WO2023048291A1 - ｔＲＮＡ、アミノアシルｔＲＮＡ、ポリペプチド合成用試薬、非天然アミノ酸の導入方法、ポリペプチドの作製方法、核酸ディスプレイライブラリの作製方法、核酸－ポリペプチド連結体及びスクリーニング方法 - Google Patents

ｔＲＮＡ、アミノアシルｔＲＮＡ、ポリペプチド合成用試薬、非天然アミノ酸の導入方法、ポリペプチドの作製方法、核酸ディスプレイライブラリの作製方法、核酸－ポリペプチド連結体及びスクリーニング方法 Download PDF

Info

Publication number
WO2023048291A1
WO2023048291A1 PCT/JP2022/035791 JP2022035791W WO2023048291A1 WO 2023048291 A1 WO2023048291 A1 WO 2023048291A1 JP 2022035791 W JP2022035791 W JP 2022035791W WO 2023048291 A1 WO2023048291 A1 WO 2023048291A1
Authority
WO
WIPO (PCT)
Prior art keywords
trna
polypeptide
nucleic acid
codon
amino acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP2022/035791
Other languages
English (en)
French (fr)
Japanese (ja)
Inventor
由紀子 石井
譲 富永
史織 木原
貴弘 芳坂
貴嘉 渡邉
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Fujifilm Corp
Original Assignee
Fujifilm Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Fujifilm Corp filed Critical Fujifilm Corp
Priority to CN202280063245.1A priority Critical patent/CN118019845A/zh
Priority to JP2023549778A priority patent/JPWO2023048291A1/ja
Priority to EP22873042.0A priority patent/EP4389890A4/en
Publication of WO2023048291A1 publication Critical patent/WO2023048291A1/ja
Priority to US18/611,981 priority patent/US20240229015A1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1044—Preparation or screening of libraries displayed on scaffold proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1062—Isolating an individual clone by screening libraries mRNA-Display, e.g. polypeptide and encoding template are connected covalently
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1096—Processes for the isolation, preparation or purification of DNA or RNA cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • 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
    • C12N15/67—General methods for enhancing the expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • 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
    • C12N2320/00—Applications; Uses
    • C12N2320/10—Applications; Uses in screening processes
    • 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
    • C12N2330/00—Production
    • C12N2330/30—Production chemically synthesised
    • C12N2330/31—Libraries, arrays

