 60°C, with a maximum ligation efficiency of 95.7%.
  • RM378RL1 performed best, achieving the highest ligation efficiency, followed by Sso7d-T4RL1 and NEB-T4RL1. These two enzymes exhibited similar ligation efficiencies at their optimal temperatures. Wild-type T4RL1 performed the worst, achieving the lowest ligation efficiency.
  • RNA ligase 1 enzymes have different preferences when connecting conventional oligonucleotides and connecting cap analogs.
  • Sso7d-T4RL1 is most suitable for connecting cap analogs
  • RM378 is most suitable for connecting conventional oligonucleotides.
  • Sso7d-T4RL1 was selected as the enzyme for the capping reaction, with a 1-hour incubation at 25°C.
  • the oligo sequences used are shown in Table 7.
  • Oligo 4 was modified with a phosphate group at the 5' end to facilitate ligation to the cap, and a biotin group was added at the 3' end to prevent self-ligation.
  • the final oligo concentration was maintained at 10 ⁇ M, and a gradient of 0-1000 ⁇ M was used for the final concentration of LzCap @ AG (3′Acm).
  • the reaction conditions are shown in Table 8.
  • the Urea-PAGE detection results are shown in Figure 7.
  • the grayscale analysis of the image was performed using Image J software, and the connection efficiency was calculated as Production grayscale value/(Oligo 4 grayscale value + Production grayscale value).
  • the results are shown in Table 9.
  • the results were analyzed using Prism 8 software, and the analysis curve was drawn, as shown in Figure 8.
  • the ligation efficiency has passed the inflection point when the final concentration of the cap analog is below 200 ⁇ M. Above 200 ⁇ M, the ligation efficiency slowly increases with the increase in the cap analog concentration, reaching a maximum value above 500 ⁇ M. The optimal ligation effect can be achieved when the final concentration of the cap analog is above 500 ⁇ M and the oligo concentration is 10 ⁇ M in the capping enzymatic reaction.
  • the ligation efficiency of this experiment was significantly higher than the previous one when the final LzCap concentration was 750 ⁇ M. Since all other conditions except the oligo sequence and modification used were the same, it is speculated that the difference in the two ligation efficiencies is due to the different oligo sequences and structures used.
  • the ligation system is shown in Table 11. Incubate at 25°C for 1 h. Terminate the reaction with 2 ⁇ RNA loading buffer and denature at 60°C for 5 min. Analyze by Urea-PAGE gel electrophoresis.
  • the Urea-PAGE test results are shown in Figure 8. Based on the size of the electrophoretic bands, it can be determined that different cap structure analogs and different receptor sequences can be successfully connected.
  • the connection efficiency of Oligo 4 is significantly higher than that of Oligo 1. Grayscale analysis of the images was performed using Image J software, and the connection efficiency was calculated as Production grayscale value/(Oligo grayscale value + Production grayscale value). The results are shown in Table 12. Oligo 4 can achieve a connection efficiency of approximately 90% for different cap analogs, while the connection efficiency of Oligo 1 is only about 30%. Different 5' bases have a significant impact on the connection efficiency, and the connection efficiency of the base G is higher than that of the base A.
  • Luciferase NanoBiT can be separated into two fragments: a short HiBiT fragment and a long LgBiT fragment. Separately, the two fragments lack luciferase activity. When mixed, they spontaneously assemble into the complete luciferase, producing fluorescence in the presence of substrate. We leveraged this property to validate our capping method and synthesize HiBiT mRNA. When the HiBiT mRNA is properly translated, the HiBiT fragment is obtained. Detectable chemiluminescence in the presence of LgBiT protein and substrate confirms that we have synthesized fully functional HiBiT mRNA.
  • the HiBiT sequence was split into three segments, namely Oligo H1 (28nt), Oligo H2 (38nt), and Oligo H3 (39nt).
  • Oligo H1 28nt
  • Oligo H2 38nt
  • Oligo H3 39nt
  • a DNA sequence that is reverse complementary to the above Oligo 1 and Oligo 2 sequences was designed and named adaptor H. The required sequence is shown in Table 13.
  • the mixture prepared according to the above system was placed in a metal bath and incubated at 25°C for 60 minutes. After completion, the reaction was terminated with 2 ⁇ RNA loading buffer, denatured at 60°C for 5 minutes, and analyzed by Urea-PAGE gel electrophoresis. The Urea-PAGE results are shown in Figure 10.
  • the next step is tailing ligation, using T4 RNA ligase 1 to connect Oligo H2 and Oligo H3 together.
  • the ligation system is shown in Table 15:
  • the mixture was incubated at 25°C for 2 h, terminated with 2 ⁇ RNA loading buffer, and denatured at 60°C for 5 min. The resulting mixture was then analyzed by Urea-PAGE gel electrophoresis.
  • the Urea-PAGE test results are shown in Figure 12, which show that Oligo H2 and Oligo H3 can be effectively connected.
  • the grayscale analysis of the image was performed using Image J software, and the connection efficiency was calculated as Production grayscale value/(Oligo grayscale value + Production grayscale value), which is about 50%.
  • T4PNK was used to add a phosphate group to the 5' end of the Oligo H2-Oligo H3 ligation product and remove the phosphate group at the 3' end of LzCap @ AG(3'Acm)-Oligo 1.
  • the reaction system is shown in Table 16:
  • the above system was prepared into a mixed solution and reacted at 37°C for 30 minutes. After the reaction, it was transferred to 65°C for 15 minutes to inactivate the T4PNK enzyme. The entire reaction mixture was put into the next ligation system.
  • the mixture prepared according to the above system was placed in a PCR instrument and programmed to repeat 5 cycles of (52°C for 30 seconds, 37°C for 5 minutes). After the reaction, 1/10 volume of DNase I and 10 ⁇ buffer were added, and the cells were incubated at 37°C for 15 minutes. The reaction was terminated with 2 ⁇ RNA loading buffer and denatured at 60°C for 5 minutes. Urea-PAGE gel electrophoresis was then used for analysis. The results, as shown in Figure 13, indicate that the target product of approximately 108 nt was obtained, and the HiBiT mRNA target band was recovered.
  • the recovered product was subjected to mass spectrometry, and the results are shown in Figure 14.
  • the predicted molecular weight of the recovered product was 35575.14, and the detected molecular weight was 35582.9, with a difference of 0.22 ⁇ , indicating that the recovered product is the desired Hibit mRNA.
  • the recovered product was subjected to Urea-PAGE, and the results are shown in Figure 15.
  • the recovered product has a single band and a purity exceeding 95%. Subsequently, cells will be transfected for activity testing.
  • oligonucleotides in the aforementioned examples can be synthesized using the method in this example.
  • RNA synthesis uses the solid-phase phosphoramidite method, which involves a four-step cycle of deprotection, coupling reaction, capping reaction, and oxidation reaction (each cycle has a synthesis efficiency ⁇ 98%). Synthesis is performed at room temperature according to set parameters. The synthesis equipment is sealed, the synthesis humidity is ⁇ 30%, and the temperature is 15-25°C. The first base at the 3' end of the oligonucleotide is bound to the solid-phase support CPG (Controlled Pore Glass). Synthesis then proceeds from 3' to 5', with adjacent nucleotides connected by 3' to 5' phosphate bonds.
  • CPG Controlled Pore Glass
  • Each cycle requires the highly efficient and chemically active nucleotide 3'-phosphite tetrazolium, which, after oxidation, becomes a stable pentavalent phosphate triester, thus forming a more stable structure.
  • a nucleic acid chain with a specific sequence is obtained, which is the oligonucleotide.
  • the deprotecting agent is a 3% (w/v) dichloroacetic acid solution in dichloromethane, used in an amount of 200 ⁇ l each time;
  • the activating coupling agent is a 0.3M ethylmercaptotetrazole solution in acetonitrile, used in an amount of 60 ⁇ l each time;
  • the capping agent CAPA is a 10% (v/v) acetic anhydride solution in tetrahydrofuran, used in an amount of 120 ⁇ l each time, and the capping agent CAPB is a 16% (v/v) 1-methylimidazole solution in tetrahydrofuran, used in an amount of 120 ⁇ l each time.
  • RNA nucleic acid was then concentrated to a dry powder state.
  • DMSO dimethyl sulfoxide
  • 125 ⁇ l of triethylamine trihydrofluoride was added and heated to 65°C for 150 minutes.
  • the white solid was dissolved in enzyme-free sterile water and purified using high-performance liquid chromatography (HPLC) with a mobile phase consisting of acetonitrile and a 0.1 M triethylamine carbonate (TEAB) solution.
  • HPLC high-performance liquid chromatography
  • a mobile phase consisting of acetonitrile and a 0.1 M triethylamine carbonate (TEAB) solution.
  • TEAB triethylamine carbonate

