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WO2023085955A1 - Enzymes de ligase d'arn et procédés de préparation et d'utilisation de ces enzymes - Google Patents

Enzymes de ligase d'arn et procédés de préparation et d'utilisation de ces enzymes Download PDF

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Publication number
WO2023085955A1
WO2023085955A1 PCT/NZ2022/050140 NZ2022050140W WO2023085955A1 WO 2023085955 A1 WO2023085955 A1 WO 2023085955A1 NZ 2022050140 W NZ2022050140 W NZ 2022050140W WO 2023085955 A1 WO2023085955 A1 WO 2023085955A1
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polypeptide
rna ligase
rna
seq
amino acid
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Wayne Patrick
Tifany Oulavallickal
Vickery Laurence Arcus
Joanna Hicks
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Victoria Link Ltd
WaikatoLink Ltd
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Victoria Link Ltd
WaikatoLink Ltd
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Publication of WO2023085955A1 publication Critical patent/WO2023085955A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1093—General methods of preparing gene libraries, not provided for in other subgroups
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/25—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving enzymes not classifiable in groups C12Q1/26 - C12Q1/66
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y605/00—Ligases forming phosphoric ester bonds (6.5)
    • C12Y605/01—Ligases forming phosphoric ester bonds (6.5) forming phosphoric ester bonds (6.5.1)
    • C12Y605/01003—RNA ligase (ATP) (6.5.1.3)
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli

Definitions

  • the present disclosure relates generally to RNA ligase enzymes, and their preparation and use. More specifically, the present disclosure relates to thermostable RNA ligase enzymes, including variant enzymes, as well as means for preparing and using these.
  • Micro RNAs are short molecules of RNA ( ⁇ 22-nucleotides) that are present in intra- and extra-cellular environments. There has been increasing awareness of the important roles of miRNA molecules in maintaining normal cellular processes. It has been recognised that miRNA populations show characteristic changes in different disease types. As such, the identification and analysis of miRNA populations has become increasingly important.
  • miRNA libraries One important step for sequencing miRNA populations is the preparation of miRNA libraries. These methods can be dependent on the use of an RNA ligase, or can be ligase-free methods, for example, where polyadenylating enzymes are used. In both cases, the enzyme-dependent steps in library preparation are affected by bias towards particular miRNA types. This leads to misrepresentation of the miRNA population when the library is sequenced.
  • the present disclosure seeks to address these needs or at least to provide the public with a useful alternative.
  • RNA ligase enzymes including variant enzymes, with significantly reduced bias rates. These enzymes are highly advantageous and have unexpected and surprising activities in relation to nucleotide substrates.
  • the present disclosure encompasses RNA ligase polypeptides, polypeptide fusion molecules comprising these polypeptides (e.g., tagged polypeptides), as well as polynucleotides encoding these polypeptides, libraries comprising these polynucleotides, methods for producing these polypeptides, and methods for ligating polynucleotides using these polypeptides.
  • Isolated polypeptides and polynucleotides are specifically noted.
  • Non-naturally occurring polypeptides and polynucleotides are also noted.
  • the present disclosure encompasses an isolated RNA ligase polypeptide: (i) comprising or consisting of a variant amino acid sequence of SEQ ID NO: 3, wherein the variant amino acid sequence includes an alanine or glycine residue substituted for a conserved lysine residue at motif I (SEQ ID NO: 43) and/or motif V (SEQ ID NO: 44), and optionally at least one other sequence variation.
  • the RNA ligase polypeptide shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to an amino acid sequence of SEQ ID NO: 3.
  • the variant amino acid sequence includes an alanine or glycine residue substituted for a conserved lysine residue at motif V and optionally at least one other sequence variation.
  • RNA ligase polypeptide comprises an amino acid sequence of SEQ ID NO: 8, or a variant amino acid sequence thereof.
  • RNA ligase polypeptide comprises the amino acid sequence of SEQ ID NO: 8.
  • RNA ligase polypeptide consists of the amino acid sequence of SEQ ID NO: 8.
  • RNA ligase polypeptide shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to an amino acid sequence of SEQ ID NO: 8.
  • the present disclosure encompasses an isolated RNA ligase polynucleotide encoding the RNA ligase polypeptide of a preceding aspect.
  • RNA polynucleotide comprises the nucleotide sequence of SEQ ID NO: 13.
  • RNA ligase polynucleotide comprises the nucleotide sequence of SEQ ID NO: 18.
  • RNA ligase polynucleotide consists of the nucleotide sequence of SEQ ID NO: 13.
  • RNA ligase polynucleotide consists of the nucleotide sequence of SEQ ID NO: 18.
  • the present disclosure encompasses:
  • a polypeptide fusion molecule comprising the RNA ligase polypeptide of a preceding aspect.
  • a nucleic acid construct which: (i) encodes an RNA ligase polypeptide of a preceding aspect; (ii) encodes a polypeptide fusion molecule of a preceding aspect.; (iii) comprises an RNA ligase polynucleotide of a preceding aspect; or (iv) comprises the fusionencoding polynucleotide of a preceding aspect.
  • a library of nucleic acid constructs wherein a nucleic acid construct in the library: (i) encodes an RNA ligase polypeptide of a preceding aspect; (ii) encodes a polypeptide fusion molecule of a preceding aspect.; (iii) comprises an RNA ligase polynucleotide of a preceding aspect; or (iv) comprises the fusion-encoding polynucleotide of a preceding aspect.
  • a host cell which: (i) expresses an RNA ligase polypeptide of a preceding aspect; (ii) expresses a polypeptide fusion molecule of a preceding aspect; (iii) comprises an RNA ligase polynucleotide of a preceding aspect; (iv) comprises a fusion-encoding polynucleotide of a preceding aspect; (v) comprises a nucleic acid construct of a preceding aspect; or (vi) comprises a library of nucleic acid constructs of a preceding aspect
  • a composition comprising: (i) an RNA ligase polypeptide of a preceding aspect; (ii) a polypeptide fusion molecule of a preceding aspect; (iii) an RNA ligase polynucleotide of a preceding aspect; (iv) a fusion-encoding polynucleotide of a preceding aspect; (v) a nucleic acid construct of a preceding aspect; (vi) a library of nucleic acid constructs of a preceding aspect; or (vii) a host cell of a preceding aspect.
  • a system or kit comprising one or more of: (i) an RNA ligase polypeptide of a preceding aspect; (ii) a polypeptide fusion molecule of a preceding aspect; (iii) an RNA ligase polynucleotide of a preceding aspect; (iv) a fusion-encoding polynucleotide of a preceding aspect; (v) a nucleic acid construct of a preceding aspect; (vi) a library of nucleic acid constructs of a preceding aspect; (vii) a host cell of a preceding aspect; or (viii) a composition of a preceding aspect.
  • a method for producing the RNA ligase polypeptide of a preceding aspect comprising incubating a cell free expression system with the polynucleotide of a preceding aspect, or the nucleic acid construct of a preceding aspect, or the library of a preceding aspect, under conditions to produce the polypeptide.
  • a method for producing the RNA ligase polypeptide of a preceding aspect comprising culturing the host cell of a preceding aspect under conditions to produce the polypeptide.
  • the present disclosure encompasses a combination of RNA ligase polypeptides, wherein the combination comprises: (i) at least one RNA ligase polypeptide of a preceding aspect, and (ii) at least one additional RNA ligase polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-2, 3, 4-7, 9-11, or 37-42, and any variant amino acid sequence thereof.
  • RNA ligase polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 3.
  • the combination includes an RNA ligase polypeptide comprising the amino acid sequence of SEQ ID NO: 8.
  • the combination includes an RNA ligase polypeptide consisting of the amino acid sequence of SEQ ID NO: 8.
  • RNA ligase polypeptide comprising an amino acid sequence of SEQ ID NO: 1, or comprising a variant amino acid sequence sharing at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 1.
  • RNA ligase polypeptide comprising an amino acid sequence of any one of SEQ ID NO: 9-11, or comprising a variant amino acid sequence sharing at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to any one of SEQ ID NO: 9-11.
  • variant amino acid sequence includes an alanine or glycine residue substituted for a conserved lysine residue at motif I (SEQ ID NO: 43) and/or motif V (SEQ ID NO: 44), and optionally at least one other sequence variation.
  • the present disclosure encompasses a method for providing RNA ligase activity in a reaction mixture, the method comprising incubating at least one RNA ligase polypeptide of a preceding aspect, or the polypeptide fusion molecule of a preceding aspect, in the reaction mixture under conditions to allow RNA ligase activity of the polypeptide.
  • RNA ligase activity allows for RNA sequencing.
  • RNA ligase activity allows RNA library preparation.
  • RNA ligase activity allows for RNA capping.
  • RNA ligase activity allows for RNA circularisation.
  • RNA ligase activity allows for one or more of: synthetic nucleotide construction; filling nicks in double stranded nucleic acids; rapid amplification of cDNA ends (RACE); or 3 '-end nucleic acid labelling.
  • the present disclosure encompasses:
  • a method for capping an RNA molecule comprising incubating the RNA molecule with at least one RNA ligase polypeptide of a preceding aspect, or the polypeptide fusion molecule of a preceding aspect, under conditions to allow capping of the RNA molecule.
  • a method for circularising an RNA molecule comprising incubating the RNA molecule with at least one RNA ligase polypeptide of a preceding aspect, or the polypeptide fusion molecule of a preceding aspect, under conditions to allow circularising of the RNA molecule.
  • a method for sequencing an miRNA molecule comprising incubating the miRNA molecule with at least one RNA ligase polypeptide of a preceding aspect, or the polypeptide fusion molecule of a preceding aspect, under conditions to allow sequencing of the miRNA molecule.
  • An RNA capping reaction mixture comprising at least one RNA ligase polypeptide of a preceding aspect or the polypeptide fusion molecule of a preceding aspect.
  • An RNA circularisation reaction mixture comprising at least one RNA ligase polypeptide of a preceding aspect or the polypeptide fusion molecule of a preceding aspect.
  • RNA sequencing reaction mixture comprising at least one RNA ligase polypeptide of a preceding aspect or the polypeptide fusion molecule of a preceding aspect.
  • RNA library preparation reaction mixture comprising at least one RNA ligase polypeptide of a preceding aspect or the polypeptide fusion molecule of a preceding aspect.
  • the present disclosure further encompasses a method for the production of an miRNA library, the method comprising the use of at least one polypeptide of a preceding aspect, the polypeptide fusion molecule of a preceding aspect, at least one polynucleotide of a preceding aspect, the nucleic acid construct of a preceding aspect, the host cell of a preceding aspect, the library of a preceding aspect, or the composition of a preceding aspect.
  • the present disclosure encompasses:
  • a method for preparing a library of miRNA molecules comprising incubating (i) a 3’ adapter and one or more miRNA molecules, and/or (ii) a 5’ adapter and one or more miRNA-3 ’ adapter molecules, with at least one RNA ligase polypeptide of a preceding aspect, or the polypeptide fusion molecule of a preceding aspect, under conditions to allow RNA ligase activity of the polypeptide, and thereby allow for ligation of the 3’ adapter to the one or more miRNA molecules and/or allow for ligation of the 5’ adapter to the one or more miRNA-3’ adapter molecules.
  • the present disclosure encompasses a non-naturally occurring RNA ligase polypeptide comprising: (i) an amino acid sequence of SEQ ID NO: 3, or (ii) a variant amino acid sequence thereof, wherein the variant amino acid sequence includes an alanine or glycine residue substituted for a conserved lysine residue at motif I (SEQ ID NO: 43) and/or motif V (SEQ ID NO: 44), and optionally at least one other sequence variation.
  • the non-naturally occurring RNA ligase polypeptide shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to an amino acid sequence of SEQ ID NO: 3.