Definitions

  • the present disclosure relates to tRNAs, aminoacyl-tRNAs, reagents for polypeptide synthesis, methods for introducing unnatural amino acids, methods for preparing polypeptides, methods for preparing nucleic acid display libraries, nucleic acid-polypeptide conjugates, and screening methods.
  • Patent document 1 discloses that an artificial aminoacylation catalyst is used to acylate tRNA with a special amino acid, and a library of special polypeptides is prepared by a cell-free translation system containing the tRNA acylated with the special amino acid, and binds to a target protein. Methods of screening for unique polypeptides are disclosed.
  • Patent Document 2 discloses a method for producing a cyclic peptide compound with excellent cell membrane permeability and a screening method.
  • Patent Document 3 discloses a tRNA obtained by modifying the tRNA for tryptophan of Mycoplasma capricolum, having CUA as an anticodon and pairing with a UAG codon, and using this tRNA to convert an unnatural amino acid into a polypeptide. A method of introducing is disclosed.
  • the present disclosure provides tRNAs and aminoacyl tRNAs that translate UAA codons into amino acids, polypeptide synthesis reagents that introduce at least two types of unnatural amino acids into polypeptides, methods for introducing unnatural amino acids, methods for producing polypeptides, and nucleic acids.
  • a display library preparation method, a nucleic acid-polypeptide conjugate having at least two types of unnatural amino acids introduced into a polypeptide, and a polypeptide having at least two types of unnatural amino acids introduced having the desired activity It is an object of the present invention to provide a screening method for discovering polypeptides.
  • ⁇ 1> A tRNA in which tRNA for tryptophan of Mycoplasma pneumoniae is modified, the tRNA having UUA as an anticodon and pairing with a UAA codon.
  • ⁇ 2> The tRNA according to ⁇ 1>, wherein the base adjacent to the 5' side of the CCA terminus is A or G.
  • ⁇ 3> The tRNA according to ⁇ 1> or ⁇ 2>, wherein the combination of the third base from the 5' end and a base that pairs with this base is UA, GU or UG.
  • ⁇ 4> The tRNA according to ⁇ 1> or ⁇ 2>, wherein the combination of the 4th base from the 5′ end and a base that pairs with this base is GU.
  • ⁇ 5> A tRNA having one base sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 15 and SEQ ID NO: 17.
  • aminoacyl-tRNA of ⁇ 6> wherein the amino acid is one selected from the group consisting of chloroacetylated lysine, N-methylalanine, N-methylphenylalanine, and fluorescently labeled amino acids.
  • amino acid is one selected from the group consisting of chloroacetylated lysine, N-methylalanine, N-methylphenylalanine, and fluorescently labeled amino acids.
  • a reagent for polypeptide synthesis comprising a tRNA in which Mycoplasma pneumoniae tryptophan tRNA is modified, the tRNA having UUA as an anticodon and pairing with a UAA codon.
  • a method for introducing at least two unnatural amino acids into a polypeptide comprising: a nucleic acid having a base sequence containing a UAG codon and a UAA codon; an aminoacyl-tRNA obtained by modifying the tRNA for tryptophan of Mycoplasma capricolum, wherein the tRNA has CUA as an anticodon and pairs with a UAG codon, and a first unnatural amino acid is bound to the tRNA; an aminoacyl-tRNA obtained by modifying the tRNA for tryptophan of Mycoplasma pneumoniae, wherein the tRNA has UUA as an anticodon and pairs with the UAA codon, and a second unnatural amino acid is bound to the tRNA;
  • a method for introducing an unnatural amino acid comprising expressing a polypeptide from a nucleic acid by a cell-free peptide synthesis system containing ⁇ 12> The method for introducing an unnatural amino acid according to ⁇ 11>, wherein the
  • a method for producing a polypeptide containing at least two unnatural amino acids in its amino acid sequence comprising: a nucleic acid having a base sequence containing a UAG codon and a UAA codon; an aminoacyl-tRNA obtained by modifying the tRNA for tryptophan of Mycoplasma capricolum, wherein the tRNA has CUA as an anticodon and pairs with a UAG codon, and a first unnatural amino acid is bound to the tRNA; an aminoacyl-tRNA obtained by modifying the tRNA for tryptophan of Mycoplasma pneumoniae, wherein the tRNA has UUA as an anticodon and pairs with the UAA codon, and a second unnatural amino acid is bound to the tRNA; expressing the polypeptide from a nucleic acid by a cell-free peptide synthesis system containing ⁇ 14>
  • the method for producing the polypeptide according to ⁇ 13>, wherein the nucleotide sequence further comprises a UUA U
  • a method of producing a nucleic acid display library comprising expressing a polypeptide from a nucleic acid by a cell-free peptide synthesis system containing a to produce a nucleic acid-polypeptide conjugate.
  • Producing a nucleic acid-polypeptide conjugate comprises producing an mRNA-polypeptide conjugate, and reverse-transcribes the mRNA of the mRNA-polypeptide conjugate to produce a cDNA-polypeptide conjugate.
  • the method for producing a nucleic acid display library according to ⁇ 15> comprising: ⁇ 17>
  • nucleic acid-polypeptide conjugate according to ⁇ 18> which is an mRNA-polypeptide conjugate or a cDNA-polypeptide conjugate.