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Abstract

L'invention concerne un procédé de coiffage d'un polynucléotide, comprenant : S1, la fourniture d'une composition, qui comprend au moins : un polynucléotide non coiffé, une enzyme de coiffage et un analogue de structure de coiffe, l'enzyme de coiffage comprenant un polypeptide de ligase de polynucléotide ; et S2, la formation d'un polynucléotide coiffé sur la base de la composition de S1.
PCT/CN2025/079459 2024-02-28 2025-02-27 Enzyme pour coiffage d'arn et son système de réaction Pending WO2025180432A1 (fr)

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WO2023085955A1 (fr) * 2021-11-09 2023-05-19 Victoria Link Limited Enzymes de ligase d'arn et procédés de préparation et d'utilisation de ces enzymes
CN116218953A (zh) * 2023-01-03 2023-06-06 南京诺唯赞生物科技股份有限公司 一种连接核酸片段与接头的方法
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CN108129571A (zh) * 2017-12-25 2018-06-08 上海捷瑞生物工程有限公司 Taq DNA连接酶融合蛋白
WO2023085955A1 (fr) * 2021-11-09 2023-05-19 Victoria Link Limited Enzymes de ligase d'arn et procédés de préparation et d'utilisation de ces enzymes
WO2023141474A1 (fr) * 2022-01-18 2023-07-27 The Broad Institute, Inc. Arnm poly-queue et poly-coiffe et ses utilisations
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