  • the non-naturally occurring RNA ligase polypeptide includes an alanine or glycine residue substituted for a conserved lysine residue at motif V and optionally at least one other sequence variation.
  • the non-naturally occurring RNA ligase polypeptide comprises an amino acid sequence of SEQ ID NO: 8, or a variant amino acid sequence thereof.
  • the non-naturally occurring RNA ligase polypeptide comprises the amino acid sequence of SEQ ID NO: 8.
  • RNA ligase polypeptide consists of the amino acid sequence of SEQ ID NO: 8.
  • RNA ligase polypeptide shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to an amino acid sequence of SEQ ID NO: 8.
  • the present disclosure encompasses a non-naturally occurring RNA ligase polynucleotide encoding the non-naturally occurring RNA ligase polypeptide of a preceding aspect.
  • the non-naturally occurring RNA polynucleotide comprises the nucleotide sequence of SEQ ID NO: 13.
  • the non-naturally occurring RNA ligase polynucleotide comprises the nucleotide sequence of SEQ ID NO: 18.
  • RNA ligase polynucleotide consists of the nucleotide sequence of SEQ ID NO: 13.
  • RNA ligase polynucleotide consists of the nucleotide sequence of SEQ ID NO: 18.
  • the present disclosure encompasses: [0075] A polypeptide fusion molecule comprising the non-naturally occurring RNA ligase polypeptide of a preceding aspect.
  • a nucleic acid construct which: (i) encodes a non-naturally occurring RNA ligase polypeptide of a preceding aspect; (ii) encodes a polypeptide fusion molecule of a preceding aspect; (iii) comprises a non-naturally occurring RNA ligase polynucleotide of a preceding aspect; or (iv) comprises the fusion-encoding polynucleotide of a preceding aspect.
  • a library of nucleic acid constructs wherein a nucleic acid construct in the library: (i) encodes a non-naturally occurring RNA ligase polypeptide of a preceding aspect;
  • (ii) encodes a polypeptide fusion molecule of a preceding aspect; (iii) comprises a non-naturally occurring RNA ligase polynucleotide of a preceding aspect; or (iv) comprises a fusionencoding polynucleotide of a preceding aspect.
  • a host cell which: (i) expresses a non-naturally occurring RNA ligase polypeptide of a preceding aspect; (ii) expresses a polypeptide fusion molecule of a preceding aspect; (iii) comprises a non-naturally occurring RNA ligase polynucleotide of a preceding aspect; (iv) comprises a fusion-encoding polynucleotide of a preceding aspect; (v) comprises a nucleic acid construct of a preceding aspect; or (vi) comprises a library of nucleic acid constructs of a preceding aspect.
  • a composition comprising: (i) a non-naturally occurring RNA ligase polypeptide of a preceding aspect; (ii) a polypeptide fusion molecule of a preceding aspect;
  • RNA ligase polynucleotide of a preceding aspect (iii) a non-naturally occurring RNA ligase polynucleotide of a preceding aspect; (iv) a fusionencoding polynucleotide of a preceding aspect; (v) a nucleic acid construct of a preceding aspect; (vi) a library of nucleic acid constructs of a preceding aspect; or (vii) a host cell of a preceding aspect.
  • a system or kit comprising one or more of: (i) a non-naturally occurring RNA ligase polypeptide of a preceding aspect; (ii) a polypeptide fusion molecule of a preceding aspect; (iii) a non-naturally occurring RNA ligase polynucleotide of a preceding aspect; (iv) a polynucleotide of a preceding aspect; (v) a nucleic acid construct of a preceding aspect; (vi) a library of nucleic acid constructs of a preceding aspect; (vii) a host cell of a preceding aspect; or (viii) a composition of a preceding aspect.
  • a method for producing the non-naturally occurring RNA ligase polypeptide of a preceding aspect comprising incubating a cell free expression system with the polynucleotide of a preceding aspect, or the nucleic acid construct of a preceding aspect, or the library of a preceding aspect, under conditions to produce the polypeptide.
  • a method for producing the non-naturally occurring RNA ligase polypeptide of a preceding aspect comprising culturing the host cell of a preceding aspect under conditions to produce the polypeptide.
  • the present disclosure encompasses a combination of RNA ligase polypeptides, wherein the combination comprises: (i) at least one non-naturally occurring RNA ligase polypeptide of a preceding aspect, and (ii) at least one additional RNA ligase polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-2, 4-7, 9-11, or 37-42, and any variant amino acid sequence thereof.
  • the combination includes an RNA ligase polypeptide comprising the amino acid sequence of SEQ ID NO: 3.
  • the combination of includes an RNA ligase polypeptide comprising the amino acid sequence of SEQ ID NO: 8.
  • the combination includes an RNA ligase polypeptide consisting of the amino acid sequence of SEQ ID NO: 8.
  • the combination includes an RNA ligase polypeptide comprising an amino acid sequence of SEQ ID NO: 1, or comprising a variant amino acid sequence sharing at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 1.
  • the combination includes an RNA ligase polypeptide comprising an amino acid sequence of any one of SEQ ID NO: 9-11, or comprising a variant amino acid sequence sharing at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to any one of SEQ ID NO: 9-11.
  • the combination includes an alanine or glycine residue substituted for a conserved lysine residue at motif I (SEQ ID NO: 43) and/or motif V (SEQ ID NO: 44), and optionally at least one other sequence variation.
  • the present disclosure encompasses a method for providing RNA ligase activity in a reaction mixture, the method comprising incubating at least one non- naturally occurring RNA ligase polypeptide of a preceding aspect, or the polypeptide fusion molecule of a preceding aspect, in the reaction mixture under conditions to allow RNA ligase activity of the polypeptide.
  • RNA ligase activity allows for RNA sequencing.
  • RNA ligase activity allows RNA library preparation.
  • RNA ligase activity allows for RNA capping.
  • RNA ligase activity allows for RNA circularisation.
  • RNA ligase activity allows for one or more of: synthetic nucleotide construction; filling nicks in double stranded nucleic acids; rapid amplification of cDNA ends (RACE); or 3 '-end nucleic acid labelling.
  • the present disclosure encompasses:
  • a method for capping an RNA molecule comprising incubating the RNA molecule with at least one non-naturally occurring RNA ligase polypeptide of a preceding aspect, or the polypeptide fusion molecule of a preceding aspect, under conditions to allow capping of the RNA molecule.
  • a method for circularising an RNA molecule comprising incubating the RNA molecule with at least one non-naturally occurring RNA ligase polypeptide of a preceding aspect, or the polypeptide fusion molecule of a preceding aspect, under conditions to allow circularising of the RNA molecule.
  • a method for sequencing an miRNA molecule comprising incubating the miRNA molecule with at least one non-naturally occurring RNA ligase polypeptide of a preceding aspect, or the polypeptide fusion molecule of a preceding aspect, under conditions to allow sequencing of the miRNA molecule.
  • RNA capping reaction mixture comprising at least one non-naturally occurring RNA ligase polypeptide of a preceding aspect or the polypeptide fusion molecule of a preceding aspect.
  • RNA circularisation reaction mixture comprising at least one non-naturally occurring RNA ligase polypeptide of a preceding aspect or the polypeptide fusion molecule of a preceding aspect.
  • RNA sequencing reaction mixture comprising at least one non-naturally occurring RNA ligase polypeptide of a preceding aspect or the polypeptide fusion molecule of a preceding aspect.
  • RNA library preparation reaction mixture comprising at least one non- naturally occurring RNA ligase polypeptide of a preceding aspect or the polypeptide fusion molecule of a preceding aspect.
  • the present disclosure further encompasses a method for the production of an miRNA library, the method comprising the use of at least one non-naturally occurring polypeptide of a preceding aspect, the polypeptide fusion molecule of a preceding aspect, at least one polynucleotide of a preceding aspect, the nucleic acid construct of a preceding aspect, the host cell of a preceding aspect, the library of a preceding aspect, or the composition of a preceding aspect.
  • the present disclosure encompasses:
  • a method for preparing a library of miRNA molecules comprising incubating (i) a 3’ adapter and one or more miRNA molecules, and/or (ii) a 5’ adapter and one or more miRNA-3’ adapter molecules, with at least one non-naturally occurring RNA ligase polypeptide of a preceding aspect, or the polypeptide fusion molecule of a preceding aspect, under conditions to allow RNA ligase activity of the polypeptide, and thereby allow for ligation of the 3 ’ adapter to the one or more miRNA molecules and/or allow for ligation of the 5 ’ adapter to the one or more miRNA-3’ adapter molecules.
  • polypeptide and polynucleotide sequences have been noted in the preceding aspects, other polypeptide and polynucleotide sequences as disclosed herein (e.g., SEQ ID NO: 1-2, 4-6, 7, 9-11, 12, 14-17, 19-26) may be substituted for or added to the polypeptide and polynucleotide sequences in the preceding aspects, and such will be understood as defining further aspects that are encompassed by the present disclosure.
  • Figure 1 Schematic showing the standard workflow for miRNA library preparation. Included are four enzyme-mediated steps with clean-up and size selection steps between the enzyme-mediated reactions: (1) ligation of the 3’ adapter, (2) ligation of the 5’ adapter, (3) first strand DNA synthesis, and (4) library amplification by PCR.
  • Figure 2 Gel electrophoresis to analyse ligation of the RNA oligonucleotide named Oligo 1, as mediated by five thermostable RNA ligase enzymes.
  • the desired products (shown by a white box) comprise two ligated molecules of Oligo 1, with and without additional adenylation.
  • the Oligo 1 band in lane 1 shows the position of the unmodified oligonucleotide on the gel. Bands that appear smaller in size are classified as circularised oligonucleotide, and bands that are slightly larger than Oligo 1 are modified by adenylation. Appearing above the desired ligation products are concatemers made of more than two ligated oligonucleotides.
  • Figure 3A Ligation of synthetic oligonucleotides with melting temperatures of 38°C (Oligo A), 65°C (Oligo G) and >80°C (Oligo J) by PfuRnl, PpRnl, orAfRnl, acting at 75°C.
  • the desired products (shown by a white box) comprise two ligated molecules.
  • the unmodified oligonucleotides (with no ligase enzyme added) are shown in lanes 1, 2 and 3. Bands that appear smaller in size are classified as circularised oligonucleotide, and bands that are slightly larger than the unmodified oligonucleotides are modified by adenylation.
  • Figure 3B Ligation of synthetic oligonucleotides with melting temperatures of 38°C (Oligo A), 65°C (Oligo G) and >80°C (Oligo J) by MfRnl or TgRnl, acting at 75°C and with T4 RNA ligase (T4Rnl) at 25°C.
  • the desired products (shown by a white box) comprise two ligated molecules.
  • the unmodified oligonucleotides (with no ligase enzyme added) are shown in lanes 1, 2 and 3. Bands that appear smaller in size are classified as circularised oligonucleotide, and bands that are slightly larger than the unmodified oligonucleotides are modified by adenylation.
  • Figure 4A Schematics showing 3’ adapter ligation reactions between Oligol- 5’B (5’ blocked RNA) and AppSRl (3’ blocked DNA), or between Oligo 1 and AppSRl (3’ blocked DNA).
  • Figure 4B Results for 3’ adapter ligation reactions between Oligol-5’B (5’ blocked RNA) and AppSRl (3’ blocked DNA), and between Oligo 1 and AppSRl (3’ blocked DNA), catalysed by the PfuRnl K92A variant.
  • Figure 4C Results for 3’ adapter ligation reactions between Oligol-5’B (5’ blocked RNA) and AppSRl (3’ blocked DNA) with variants PfuRnl K92A, AfRnl K92A, PpRnl K92A, and AfRnl K96A.
  • Figure 5A Schematic showing 5’ adapter ligation reaction between Oligo 1- 5’B (RNA with a 5 ’-block due to the absence of a phosphate group) and an RNA molecule (SRI) that was blocked with an amino modifier at its 3’ end.
  • Figure 5B Results for 5’ adapter ligation reactions between Oligo 1-5’B (5’- blocked RNA) and SRI (3 ’blocked RNA) using PfuRnl and its variants (K238A and K238G), in the presence of ATP.
  • Figure 5C Results for 5’ adapter ligation reactions between Oligo 1-5’B and SRI using the mutated ligase variants PfuRnl K238G, MfRnl K243G, PpRnl K238G and AfRnl K242G.
  • Figure 6A Ligation of RNA oligonucleotides with different melting temperatures using PpRnl K238G. Reactions were performed at 50°C. Successful ligation is demonstrated by the appearance of bands in the white boxes, corresponding to two oligonucleotides ligated together. The melting temperature (T m ) of each oligonucleotide (Oligo A to Oligo J) is shown at the bottom of the figure.
  • Figure 6B Ligation of RNA oligonucleotides with different melting temperatures using PpRnl K238G. Reactions were performed at 75°C.
  • Figure 7 Ligation reactions using PpRnl K238G and T4 Rnl enzymes with synthetic RNA oligonucleotides from Table 2. Within the box are ligation products comprising two ligated molecules.
  • Figure 8 Amino acid sequence information and Clustal amino acid sequence alignments for PfuRnl (Pyrococcus furiosus DSM 3638 RNA ligase; GenBank: QEK78102.1; SEQ ID NO: 1), TgRnl (Thermococcus gorgonarius RNA ligase; NCBI Reference Sequence: WP_088884437.1; SEQ ID NO: 2), PpRnl (Palaeococcus pacificus RNA ligase; NCBI Reference Sequence: WP_048164472.1; SEQ ID NO: 3), GaRnl (Geoglobus ahangari RNA ligase; NCBI Reference Sequence: WP_048094670.1; SEQ ID NO: 4), MfRnl (Methanotorris formicus RNA ligase; NCBI Reference Sequence: WP_007044380.1; SEQ ID NO: 5), AfRnl (Arch
  • Figure 9A Amino acid sequence information for PpRnl (Palaeococcus pacificus') RNA ligase (NCBI Reference Sequence: WP_048164472.1; SEQ ID NO: 3). Key lysine residues shown by boxing and bold text. Sequence motifs I and V shown with underlining and bold text.
  • Figure 9B Amino acid sequence information for PpRnl variant (K92A; SEQ ID NO: 7). Key lysine/altered residues shown by boxing and bold text. Sequence motifs I and
  • Figure 9C Amino acid sequence information for PpRnl variant (K238G; SEQ ID NO: 8). Key lysine/altered residues shown by boxing and bold text. Sequence motifs I and
  • Figure 9D Amino acid sequence information for PfuRnl (Pyrococcus furiosus DSM 3638) RNA ligase (GenBank Sequence: QEK78102.1; SEQ ID NO: 1). Key lysine/altered residues shown by boxing and bold text. Sequence motifs I and V shown with underlining and bold text.
  • Figure 9E Amino acid sequence information for PfuRnl variant (K92A; SEQ ID NO: 9). Key lysine/altered residues shown by boxing and bold text. Sequence motifs I and
  • Figure 9F Amino acid sequence information for PfuRnl variant (K238A; SEQ ID NO: 10). Key lysine/altered residues shown by boxing and bold text. Sequence motifs I and
  • Figure 9G Amino acid sequence information for PfuRnl variant (K238G; SEQ ID NO: 11). Key lysine/altered residues shown by boxing and bold text. Sequence motifs I and
  • Figure 10A Overlay of the 3D models for PfuRnl, A/Rnl, GaRnl, AfRnl, PpRnl and TgRnl generated by PyMOL molecular visualisation software. Key lysine residues are circled.
  • Figure 10B Close-up view of the 3D model in Figure 10A showing the two key lysine residues in PfuRnl, A/Rnl, GaRnl, AfRnl, PpRnl and TgRnl. These lysine residues are numbered K92 and K238 in PfuRnl.
  • Figure 11 conserved domains and amino acid residues illustrated for PfuRnl polypeptide sequence.
  • Figures 12A-12E Nucleotide sequence information for PfuRnl, PpRnl, AfRnl, MfRnl, TgRnl.
  • Figures 13A-13K Nucleotide sequence information for variant polypeptides. Key altered codons shown in bold text with underlining.
  • Figures 14A-14F Amino acid sequence information for variant polypeptides. Key altered residues shown in bold text with underlining.
  • Figures 15A-15B Activity screening of ATP-independent RNA ligase mutants with non-adenylated RNA substrates. Assays were resolved using denaturing urea-PAGE gels.
  • Figure 15A TgRnl mutants K92A and K238G show no ligation activity compared to the wild-type enzyme.
  • Figure 15B The PpRnl K92A mutant shows reduced activity compared to wild-type PpRnl, whereas the PpRnl K238G mutant displays more efficient ligation activity.
  • Figures 16A-16B The choice of nucleotide cofactor affects the ligation activities of PpRnl ( Figure 16A) andTgRnl ( Figure 16B).
  • -Lig refers to no ligase added to the reaction.
  • NC refers to no cofactor added to the reaction.
  • the graphs indicate the relative ligation-based activity (%) of the enzyme. Error bars represent standard error of two replicate reactions.
  • Figure 17 Results of a deep sequencing experiment, conducted on an Illumina MiSeq instrument, to assess the bias in adapter ligation during library preparation.
  • each range that is specified includes all possible combinations of numerical values between the lowest value and the highest value enumerated (e.g., 1, 1.1, 2, 3, 3.3, 4, 5.5, 6, 7, 8.9, 9 and 10) and also any range of rational numbers within that range (e.g., 2 to 8, 1.5 to 5.5, and 3.1 to 4.9), and, therefore, all sub-ranges of all ranges expressly disclosed herein are hereby expressly disclosed.
  • the numeric values provided in parentheses here are only examples of what is specifically intended and all possible combinations of numerical value between the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure in a similar manner.
  • an “isolated” component refers to a component that has been purified from (e.g., separated from) other components.
  • An isolated component may be removed from its originating environment, e.g., natural cellular environment or synthetic environment.
  • the isolated component of this disclosure may be prepared by at least one purification step.
  • An isolated component may have: about 70% purity or greater, about 80% purity or greater, about 90% purity or greater; or, in particular aspects, about 99% purity or greater.
  • An isolated component may be obtained by any method or combination of methods as known and used in the art, including biochemical, recombinant, and synthetic techniques.
  • isolated when used herein in reference to a cell or host cell describes to a cell or host cell that has been obtained or removed from an organism or from its natural environment or from an artificial environment.
  • the term encompasses single cells, per se, as well as cells or host cells comprised in a cell culture and can include a single cell or single host cell.
  • RNA capping refers to modification of an RNA molecule to include a 5’ cap structure. This can involve transfer of a nucleotide moiety to the 5’ end of the RNA molecule, the moiety being obtained, for example, from a high-energy cofactor such as, ATP, UTP, NADH, or other.
  • construct refers to a polynucleotide molecule, usually double- stranded DNA, which may have cloned or inserted into it another polynucleotide molecule.
  • a construct may have an unidentified polynucleotide insert that is prepared from an environmental sample or as a cDNA, but not limited thereto.
  • a construct may contain the necessary elements that permit transcription of a cloned or inserted polynucleotide molecule, and, optionally, for translating the transcript into a peptide or polypeptide.
  • the inserted polynucleotide molecule may be derived from the host cell, or may be derived from a different cell or organism. Once inside the host cell the construct may become integrated in the host chromosomal DNA. The construct may be linked to a vector.
  • vector refers to a polynucleotide molecule, usually double stranded DNA, which is used to replicate or express a construct.
  • the vector may be used to transport a construct into a given host cell.
  • polynucleotide(s), means a single or double-stranded deoxyribonucleotide or ribonucleotide polymer of any length, and include as non-limiting examples, coding and non-coding sequences of a gene, genomic DNA, recombinant polynucleotides, isolated and purified naturally occurring DNA or RNA sequences, synthetic RNA and DNA sequences, fragments, constructs, and vectors. Reference to nucleic acids, nucleic acid molecules, nucleotide sequences, and polynucleotide sequences is to be similarly understood.
  • polypeptide encompasses amino acid chains of any length, wherein the amino acid residues are linked by covalent peptide bonds.
  • Polypeptide may refer to a polypeptide that is a purified natural product, or that has been produced partially or wholly using recombinant or synthetic techniques. The term may refer to an aggregate of a polypeptide such as a dimer or other multimer, a fusion polypeptide, a polypeptide fragment, a polypeptide variant, fragment, or derivative thereof.
  • polypeptide is used interchangeably herein with the terms “protein” and “enzyme”.
  • a “fragment” of a polypeptide is a subsequence of a particular polypeptide, i.e., truncation.
  • the fragment is a functional fragment.
  • a functional fragment performs a function that is required for a biological activity or substrate binding and/or provides three dimensional structure of the polypeptide.
  • the term may refer to a polypeptide fragment, an aggregate of a polypeptide fragment, a fusion polypeptide fragment, a fragment of a polypeptide variant, or a fragment of a polypeptide derivative thereof that is capable of performing the polypeptide activity.
  • full length as used herein with reference to a sequence means a peptide or polypeptide that comprises a contiguous sequence of amino acid residues where each amino acid residue has been expressed from each of its corresponding codons in the polynucleotide over the entire length of the coding region and resulting in a fully functional polypeptide, peptide, or protein.
  • a “full length” sequence contains the amino acid sequence that corresponds to and has been expressed from each and every codon encoded by the polynucleotide comprising the entire coding region of the polypeptide, wherein each of said codons is located between the start codon and the termination codon normally associated with that coding region.
  • expressing refers to the expression of a nucleic acid transcript from a nucleic acid template and/or the translation of that transcript into a peptide or polypeptide, and is used herein as commonly used in the art.
  • endogenous refers to a constituent of a cell, tissue or organism that originates or is produced naturally within that cell, tissue or organism.
  • An “endogenous” constituent may be any constituent including but not limited to a polynucleotide, a polypeptide, and a peptide.
  • exogenous refers to any constituent of a cell, tissue or organism that does not originate or is not produced naturally within that cell, tissue or organism.
  • An exogenous constituent may be, for example, a polynucleotide sequence that has been introduced into a cell, tissue or organism, or a peptide or polypeptide expressed in that cell, tissue or organism from that polynucleotide sequence.
  • Naturally occurring refers to a sequence that is found in nature.
  • a synthetic sequence that is identical to a wild-type sequence is, for the purposes of this disclosure, considered a naturally occurring sequence.
  • a naturally occurring sequence also refers to a variant sequence as found in nature that differs from wild-type. For example, allelic variants and naturally occurring sequences due to hybridisation or horizontal gene transfer, and variants arising out of other natural processes. What is important for a naturally occurring sequence is that the actual sequence (e.g., nucleotide or amino acid sequence) is found or known from nature.
  • Non-naturally occurring refers to a sequence that is not found in nature. Examples of non- naturally occurring sequences include artificially produced and variant sequences, made for example by recombination, domain swapping, point mutation, insertion, deletion, or other methods, or combinations of these methods. Non-naturally occurring sequences also include chemically evolved sequences. What is important for a non-naturally occurring sequence is that the actual sequence (e.g., nucleotide or amino acid sequence) is not found or known from nature.
  • wild-type when used herein with reference to a polynucleotide refers to a naturally occurring, non-mutant form of a polynucleotide, peptide, polypeptide, or organism.
  • a wild-type peptide or polypeptide is capable of being expressed from a wild-type polynucleotide.
  • a wild-type polypeptide is a wild-type RNA ligase that is expressed from a wild-type polynucleotide.
  • homologous as used herein with reference to polynucleotide regulatory elements, means a polynucleotide regulatory element that is a native and naturally-occurring polynucleotide regulatory element.
  • a homologous polynucleotide regulatory element may be operably linked to a polynucleotide of interest such that the polynucleotide of interest can be expressed from a, vector, construct, or expression cassette according to this disclosure.
  • Heterologous as used herein with reference to polynucleotide regulatory elements, means a polynucleotide regulatory element that is not a native and naturally- occurring polynucleotide regulatory element.
  • a heterologous polynucleotide regulatory element is not normally associated with the coding sequence to which it is operably linked.
  • a heterologous regulatory element may be operably linked to a polynucleotide of interest such that the polynucleotide of interest can be expressed from a vector, construct, or expression cassette according to this disclosure.
  • promoters may include promoters normally associated with other genes, ORFs or coding regions, and/or promoters isolated from any other bacterial, viral, eukaryotic, or mammalian cell.
  • recombinant refers to a polynucleotide sequence that is removed from sequences that surround it in its natural context and/or is recombined with sequences that are not present in its natural context.
  • a “recombinant” peptide or polypeptide sequence is produced by translation from a “recombinant” polynucleotide sequence.
  • modified refers to a component that is not a naturally occurring.
  • a “modified polypeptide” or “modified enzyme” refers to a polypeptide that is not a naturally occurring. Modification may be carried out in accordance with the disclosed methods, e.g., sequence variants or other modifications, e.g., post-translational modifications. For example, various methods of recombination may be used to achieve modification. Modified enzymes and polypeptides useful in this disclosure may have biological activities, stability, and/or production levels that are the same or similar to those of a corresponding wildtype molecule i.e., functional modifications.
  • modified enzymes and polypeptides may have biological activities that differ from their corresponding wild-type molecules. In certain embodiments, the differences are altered activity stability, and/or production levels.
  • a functional modification may produce a particular type of product or prefer a particular substrate. In certain embodiments, the levels of product produced by the functional modification may be higher or lower than produced by the wild-type molecule.
  • a modified enzyme may comprise a recombinant enzyme, a modified polypeptide may comprise a recombinant polypeptide, and a modified enzyme may comprise a recombinant enzyme, as set out in this description.
  • variant refers to polynucleotide, peptide, or polypeptide sequences different from the specifically identified sequences, wherein one or more nucleotides or amino acid residues is deleted, transposed, substituted, or added. Variants may be naturally occurring allelic variants, or non-naturally occurring variants. Variants may be from the same or from other species and may encompass homologues, paralogues, and orthologues.
  • the variants useful in this disclosure have biological activities that are the same or similar to those of a corresponding wild-type molecule; i.e., functional variants of the parent polypeptide or polynucleotide. In certain embodiments, the variants have biological activities that differ from their corresponding wild-type molecules. In certain embodiments, the differences are altered activity, stability, and/or production levels.
  • variant polypeptides encompasses all forms of polynucleotides, peptides, and polypeptides as defined herein.
  • a variant polypeptide, including an enzyme variant, as described herein will retain functionality of the reference polypeptide, for example, enzymatic activity will be retained.
  • an enzyme variant will have new and/or enhanced activities compared to the reference polypeptide.
  • mutagenesis refers to methods to alter a polynucleotide sequence either in vitro or in vivo, most commonly to change the sequence of one or more polypeptides encoded therein. Mutagenesis methods include as non-limiting examples, site-directed mutagenesis, de novo synthesis of sequences carrying mutations, error- prone PCR, DNA shuffling, chemical mutagenesis, application of ultraviolet radiation, genome shuffling, and use of mutator strains.
  • the term in vitro refers to a reaction performed outside of the confines of a living cell or a host organism.
  • the term in vivo refers to a reaction performed within a living cell and/or within a host organism.
  • ligate refers to the formation of phosphodiester bonds between the ribose and/or deoxyribose moieties of nucleic acid molecules, and particularly, the 3'-OH and 5'-P groups of these nucleic acid molecules. These may be RNA or DNA molecules.
  • ligase refers to an enzyme that catalyses the formation of phosphodiester bonds between the ribose and/or deoxyribose moieties of nucleic acid molecules, as described herein. Specifically included are thermostable RNA ligases.
  • RNA ligases from (e.g., originated from or modified from) Palaeococcus, Pyrococcus, Thermococcus, Geoglobus, Methanotorris, Methanobacterium, or Archaeoglobus organisms.
  • enzymes from (e.g., originated from or modified from) Palaeococcus pacificus, Pyrococcus furiosus, Thermococcus gorgonarius, Methanotorris formicus, or Archaeoglobus fulgidus organisms.
  • enzymes that possess ssRNA ligase activity, but not ssDNA ligase activity. Variants of these enzymes, for example, point mutations, and also truncations, are specifically encompassed.
  • thermostable refers to a component, such as a polypeptide or enzyme, that retains one or more functions at elevated temperatures.
  • a thermostable enzyme including an enzyme variant, as described herein will retain functionality at temperatures of 50°C and higher.
  • PfuRnl refers to Pyrococcus furiosus RNA ligase (e.g., SEQ ID NO: 1)
  • TgRnl refers to Thermococcus gorgonarius RNA ligase (e.g., SEQ ID NO: 2)
  • PpRnl refers to Palaeococcus pacificus RNA ligase (e.g., SEQ ID NO: 3)
  • GaRnl refers to Geoglobus ahangari RNA ligase (e.g., SEQ ID NO: 4)
  • AfRnl refers to Methanotorris formicus RNA ligase (e.g., SEQ ID NO: 5)
  • AfRnl refers to Archaeoglobus fulgidus RNA ligase (e.g., SEQ ID NO: 6).
  • thermostable RNA ligase enzymes including novel variant enzymes. We demonstrate the ability of these enzymes to perform ligation reactions with significantly less bias compared to industry standards.
  • the RNA ligases that may be utilised in the present methods include but are not limited to archaeal polypeptides, such as Palaeococcus, Pyrococcus, Thermococcus or Archaeoglobus polypeptides. In particular, Palaeococcus pacificus, Pyrococcus furiosus, Thermococcus gorgonarius and Archaeoglobus fulgidus polypeptides are noted.
  • Specific exemplifications include polypeptides that comprise an amino acid sequence of any one of SEQ ID NO: 1-6, and any variants of these sequences; as well as polypeptides that consist of an amino acid sequence of any one of SEQ ID NO: 1-6.
  • Modified RNA ligase enzymes are particularly noted. Of interest are variant polypeptides comprising substitution of one or more of the two conserved lysine residues, as illustrated in Figure 8.
  • polypeptides that comprise an amino acid sequence of any one of SEQ ID NO: 7-11 or 37-42, and any further variants of these sequences; as well as polypeptides that consist of an amino acid sequence of any one of SEQ ID NO: 7-11 or 37-42. See, e.g., Figures 9B-9C, 9E-9G, 14A-14F.
  • At least one of the conserved lysine residues is substituted with alanine. In other aspects, at least one of the conserved lysine residues is substituted with glycine. In yet other aspects, both of the conserved lysine residues is substituted with either alanine or glycine. In still other embodiments, both of the conserved lysine residues are substituted, one being replaced by alanine and one being replaced by glycine.
  • Nonlimiting examples of these substitutions include: PfuRnl K92A, PfuRnl K92G, PpRnl K92A, PpRnl K92G, AfRnl K96A, AfRnl K96G, MfRnl K92A, AfRnl K92G, TgRnl K92A, and TgRnl K92G.
  • non-conserved regions of the RNA ligase polypeptide may be modified by substitutions or truncations, or may be deleted entirely.
  • Non-conserved regions include amino acids 1-9 and 379 in PfuRnl (see Figure 11), amino acids 1-9 and 380 in PpRnl , amino acids 1-9 and 380 in TgRnl , amino acids 1-37 in AfRnl, and amino acids 1-4 and 378 in A/Rnl.
  • variant polypeptides with one or more substitutions of conserved lysines may be further modified in their non-conserved regions to include one or more additional substitution(s), and/or truncations or deletions.
  • RNA ligase polypeptides can be understood in terms of the corresponding nucleotide sequences. Therefore, where wild-type and variant polypeptides are encompassed and disclosed, the correlating wild-type and variant polynucleotides are considered to be encompassed and disclosed.
  • Specific exemplifications include polynucleotides that encode an amino acid sequence of any one of SEQ ID NO: 1-6, or encode any variants of these, such as SEQ ID NO: 7-11; as well as polynucleotides that comprise a nucleotide sequence of any one of SEQ ID NO: 12-26, or any variants as these; along with polynucleotides that consist of a nucleotide sequence of any one of SEQ ID NO: 12-26. See, e.g., Figures 13A-13K.
  • polynucleotide variants encompass naturally occurring or non-naturally occurring (e.g., recombinantly or synthetically produced) polynucleotides.
  • variant polynucleotide sequences exhibit at least 50%, at least 60%, at least 70%, at least 71%, at least 72%, at least 73%, 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 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a sequence of the present disclosure.
  • a polynucleotide sequence of an RNA ligase or an RNA ligase domain may be modified to include the above noted levels of sequence identity.
  • a fragment of a polynucleotide sequence includes a subsequence of contiguous nucleotides.
  • the polynucleotide fragment allows expression of at least a portion of an RNA ligase polypeptide, e.g., expression of one or more functional domains of the polypeptide.
  • Variant polynucleotides include polynucleotides that differ from the disclosed sequences but that, as a consequence of the degeneracy of the genetic code, encode a polypeptide having similar activity to a polypeptide encoded by a disclosed polynucleotide.
  • a sequence alteration that does not change the amino acid sequence of the polypeptide is termed a silent variation. Except for ATG (methionine) and TGG (tryptophan), other codons for the same amino acid may be changed by art recognised techniques, e.g., to optimise codon expression in a particular host organism.
  • sequence identity may be found over a comparison window of at least 1500 nucleotide positions, at least 2000 nucleotide positions, at least 2500 nucleotide positions, at least 3000 nucleotide positions, at least 3500 nucleotide positions, at least 3800 nucleotide positions, or over the entire length of a polynucleotide used according to a method of this disclosure.
  • shorter regions may be compared, for example, at least 50 nucleotide positions, at least 100 nucleotide positions, at least 200 nucleotide positions, at least 300 nucleotide positions, at least 400 nucleotide positions, at least 500 nucleotide positions, at least 600 nucleotide positions, at least 700 nucleotide positions, at least 800 nucleotide positions, at least 900 nucleotide positions, or at least 1000 nucleotide positions.
  • Polynucleotide sequence identity and similarity can be determined in the following manner.
  • the subject polynucleotide sequence is compared to a candidate polynucleotide sequence using sequence alignment algorithms and sequence similarity search tools such as in GenBank, EMBL, Swiss-PROT and other databases.
  • sequence alignment algorithms and sequence similarity search tools such as in GenBank, EMBL, Swiss-PROT and other databases.
  • Nucleic Acids Res 29:1- 10 and 11-16, 2001 provides examples of online resources.
  • polypeptide variants encompass naturally occurring or non-naturally occurring (e.g., recombinantly or synthetically produced) polypeptides.
  • variant polypeptide sequences exhibit at least 50%, at least
  • RNA ligase or an RNA ligase domain may be modified to include the above noted levels of sequence identity.
  • a fragment of a polypeptide sequence includes a subsequence of contiguous amino acids.
  • a polypeptide fragment is a functional fragment, e.g., a fragment capable of carrying out ligation.
  • an RNA ligase polypeptide fragment may be capable of producing a particular nucleic acid dimer.
  • the polypeptide fragment may include at least one functional domain.
  • a fragment would include a ligase domain, or a modification thereof, as described herein. See, e.g., Figure 11.
  • an amino acid sequence may differ from a polypeptide disclosed herein by one or more conservative amino acid substitutions, deletions, additions or insertions which do not affect the biological activity of the peptide.
  • Conservative substitutions typically include the substitution of one amino acid for another with similar characteristics, e.g., substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
  • Non-conservative substitutions will entail exchanging a member of one of these classes for a member of another class.
  • variants include peptides with modifications which influence peptide stability.
  • Such analogues may contain, for example, one or more non-peptide bonds (which replace the peptide bonds) in the peptide sequence.
  • analogues that include residues other than naturally occurring L-amino acids, e.g. D-amino acids or non-naturally occurring synthetic amino acids, e.g. beta or gamma amino acids and cyclic analogues.
  • substitutions, deletions, additions, or insertions may be made by mutagenesis methods known in the art.
  • a skilled worker will be aware of methods for making phenotypically silent amino acid substitutions. See, for example, Bowie et al. 1990.
  • a polypeptide may be modified during or after synthesis, for example, by biotinylation, benzylation, glycosylation, phosphorylation, amidation, by derivatisation using blocking/protecting groups and the like. Such modifications may increase stability or activity of the polypeptide.
  • sequence identity may be found over a comparison window of at least 600 amino acid positions, at least 700 amino acid positions, at least 800 amino acid positions, at least 900 amino acid positions, at least 1000 amino acid positions, at least 1100 amino acid positions, at least 1200 amino acid positions, or over the entire length of a polypeptide used in or identified according to a method of this disclosure.
  • shorter regions may be compared, for example, at least 8 amino acid positions, at least 10 amino acid positions, at least 20 amino acid positions, at least 30 amino acid positions, at least 40 amino acid positions, at least 50 amino acid positions, at least 60 amino acid positions, at least 70 amino acid positions, at least 80 amino acid positions, at least 90 amino acid positions, or at least 100 amino acid positions.
  • Polypeptide variants also encompass those that exhibit a similarity to one or more of the specifically identified sequences that is likely to preserve the functional equivalence of those sequences and which could not reasonably be expected to have occurred by random chance.
  • exemplary sequence alignment platforms include but are not limited to: homology alignment algorithms (Needleman and Wunsch (1970) J Mol Biol 48: 443); local homology algorithms (Smith and Waterman (1981) Adv Appl Math 2: 482); searches for similarity (Pearson and Lipman (1988) PNAS USA 85: 2444).
  • the BLAST algorithm may be used (Altschul et al. (1990) J Mol Biol 215: 403-410; Henikoff and Henikoff. (1989) PNAS USA 89: 10915; Karlin and Altschul (1993) PNAS USA 90: 5873-5787).
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.
  • Other examples of alignment software include GAP, BESTFIT, FASTA, PILEUP, and TFASTA provided by Wisconsin Genetics Software Package (Genetics Computer Group), and CLUSTAL programs such as ClustalW, ClustalX, and Clustal Omega (see, e.g., Thompson et al. (1994) Nuc Acids Res 22: 4673-4680).
  • an RNA ligase polypeptide (e.g., a wild-type or variant polypeptide) is expressed using a nucleic acid construct.
  • a nucleic acid construct e.g., a nucleic acid construct.
  • Specific exemplifications include polypeptides that comprise an amino acid sequence of any one of SEQ ID NO: 1-6, and any variants of these sequences (such as SEQ ID NO: 7-11 and any further variants of these); as well as polypeptides that consist of an amino acid sequence of any one of SEQ ID NO: 1-6, or SEQ ID NO: 7-11, or SEQ ID NO: 37-42.
  • RNA ligase constructs may be used, i.e., a nucleic acid expression construct that comprises a polynucleotide sequence that encodes the desired polypeptide.
  • This polynucleotide sequence can be operatively linked to a promoter that allows expression of the polynucleotide sequence to produce the RNA ligase polypeptide.
  • this process produces an active enzyme, or a functional variant of this enzyme.
  • An expression cassette may be used to include the necessary elements that permit the transcription of a polynucleotide molecule that has been cloned or inserted into the construct.
  • the expression cassette may comprise some or all of the necessary elements for translating the transcript produced from the expression cassette into a polypeptide.
  • An expression cassette may include RNA ligase coding regions. It may also include any necessary noncoding regions.
  • a polynucleotide sequence encoding the RNA ligase polypeptide may be any suitable polynucleotide sequence from any organism.
  • the organism may be an archaea strain.
  • Exemplifications include but are not limited to: Palaeococcus, Pyrococcus, Thermococcus, and Archaeoglobus strains, and related organisms. Further exemplifications include: Pyrococcus furiosus, Thermococcus gorgonarius, Palaeococcus pacificus, and Archaeoglobus fulgidus strains.
  • the polynucleotide sequence encoding the RNA ligase polypeptide may be a naturally occurring (i.e., wild-type) or modified (e.g., variant) polynucleotide sequence.
  • a wild-type or a modified polynucleotide sequence for one or more RNA ligase enzymes may be used.
  • the polynucleotide sequence encoding the RNA ligase polypeptide may be a wild-type or modified polynucleotide sequence, as described herein.
  • Specific exemplifications include polynucleotides that encode an amino acid sequence of any one of SEQ ID NO: 1-6, or encode any variants of these (such as SEQ ID NO: 7-11, or SEQ ID NO: 37-42, and any further variants of these); as well as polynucleotides that comprise a nucleotide sequence of any one of SEQ ID NO: 12-26, or any variants as these; along with polynucleotides that consist of a nucleotide sequence of any one of SEQ ID NO: 12-26.
  • a construct is made by cloning a polynucleotide sequence encoding a wild-type or modified polypeptide as above into an appropriate vector.
  • An appropriate vector is any vector that comprises a promoter operatively linked to the cloned, inserted polynucleotide sequence that allows expression of the polypeptide from the vector.
  • a skilled worker appreciates that different vectors may be employed in the methods of this disclosure.
  • methods for constructing vectors including the choice of an appropriate vector, and the cloning and expression of a polynucleotide sequence inserted into an appropriate vector as described above is believed to be within the capabilities of a person of skill in the art (Sambrook et al. 2003).
  • the expressed RNA ligase polypeptide comprises a functional catalytic domain. Expression may be inducible, for example, with IPTG. Similar approaches may be used for the polypeptides disclosed herein, and any functional variants thereof. The person of skill in the art recognises that there are also many suitable alternative expression systems available that may be used in the methods of this disclosure to express an RNA ligase polypeptide.
  • expression is obtained in a suitable host cell or strain or cell free expression system.
  • the host cell or strain may be a cell or strain of E. coli. Particularly of interest are the BAP1, BL21 (e.g., BL21(DE3)), and E. cloni®10G strains of E. coli or any variant of these strains (see, e.g., Pfeifer et al. 2001).
  • the expression vector is chosen to allow inducible expression in a non-E. coli host cell or strain. Expression may also be obtained using in vitro expression systems; such systems are well known in the art.
  • the RNA ligase polynucleotide may be adapted for use in such strains or systems. For example, the nucleotide sequence may be modified to include appropriate codon usage.
  • RNA ligase polypeptides are co-expressed in the same host cell or strain or expression system.
  • the nucleotide sequences encoding the polypeptides may be cloned into one or more suitable expression vectors.
  • suitable vectors may have the same or compatible origins of replication in order to be stably maintained in the same host cell or strain.
  • an expression vector can encode at least one RNA polypeptide (e.g., one or more of SEQ ID NO: 1-6) and/or at least one functional variant thereof (e.g., one or more of SEQ ID NO: 7-11, or SEQ ID NO: 37-42, or further variants of these).
  • one or more polynucleotide sequences encoding a RNA ligase polypeptide may be integrated into the chromosome of an appropriate host organism as described herein, to produce a strain useful in accordance with the present disclosure.
  • an RNA ligase construct comprises at least one nucleotide sequence encoding an RNA ligase polypeptide and suitable regulatory promoter sequences that are integrated into the chromosome of E. coli or other host organism in an appropriate orientation to allow expression of the polypeptide or polypeptides in the cell.
  • a construct encoding one or more RNA ligase enzyme or variant enzyme is integrated into a host cell.
  • an RNA ligase construct may be integrated and then expressed in vivo.
  • the constructs may allow co-expression of wild- type polypeptides or functional variants.
  • a construct that encodes a single or multiple RNA ligase polypeptides is expressed in a host cell or strain, or cell free expression system.
  • RNA ligase nucleic acids may include at least 15, at least 25, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, or at least 1000 RNA ligase nucleic acids.
  • Libraries of RNA ligase polynucleotides, and specifically variant RNA ligase polynucleotides, may be generated using standard methods.
  • nucleic acid libraries may be generated to include a plurality of RNA ligase polynucleotides with modified sequences.
  • a nucleic acid library may include RNA ligase polynucleotides with substitutions or swapped domains.
  • libraries of RNA ligase polynucleotides may be generated using random mutagenesis of one or more domains (e.g., conserved sequence or regions). For example, error prone PCR may be utilised (see, e.g., Beaudry and Joyce (1992) Science 257: 635 and Bartel and Szostak (1993) Science 261: 1411).
  • mutagenesis may be used, for example, chemical mutagens, radiation, amongst others.
  • kits are also available, e.g., GeneMorph® II EZClone domain mutagenesis kit (Agilent) and DiversifyTM PCR random mutagenesis kit (Clontech Laboratories, Inc).
  • the library may be provided as a mixture of polynucleotides, or may be provided via a host cell or strain.
  • a system or kit is provided. This may include one or more RNA ligase polynucleotides or polypeptides.
  • the one or more polynucleotides or polypeptides may be a modified component as described herein.
  • the one or more polynucleotide or polypeptide may be provided in one or more containers in the system or kit. Additional components may also be provided with the system or kit, for example, one or more components to obtain expression, one or more ligation substrates, one or more ligation cofactors, and/or one or more components to measure activity, which are intended for use with the polynucleotide(s) or polypeptide(s).
  • the system or kit may include a first single stranded nucleic acid sequence with a 5' end base that is adenylated (or will be adenylated during the reaction) and/or a second single stranded nucleic acid sequence with a 3' hydroxyl group but no 5' phosphate group.
  • instructions may be provided with the kit, as well as any other item, such as any number of containers, labels, or measurement tools.
  • the one or more polynucleotide or polypeptide of the system or kit may be provided as isolated components, or as mixtures, or may be provided via a host cell or strain.
  • compositions may include one or more of the RNA ligase polynucleotides, polypeptides, fusion polypeptides (e.g., tagged polypeptides), nucleic acid constructs, nucleic acid libraries, or host cells, as described herein.
  • the composition may further include one or more buffers, co-factors, cryoprotectors, reaction reagents, preservatives, salts, stabilisers, diluents, excipients, or any other components.
  • RNA ligase enzymes may be provided in a liquid composition (e.g., storage solution) comprising: 20 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1 mm DTT, 0.1 mM EDTA and 50% glycerol.
  • a liquid composition e.g., storage solution
  • Other compositions may be prepared in accordance with standard methods.
  • RNA ligase polypeptide e.g., a variant polypeptide
  • methods of production for an RNA ligase polypeptide are provided, and methods of use of these polypeptides are also provided.
  • Specific exemplifications include polypeptides that comprise an amino acid sequence of any one of SEQ ID NO: 1-6, and any variants of these sequences (such as SEQ ID NO: 7-11, or SEQ ID NO: 37-42 and any further variants of these); as well as polypeptides that consist of an amino acid sequence of any one of SEQ ID NO: 1-6, or SEQ ID NO: 7-11, or SEQ ID NO: 37-42.
  • Host cells and their use for such production are set out in detail in this description.
  • RNA ligase polypeptide By use of a host cell comprising an RNA ligase polypeptide, this allows production of ligation products.
  • the expression of an RNA ligase polypeptide e.g., a modified RNA ligase polypeptide
  • In vivo expression may be carried out in a suitable host cell or strain.
  • a suitable host cell or strain may be any suitable prokaryotic or eukaryotic cell in which an RNA ligase polypeptide, or any functional variants thereof, may be expressed.
  • the host cell or strain is a fungal or bacterial, preferably bacterial, host cell or strain, but not limited thereto.
  • the bacterial cell or strain may be a Gram negative bacterial cell or strain.
  • the bacterial cell or strain may be a cell or strain of E. coli.
  • the host strain may be a Bacillus (e.g., Brevibacillus). Streptomyces, or Pseudomonas strain, or a strain of Saccahromyces, Pichia or Aspergillus, or another bacterial or fungal strain as set out herein, or any functional variant thereof.
  • RNA ligase polynucleotide or RNA ligase construct into an appropriate host cell or strain may be achieved using any of a number of available standard protocols and/or as described herein as known and used in the art (Sambrook et al. 2003).
  • the RNA ligase construct may be a construct for enzyme production as set out herein.
  • the construct may be introduced via transformation, transduction, transfection, or other techniques (see, e.g., Sambrook et al. 2003).
  • the expressed polypeptide is an exogenous polypeptide in the host cell, strain, or cell free expression system, which is expressed from a construct according to this disclosure.
  • the polypeptide is expressed from the genome of the host cell or strain.
  • the polypeptide may be endogenous or exogenous, naturally occurring or non-naturally occurring with respect of the host cell or strain.
  • the polypeptide so expressed may be a functional RNA ligase as set out herein, or a functional variant thereof.
  • a single host organism or cell free expression system could be utilised to allow expression of multiple polypeptides (e.g., two or more of RNA ligase polypeptides, two or more variant polypeptides, or any combination of these), to maximise production.
  • multiple polypeptides e.g., two or more of RNA ligase polypeptides, two or more variant polypeptides, or any combination of these
  • wild-type and variant enzymes may be co-expressed.
  • specific examples include co-expression expression for: (1) two or more wild-type polypeptides as described herein, e.g., PfuRnl, PpRnl , TgRnl , AfRnl; (2) two or more variant polypeptides as described herein, e.g., PfuRnl K92A, PfuRnl K238A, PfuRnl K238G, PpRnl K238G; or (3) two or more polypeptides, these being a combination of at least one wild-type polypeptide (e.g., PfuRnl, PpRnl , TgRnl , AfRnl) and at least one variant polypeptide (PfuRnl K92A, PfuRnl K238A, PfuRnl K238G, PpRnl K238G).
  • RNA ligase polypeptide may be carried out in vitro or in vivo.
  • Cell free expression systems and cell free synthesis systems may be used in accordance with standard methodology.
  • the choice host strain or cell free expression system may be made based on the expression componentry or other factors. For example, host cells or cell free systems may be chosen based on promoter activity, codon bias, protein solubility, or other factors. Therefore, the use of different host strains and systems provides alternative means suitable for use in production of any polypeptides of interest.
  • the RNA ligase polypeptide (expressed or otherwise produced) may be isolated using various biochemical techniques. These techniques include but are not limited to filtration, centrifugation, and various types of chromatography, such as ionexchange, affinity, hydrophobic interaction, size exclusion, and reverse-phase chromatography. In one particular embodiment, Ni-affinity chromatography is used. As exemplifications, the polypeptides may be linked to a solid substrate such as beads, filters, fibres, paper, membranes, chips, and plates such as multiwell plates.
  • polypeptides may also be prepared as a polypeptide conjugates or polypeptide fusion molecules in accordance with known methods.
  • Protein affinity tags may be used to assist with purification, for example, albumin-binding protein (ABP), biotin-carboxy carrier protein (BCCP), calmodulin binding peptide (CBP), cellulose binding domain (CBP), chitin binding domain (CBD), galactose-binding protein (GBP), glutathione S-transferase (GST), HaloTag®, LacZ, polyhistidine (His-tag), polyphenylalanine (Phe-tag), S-tag, small ubiquitin-like modifier (SUMO), Staphylococcal protein A (Protein A), Staphylococcal protein G (Protein G), Strep-tag, streptavadin, thioredoxin (Trx), tandem affinity purification (TAP), and ubiquitin protein tags.
  • RNA ligase polypeptides of this disclosure may be utilised in ligation methods, in the preparation of miRNA libraries, as well as other useful procedures.
  • these include, for example: synthetic nucleotide construction (e.g., RNA synthesis); sequencing (e.g., RNA sequencing, deep sequencing); filling nicks in double stranded nucleic acids; rapid amplification of cDNA ends (RACE); and 3 '-end nucleic acid labelling (e.g., RNA labelling), such as 3 '-end biotin and fluorophore labelling.
  • Other examples include the circularisation of RNA molecules (e.g., for vaccine preparation or other therapeutic methods), as well as the modification of the 5’ ends of RNA or DNA molecules by adenylation, guanidinylation and/or uridinylation.
  • the ligation of single- stranded nucleic acids may comprise: combining (i) one or more RNA ligase of this disclosure (e.g., the noted enzymes and/or their variants), (ii) a first single stranded nucleic acid sequence with a 5' end base that is adenylated (or becomes adenylated while in the reaction mixture), and (iii) a second single stranded nucleic acid sequence with a 3' hydroxyl group; in a ligation reaction mixture and incubating the ligation reaction mixture at a temperature of at least 50°C (e.g., at least 60°C, at least 65°C, at least 70°C, at least 75°C, at least 85°C, or at least 90°C, or about 65°C to about 85°C) such that the one or more RNA ligase ligates the 5' adenylated end of
  • the adenylation of a single- stranded nucleic acid may comprise: combining (i) one or more RNA ligase of this disclosure (e.g., the noted enzymes and/or their variants), and (ii) a single stranded nucleic acid sequence with a 5' end base that requires adenylation, in an adenylation reaction mixture and incubating the adenylation reaction mixture at a temperature of at least 50°C (e.g., at least 60°C, at least 65°C, at least 70°C, at least 75°C, at least 85°C, or at least 90°C, or about 65°C to about 85°C) such that the one or more RNA ligase adenylates the 5' end of the single stranded nucleic acid sequence to form an adenylated nucleic acid sequence.
  • RNA ligase of this disclosure e.g., the noted enzymes and/or their variants
  • the circularisation of a single- stranded nucleic acid may comprise: combining (i) one or more RNA ligase of this disclosure (e.g., the noted enzymes and/or their variants), and (ii) a single stranded nucleic acid sequence with a 5' end base that is adenylated (or becomes adenylated while in the reaction mixture) and also a 3’ end base that has a 3' hydroxyl group, in a ligation reaction mixture and incubating the ligation reaction mixture at a temperature of at least 50°C (e.g., at least 65°C, at least 70°C, at least 75°C, at least 85°C, or at least 90°C, or about 65°C to about 85°C) such that the one or more RNA ligase ligates the 5' end of the single stranded nucleic acid sequence to the 3’ end of the single RNA ligase of this disclosure (e.g., the noted enzymes and
  • the preparation of a miRNA library for subsequent deep sequencing may comprise:
  • Step 1 Ligation of a 3’ adapter to miRNA molecules:
  • the adapter used in this step is a DNA oligonucleotide that is adenylated at its 5’ end (5’-App) and blocked at its 3’ end.
  • Most commercially available kits typically utilise a truncated version of T4-RNA ligase 2 that is deficient in the ability to adenylate and deadenylate nucleic acids to achieve this ligation.
  • Use of this enzyme variant (or an equivalent of this) ensures that only the 3’ ends of the miRNA molecules are ligated to the 5’ end of the adapter.
  • Unwanted circularisation of the adapter is prevented by blocking its 3’ end and use of a truncated RNA ligase that requires a pre- adenylated 5’ end (which the miRNA does not have) prevents unwanted circularisation of miRNA.
  • Step 2 Ligation of a 5’ adapter to the miRNA-3’ adapter molecules created in step 1: This step is typically mediated by a wild-type RNA ligase (typically T4 RNA ligase 1). Use of an adapter that is dephosphorylated at its 5’ end prevents its unwanted circularisation. The fact that the miRNA-3 ’ adapter molecule created in step 1 is blocked at its 3 ’ end prevents unwanted circularisation of this molecule.
  • Step 3 - First strand synthesis This is typically a standard first strand DNA synthesis reaction mediated by a reverse transcriptase enzyme.
  • Step 4 - Library amplification This a standard PCR reaction mediated by a thermostable DNA polymerase. See, also, Figure 1. It will be understood that the adaption and modification of the method steps are also possible.
  • RNA ligase polypeptides of this disclosure may be used in the initial and/or second ligation steps, e.g., for ligation of a 3’ adapter to miRNA molecules and/or for ligation of a 5’ adapter to the miRNA-3’ adapter molecules. It was surprisingly found that the ability of enzymes to avoid bias in these two ligation steps was not entirely correlated to reaction temperature, but rather appeared to be a function of the enzymes themselves. Without wishing to be bound by theory, it is suggested that the novel enzymes may themselves assist in making the miRNA available for ligation.
  • RNA ligase enzymes may be used in combination to maximise ligation efficiency and minimise bias (e.g., in miRNA library production).
  • specific examples include combinations for: (1) two or more wild-type polypeptides as described herein, e.g., PfuRnl, PpRnl , TgRnl , A/Rnl; (2) two or more variant polypeptides as described herein ,e.g., PfuRnl K92A, PfuRnl K238A, PfuRnl K238G, PpRnl K238G; or (3) two or more polypeptides, these being a combination of at least one wild-type polypeptide (e.g., PfuRnl , PpRnl , TgRnl , AfRnl) and at least one variant polypeptide (PfuRnl K92A, PfuRnl K238A, Pf
  • an RNA ligase from the bacteriophage T4 may be combined for use with one or more wild-type polypeptides, one or more variant polypeptides, or combined wild-type and variant polypeptides, as disclosed herein.
  • These include polypeptides that comprise an amino acid sequence of any one of SEQ ID NO: 1-6, and any variants of these sequences (such as SEQ ID NO: 7-11, or SEQ ID NO: 37-42, and any further variants of these); as well as polypeptides that consist of an amino acid sequence of any one of SEQ ID NO: 1-6, or SEQ ID NO: 7-11, or SEQ ID NO: 37-42.
  • Any T4 RNA ligase may be utlised, including modified and variant T4 ligases.
  • reaction conditions may be used to enhance functionality of the thermostable RNA ligase enzymes disclosed herein, including the variant enzymes that have been described.
  • reactions are carried out at 37°C or higher, 40°C or higher, 45°C or higher, 50°C or higher, 55°C or higher, 60°C or higher, 65°C or higher, 70°C or higher, 75°C or higher, 80°C or higher, 85°C or higher, or 90°C or higher, or in a range of about 40°C to about 85°C, or about 50°C to about 85°C, or about 55°C to about 80°C, or about 60°C to about 85°C, or about 65°C to about 85°C.
  • reaction temperatures and reagents may be modified as appropriate.
  • the examples provided herein are provided for the purpose of illustrating specific embodiments and aspects and are not intended to limit this disclosure in any way. Persons of ordinary skill can utilise the disclosures and teachings herein to produce other embodiments, aspects, and variations without undue experimentation. All such embodiments, aspects, and variations are considered to be part of this disclosure.
  • RNA ligases catalyse the formation of phosphodiester bonds between RNA molecules. Thermostable RNA ligases play an important role as molecular tools, however their precise physiological role is not completely understood. With the biotechnological potential that thermophilic RNA ligases hold and the extent of cellular function and evolution to uncover, we set out to characterise novel archaeal RNA ligase enzymes. To begin, we focused on the RNA ligase from Pyrococcus furiosus (Pfu), as this is an accepted model organism for elucidating the structures, functions and evolution of thermostable archaeal proteins.
  • Pyrococcus furiosus Pyrococcus furiosus
  • the active site was further investigated by creating mutants K92A and K238A based on the previously-described Methanobacterium thermoautotrophicum K97A and K246A mutants respectively. Additional mutants having significant utility were generated and studied. This is described in detail as follows.
  • Pfu RNA ligase PfuRnl
  • PfuRnl PfuRnl
  • the Pp RNA ligase (Palaeococcus pacificus; PpRnl ) polynucleotide sequences, including the wildtype sequence and the variants encoding PpRnl K92A and PpRnl K238G were codon optimised for recombinant expression in E. coli and ordered from Twist Bioscience. The sequences are provided herein.
  • the Af RNA ligase (Archaeoglobus fulgidus; A/Rnl) polynucleotide sequence, Mf RNA ligase (Methanotorris formicus; AfRnl) polynucleotide sequence, Ga RNA ligase (Geoglobus ahangari; GaRnl) polynucleotide sequence, and Tg RNA ligase (Thermococcus gorgonarius; TgRnl ) polynucleotide sequence, as well as mutated forms of these ligases, were also codon optimised for recombinant expression in E. coli and ordered from Twist Bioscience. The sequences are set out herein.
  • PfuRnl polynucleotide sequences including wildtype and variants encoding PfuRnl K92A and PfuRnl K238G, and also PfuRnl K238A, were cloned into the vector pCA24N and this was used to transform E. coli strain E. cloni® 10G for protein expression.
  • PpRnl , TgRnl , AfRnl, GaRnl and AfRnl polynucleotide sequences were cloned into the vector pET28b and used to transform E. coli strain BL21(DE3) for protein expression.
  • All E. coli strains containing PpRnl , TgRnl , AfRnl, GaRnl and AfRnl polynucleotides in the pET28b vector were grown in 500 mL of LB medium supplemented with kanamycin and incubated at 37°C, 200 rpm. Growth of cells was monitored and at an OD 600 of 0.5-0.7, protein expression was induced by the addition of 0.75 mM IPTG followed by incubation overnight at 18°C, 200 rpm. The cells were harvested by centrifugation in 50 mL conical tubes at 7068 x g, 4°C, for 30 min. The supernatant was discarded, and cell pellets were stored at -20 °C until required.
  • samples were transferred to high speed centrifuge tubes and spun for 20 minutes at 30,000 x g in a Sorvall LYNX 4000 Superspeed Centrifuge. The supernatant was taken out and heat treated at 70°C for 30-45 minutes to remove the bulk of the endogenous E. coli protein and further centrifuged for 20 minutes at 30,000 x g in a Sorvall LYNX 4000 Superspeed Centrifuge. The supernatant was filtered through a 0.22 pm syringe-driven filter prior to loading onto a HisTrap column.
  • a 1 ml HisTrap HP nickel affinity column (Cytiva) was washed with five column volumes (CV) of MQ water and primed with five CV of Lysis Buffer (50 mM Tris. 200 mM NaCl, 10 mM imidazole, pH 7.4) using an AKTA Start or Pure (Cytiva).
  • the filtered supernatant from the lysed cells was in turn loaded onto the HisTrap column. Unbound protein was removed by washing the column with Lysis Buffer at a flow rate of 1 ml/min until the absorbance at 280 nm dropped to baseline.
  • Nucleic acid bound to the protein was eluted out with ten CV of salt wash buffer (50 mM Tris, 4 M NaCl, 10 mM imidazole, pH 7.4) while the protein was bound to the column.
  • the protein bound column was then re-equilibrated with five CV of Lysis Buffer.
  • Bound proteins were eluted with a gradient from 0-100 % elution buffer (50 mM Tris-HCl, 200 mM NaCl and 500 mM imidazole, pH 7.4) at a flow rate of 1 ml/min over 20 ml and collected in 1 ml fractions. The fractions corresponding to the peak/s at 280 nm on the resulting chromatogram were analysed using SDS-PAGE for the protein of interest.
  • Size exclusion chromatography was used to further purify proteins following nickel affinity chromatography.
  • the size exclusion column to be used a Superdex 200 Increase 10/300 GL, was equilibrated with SEC buffer (50 mM Tris-HCl, 200 mM NaCl, pH 7.4).
  • SEC buffer 50 mM Tris-HCl, 200 mM NaCl, pH 7.4.
  • the protein fractions from the nickel purification were concentrated by centrifugation in 15 mL Amicon ultra centrifugal filters with a membrane nominal molecular weight cut-off (NMWL) of 10 kDa and passed through a 0.2 pm filter prior to loading onto the size exclusion column.
  • NMWL membrane nominal molecular weight cut-off
  • Ligation in the presence of a single oligo Ligation reactions with single oligonucleotides were carried out to test the general activity of an RNA ligase or to test the enzyme’s activity with different oligos.
  • Ligation reactions contained a final concentration of 1 X NEBuffer 1 (New England Biolabs; NEB), 5-10 ⁇ M of desired oligonucleotide, 15 ⁇ M ATP, 0.5 U/ ⁇ l of SUPERaseInTM RNase inhibitor (20 U/ ⁇ l ), 10 ⁇ M of respective thermophilic RNA ligase enzyme and nuclease-free water to a total volume of 20 ⁇ L.
  • the reaction mixture was pre-equilibrated at 75 °C for a few minutes before adding the enzyme and incubated for a further 90 minutes.
  • T4 RNA ligase reactions were carried as per the manufacturer’s protocol and the reactions were incubated at 25°C for 90 minutes.
  • the T4 RNA ligase enzyme was obtained from NEB (catalogue number M0204S: T4 RNA Ligase 1 (ssRNA Ligase)).
  • 3 ’ ligation reaction Ligation reactions corresponding to the 3 ’ ligation reaction in the workflow for preparing miRNA sequencing libraries consisted of an RNA oligonucleotide with or without a 5’ phosphate and a pre-adenylated DNA oligonucleotide blocked at 3’ end with an amino modifier.
  • NEB 5' DNA Adenylation Kit
  • Ligation reactions contained a final concentration of 1 X NEBuffer 1 (NEB), 5 ⁇ M of RNA oligonucleotide, 5 ⁇ M of pre-adenylated DNA oligonucleotide, 0.5 U/ ⁇ l of SUPERase*InTM RNase Inhibitor (20 U/ ⁇ l ), 10 ⁇ M of respective thermophilic RNA ligase enzyme and nuclease-free water to a total volume of 20 ⁇ L.
  • the reaction mixture was pre- equilibrated at 75 °C for few minutes before adding the enzyme and incubated for a further 90 minutes.
  • Ligation reactions corresponding to the 5’ ligation reaction in the workflow for preparing miRNA sequencing libraries consisted of an RNA oligonucleotide without a 5’ phosphate and an RNA oligonucleotide blocked at its 3 ’end with an amino modifier.
  • Ligation reactions contained a final concentration of IX NEBuffer 1 (NEB), 15 ⁇ M ATP, 5 ⁇ M of each RNA oligonucleotide, 0.5 U/ ⁇ l of SUPERaseInTM RNase Inhibitor (20 U/ ⁇ l ), 10 ⁇ M of respective thermophilic RNA ligase enzyme and nuclease-free water to a total volume of 20 ⁇ L.
  • the reaction mixture was pre-equilibrated at 75°C for few minutes before adding the enzyme and incubated for a further 90 minutes.
  • thermophilic microorganisms Identification of candidate RNA ligases from thermophilic microorganisms
  • RNA ligase enzyme from the thermophilic microorganism Pyrococcus furiosus has been the focus of research in our laboratory for a number of years.
  • Putative RNA ligases were identified in publicly available (NCBI) genomes of thermophilic bacteria and archaea using the amino acid sequence of the Pfu enzyme as a search 'string'. Sequences were mainly found in species of Euryarcheota and in some Crenarchaeota.
  • Table 1 Putative RNA ligases selected for investigation
  • key motifs as defined by Zhelkovsky and McReynolds (2012), are shown in bold and underlined: motif I (EKXXGYN; SEQ ID NO: 43) and motif V (XXKYXTX; SEQ ID NO: 44). Key lysine residues are shown in bold and also boxed.
  • Codon-optimised coding sequences of PfuRnl and the five putative RNA ligases were expressed in E. coli to enable functional characterisation of these enzymes. GaRnl was omitted from the study due to technical difficulties with large-scale expression and purification.
  • RNA oligonucleotide (Oligo 1)
  • possible reaction products include unmodified oligo, adenylated oligo, circularised products containing one or more copies of the oligo, “dimers” that are two copies of the oligonucleotide ligated together, adenylated dimers, and adenylated and non-adenylated products that contain more than two ligated oligonucleotides.
  • RNA ligases displayed better activity than PfuRnl.
  • PpRnl displayed high adenylation activity.
  • A/Rnl produced notable levels of concatemers.
  • AfRnl displayed very high levels of ligase activity, but moderate levels were also displayed by PfuRnl, PpRnl , and TgRnl . Therefore, each of A/Rnl PfuRnl, PpRnl , and TgRnl are candidate enzymes for miRNA library preparation.
  • PpRnl had extremely high adenylation activity with almost all of the oligonucleotide adenylated and a small proportion converted to a dimer, which was then adenylated as well.
  • AfRnl was extremely active, with most of the oligonucleotide converted to dimerised product or circularised product with some multi-oligonucleotide (>2) concatemers. Some adenylated oligonucleotide was also evident.
  • AfRnl had only moderate/low activity with most of the oligonucleotide remaining unmodified.
  • TgRnl The activity of TgRnl was very similar to that of PfuRnl.
  • RNA oligonucleotides with a range of melting temperatures Experiments were performed to analyse the effectiveness of novel RNA ligases against miRNAs that form secondary structures at different temperatures.
  • a range of synthetic RNA oligonucleotides were designed and synthesised. These were predicted to have a range of unfolding temperatures (i.e., T m values) from 40°C to 85°C as shown in Table 2.
  • T m values i.e., T m values
  • RNA ligase activity for the wild-type enzymes.
  • each enzyme assessed each enzyme’s ability to ligate a variety of synthetic oligonucleotides with different melting temperatures was tested.
  • RNA oligonucleotides with T m values of 38°C, 65°C and >80°C.
  • enzyme and oligonucleotide were incubated at 75°C and successful ligation was determined by the appearance of concatemers of two or more oligos. Similar reactions were performed using the industry standard T4 RNA ligase at a reaction temperature of 25°C.
  • thermostable RNA ligases PfuRnl, PpRnl , MfRnl, and TgRnl , were significantly more consistent in their ligation of the various oligonucleotides.
  • thermostable RNA ligases all showed more activity towards Oligo J (which is folded at the assay temperature of 75°C) than towards Oligo A (which is unfolded at 75°C). This is very surprising and indicates that parameters other than temperature- induced unfolding may contribute to ligation bias.
  • a pre-adenylated DNA adapter is ligated to the 3’ end of the miRNA molecules.
  • Pre-adenylating the adaptor means that the cofactor, ATP, is no longer required by the RNA ligase.
  • omitting the ATP means that two miRNA molecules, which have 5 ’-phosphate groups but are not adenylated, cannot be ligated to each other.
  • mutated variants of enzymes were engineered to disrupt adenylation/deadenylation activity and allow the use of pre-adenylated DNA adapters in an ATP-independent ligation reaction. Mutated versions of the enzymes were tested for their ability to perform this step.
  • the two substrates for ligation were a pre-adenylated DNA oligonucleotide that was blocked with an amino modifier (https://sg.idtdna.com/site/Catalog/Modifications/ Product/3299) at its 3’ end (AppSRl) and the synthetic oligonucleotide Oligo 1 that lacked a phosphate group at its 5' end (Oligo 1-5'B) ( Figure 4A).
  • the first enzyme to be tested was PfuRnl K92A.
  • PfuRnl K92A To test this enzyme’s ability to function in a normal library preparation workflow, a second reaction was also performed using the phosphorylated version of Oligo 1 ( Figure 4A). In both cases, ligation of the RNA and DNA oligonucleotides yielded only the expected products in addition to unreacted oligonucleotides ( Figure 4B).
  • the second step in miRNA library preparation protocols is ligation of an RNA oligonucleotide adapter that is blocked at its 5’ end (via removal of its 5’ phosphate) to the 5’ end of the RNA-DNA hybrid molecule created by 3’ ligation ( Figure 1).
  • This step requires an enzyme that is capable of adenylating RNA in the presence of ATP.
  • an RNA molecule that was blocked with the amino modifier at its 3’ end was used as a model substrate, in place of the RNA-DNA hybrid ( Figure 5A).
  • K238A and K238G variants of PfuRnl enzyme were tested for their ability to perform this step compared with the wild-type enzyme ( Figure 5B). Both variants out- performed the wild-type enzyme for producing the desired ligated product. Wild-type PfuRnl predominantly produced adenylated oligonucleotide and yielded less of the ligated product.
  • T4 RNA ligase displayed significantly greater bias in its ligation of the different RNA oligonucleotides than was seen for PpRnl K238G as shown in Figure 7.
  • oligonucleotides that had somewhat reduced ligation levels using PpRnl K238G had somewhat increased ligation levels using by T4 Rnl, suggesting that use of an enzyme combination may be used to further decrease the ligation bias, beyond what can be achieved by a single enzyme or enzyme variant.
  • RMSD scores root mean square deviations
  • GaRnl to PfuRnl 0.710 A (291 to 291 atoms)
  • thermostable RNA ligases including variant RNA ligases, which find significant utility in miRNA library preparation protocols, amongst other uses.
  • AfRnl (which has a highly divergent sequence from PfuRnl, PpRnl or TgRnl) shows high levels of ligation, adenylation, and circularisation activities. See, e.g., Figure 2. Importantly, it is noted that different levels of activities are observed even though key sequence motifs I and V are conserved amongst these enzymes. See, e.g., Figure 8.
  • RNA ligases were expressed and purified following methods in Example 1. Protein concentration was measured by absorbance at 280 nm using NanodropTM. All RNA ligase enzymes used in assays were taken from 2 mg.ml -1 stocks stored at 4 °C, and purifications were performed at similar timeframes to account for between - enzyme differences. Enzyme stocks are aliquoted and diluted using 50 mM Tris pH 7.4, 200 mM NaCl, 10% glycerol into 20 ⁇ M stocks for assay reactions. Master stocks for substrates and cofactors were prepared in Ultrapure DNase/RNase-free distilled water.
  • Standard reaction mixtures (20 pl) containing NEBuffer 1.1 (New England BioLabs), 5 ⁇ M 5’-phosphyated RNA Oligo 1 and 10 ⁇ M wild-type or mutant RNA ligase were set up as per Table 4, below, with or without cofactor (for a final concentration of 70 ⁇ M of cofactor). Reactions were incubated for 90 minutes at 65°C in a T100 Thermal Cycler (BioRad). Variations in substrates, high energy cofactors or temperature are indicated, and reactions use oligonucleotide 1 (5’phos-GAGCUAGCAUUAACUUGG; SEQ ID NO: 45) unless otherwise specified.
  • reaction mixture by adding 2X Formamide loading dye (0.5 M EDTA, 95% Formamide, 2% Bromophenol Blue, 1% Xylene Cyanol, 1% Orange G). Reactions were stored at 4°C overnight or frozen at 20°C.
  • 2X Formamide loading dye 0.5 M EDTA, 95% Formamide, 2% Bromophenol Blue, 1% Xylene Cyanol, 1% Orange G. Reactions were stored at 4°C overnight or frozen at 20°C.
  • the gels were incubated and protected from light (wrapped in tinfoil) with agitation for approximately 15 minutes to avoid sample leeching.
  • Gel bands were visualised using an iBRIGHTTM gel imager (Invitrogen, Thermo Fisher Scientific).
  • ImageJ (Rasband et al., 1997-2018; NIH) was used to compare the intensity of bands on the urea-PAGE gel images to quantify ligation by-products between reaction samples. Brightness and contrast adjustments were made using the automatic function of ImageJ. The image background was subtracted using the rolling ball radius method, with a rolling ball radius size set to 50.0 pixels. Each band was selected and circumscribed, producing a user-defined area around the band (region of interest; ROI). Once defined, the same ROI dimensions were used to measure all bands on the gel images. The Gel Analyser tool of ImageJ was used to generate profiles of the bands.
  • the resulting histograms were used to manually create fluorescence profiles for the gel images by drawing a straight line across the baseline of the histogram. Data were analysed using Microsoft® Excel® (Version 2205) to calculate the relative density of the peaks. The extent of ligation and adenylation was quantified using ImageJ (intensity of ligation/adenylation band/full lane band intensity* 100). Graphs depicting ligation or adenylation were made using GraphPad Prism.
  • the PpRnl K283G mutant showed specific increases in ligation activity (end-to-end) and circularisation activity (Figure 15B).
  • the TgRnl K238G mutation shows the opposite effect; this mutant has its enzymatic activity dramatically reduced ( Figure 15A).
  • Figure 15A shows that mutation of the motif I lysine (K92A) in
  • TgRnl abolished ligase self-adenylation, evident by no observable adenylation or ligation of the input RNA substrate. Mutation of the corresponding lysine in PpRnl (K92A) also abolished ligation activity, with no ligation observable with non-adenylated RNA substrate ( Figure 15B).
  • TgRnl (without mutation) displays efficient ligation using both ATP and NAD + cofactors, ligating approximately 40% ( ⁇ 1.5%) and 43% ( ⁇ 7%) of the input RNA substrate, respectively ( Figure 16 B).
  • the ligases of this disclosure find use in various methods, including miRNA library preparation and subsequent deep sequencing, circularisation of RNA for vaccines and other therapeutics, and capping of oligonucleotides through adenylation, uridylation, or other similar modifications.
  • thermophilic RNA ligases as developed herein in the library preparation steps. In particular, we sought to perform ligations at elevated temperatures and quantify the effect on this bias.
  • the disclosed enzyme variant PfuRnl K92A replaced the ligase enzyme from the commercial NEXTFLEX kit for the 3' adapter ligation step.
  • PfuRnl K92A (but not the NEXTFLEX ligase enzyme) was added at the appropriate step in the protocol, to a final concentration of 10 ⁇ M.
  • the 3' adapter ligation reaction was then carried out at 65°C for 2 hours. All other steps in the library preparation procedure were carried out using the reagents in the NEXTFLEX kit, according to the manufacturer’s protocols.
  • the disclosed enzyme variant PfuRnl K238G replaced the ligase enzyme from the commercial NEXTFLEX kit for the 5' adapter ligation step.
  • PfuRnl K238G (but not the NEXTFLEX ligase enzyme) was added at the appropriate step in the protocol, to a final concentration of 8 ⁇ M.
  • the 5' adapter ligation reaction was then carried out at 65°C for 90 minutes. The remainder of the library preparation procedure was carried out using the reagents in the NEXTFLEX kit, according to the manufacturer’s protocols.
  • PfuRnl K92A replaced the ligase enzyme from the commercial NEXTFLEX kit for the 3' adapter ligation step and PfuRnl K238G replaced the ligase enzyme from the commercial NEXTFLEX kit for the 5' adapter ligation step.
  • PfuRnl K92A and PfuRnl K238G (but not the NEXTFLEX ligase enzymes) were added at the appropriate steps in the protocol, to a final concentration of 10 ⁇ M for PfuRnl K92A and 8 ⁇ M for PfuRnl K238G.
  • the 3' adapter ligation reaction was carried out at 65°C for 2 hours and the 5' adapter ligation reaction was carried out at 65°C for 90 minutes.
  • the remainder of the library preparation procedure was carried out using the reagents in the NEXTFLEX kit, according to the manufacturer’s protocols.
  • the ligase enzymes of this disclosure can be used to decrease sequence bias during library preparation, and have notable utility in various molecular biology protocols.
  • RNA ligase 2 (gp24.1) exemplifies a family of RNA ligases found in all phylogenetic domains. Proc Natl Acad Sci USA. 2002, 99(20): 12709-14.
  • Torchia et al., Archaeal RNA ligase is a homodimeric protein that catalyzes intramolecular ligation of single-stranded RNA and DNA. Nucleic Acids Res. 2008, 36(19):6218-27.
  • SEQ ID NO: 1 to SEQ ID NO: 45 correspond to the various polypeptide and polynucleotide sequences set out herein.
  • the database sequence information (including sequences and accession numbers) provided with this description corresponds to the information accessed online as of 9 November 2021.
  • Any references cited in this specification are hereby incorporated by reference. All amino acid and nucleotide sequences in the references cited in this specification are hereby incorporated into this disclosure. No admission is made that any reference constitutes prior art. Nor does discussion of any reference constitute an admission that such reference forms part of the common general knowledge in the art, in Australia or in any other country.

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Abstract

La présente divulgation concerne des enzymes d'ARN-ligase, en particulier des enzymes d'ARN-ligase thermostables, et comprenant des enzymes variantes, ainsi que leur préparation et leur utilisation. L'invention concerne spécifiquement des procédés de préparation des enzymes d'ARN-ligase, des procédés de ligature utilisant ces enzymes, ainsi que des polynucléotides utilisés pour produire ces enzymes, des banques comprenant ces polynucléotides, et des souches microbiennes comprenant ces polynucléotides.
PCT/NZ2022/050140 2021-11-09 2022-11-09 Enzymes de ligase d'arn et procédés de préparation et d'utilisation de ces enzymes Ceased WO2023085955A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025124917A1 (fr) * 2023-12-13 2025-06-19 Hummingbird Diagnostics Gmbh Séquençage par nanopores de petits arn non codants
WO2025180432A1 (fr) * 2024-02-28 2025-09-04 图维生物医药科技(苏州)有限公司 Enzyme pour coiffage d'arn et son système de réaction

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DATABASE Protein 23 September 2015 (2015-09-23), ANONYMOUS : "ATP dependent DNA ligase [Palaeococcus pacificus DY20341] ", XP093068108, retrieved from NCBI Database accession no. AIF68870 *
DATABASE Protein 28 February 2022 (2022-02-28), ANONYMOUS : "RNA ligase [Palaeococcus pacificus] ", XP093068111, retrieved from NCBI Database accession no. WP048164472 *
ZHELKOVSKY ALEXANDER M, MCREYNOLDS LARRY A: "Structure-function analysis of Methanobacterium thermoautotrophicum RNA ligaseengineering a thermostable ATP independent enzyme", BMC MOLECULAR BIOLOGY, BIOMED CENTRAL LTD., GB, vol. 13, no. 1, 1 January 2012 (2012-01-01), GB , pages 1 - 10, XP093068116, ISSN: 1471-2199 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025124917A1 (fr) * 2023-12-13 2025-06-19 Hummingbird Diagnostics Gmbh Séquençage par nanopores de petits arn non codants
WO2025180432A1 (fr) * 2024-02-28 2025-09-04 图维生物医药科技(苏州)有限公司 Enzyme pour coiffage d'arn et son système de réaction

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