  • a tRNA and an aminoacyl tRNA that translate a UAA codon into an amino acid a reagent for polypeptide synthesis that introduces at least two types of unnatural amino acids into a polypeptide, a method for introducing an unnatural amino acid, and a method for producing a polypeptide and a method for preparing a nucleic acid display library, a nucleic acid-polypeptide conjugate having at least two types of non-natural amino acids introduced into a polypeptide, and a polypeptide having at least two types of non-natural amino acids introduced, and the activity of interest and screening methods for finding polypeptides having
  • FIG. 11 shows an example embodiment of a tRNA of the present disclosure and is a schematic representation of a cloverleaf structure.
  • Electrophoresis gel of formamide-fixed tRNA It is an electrophoresis gel of a polypeptide into which fluorescent-labeled amino acids and N-methylphenylalanine have been introduced multiple times. It is an electrophoresis gel of mRNA display in which fluorescently-labeled amino acids and N-methylphenylalanine are multiply introduced.
  • a numerical range indicated using “to” indicates a range including the numerical values before and after "to” as the minimum and maximum values, respectively.
  • the upper limit or lower limit of one numerical range may be replaced with the upper or lower limit of another numerical range described step by step.
  • the upper or lower limits of the numerical ranges may be replaced with the values shown in the examples.
  • each component may contain multiple types of applicable substances.
  • the amount of each component in the composition in this disclosure if there are multiple types of substances corresponding to each component in the composition, unless otherwise specified, multiple types of substances present in the composition means the total amount of
  • a polypeptide refers to a molecule in which amino acids are linked by peptide bonds. There is no limit to the number of amino acid residues in a polypeptide, and polypeptide is a term that includes proteins.
  • the polypeptide in the present disclosure preferably has 6 or more amino acid residues.
  • Polypeptides include polypeptides with post-translational modifications of amino acids. Post-translational modifications of amino acids include phosphorylation, methylation, acetylation, and the like.
  • nucleic acid refers to a molecule that carries information for synthesizing a polypeptide.
  • Nucleic acids include all nucleic acids (e.g., DNA, RNA, analogues thereof, natural products, and artificial products), and all nucleic acids linked to low-molecular-weight compounds, groups, molecules other than nucleic acids, structures, etc. It is a term that includes A nucleic acid may be a single-stranded nucleic acid or a double-stranded nucleic acid.
  • Table 1 shows the base sequences of tRNAs identified by SEQ ID NOs in the present disclosure.
  • SEQ ID NO: 2 and SEQ ID NOS: 5-17 are variants of Mycoplasma pneumoniae tRNA for tryptophan.
  • SEQ ID NO: 18 is tRNA for wild-type tryptophan of Mycoplasma pneumoniae.
  • SEQ ID NO: 1 and SEQ ID NO: 19 are variants of Mycoplasma capricolum tryptophan tRNA.
  • SEQ ID NO: 3 is a variant of Staphylococcus aureus tRNA for tryptophan.
  • SEQ ID NO: 4 is a variant of the Shewanella oneidensis tRNA for tryptophan.
  • the anticodon is underlined and the bases that differ from the wild-type tryptophan tRNA for each microorganism are shown in italics.
  • the tRNAs found in nature range in number from 70 to 90 bases.
  • a typical tRNA has 76 bases.
  • the base positions described in the remarks column of Table 1 are positions specified in comparison with a typical tRNA having 76 bases. Taking SEQ ID NO: 2 as an example, the number of bases is 74, and the position of the 3′ end is specified as base number 76, and the position adjacent to the 5′ side of the CCA end is specified as base number 73.
  • the tRNA of the present disclosure is a modified tRNA for Mycoplasma pneumoniae for tryptophan, has UUA as an anticodon, and pairs with a UAA codon.
  • the tRNAs of this disclosure translate UAA codons into amino acids.
  • Mycoplasma pneumoniae has two types of tRNA for wild-type tryptophan (Nucleic Acids Res. 1993 Oct 25; 21(21): 4967-4974).
  • the tRNAs of the present disclosure include variants of either of the two wild-type tRNAs for tryptophan.
  • the tRNA of the present disclosure is preferably a modified tRNA of the wild-type tryptophan tRNA shown in SEQ ID NO:18.
  • the tRNA of the present disclosure is a tRNA in which the anticodon of Mycoplasma pneumoniae wild-type tryptophan tRNA is modified from CCA to UUA. With this modification, the tRNA of the present disclosure has the function of translating UAA codons into amino acids.
  • the inventors of the present disclosure modified wild-type tryptophan tRNAs of various microorganisms and, as a result, found that modified Mycoplasma pneumoniae tryptophan tRNAs efficiently translated UAA codons into amino acids.
  • the tRNA of the present disclosure is preferably a variant of Mycoplasma pneumoniae wild-type tryptophan tRNA in which not only the anticodon but also the acceptor stem is modified. Modification of the acceptor stem is presumed to affect the higher-order structure of tRNA and increase the efficiency of the peptidyl transfer reaction in the ribosome. Examples of modification of the acceptor stem include modification of the base adjacent to the 5' side of the CCA end; modification of the combination of the third base from the 5' end and the base that pairs with this base; modification of the combination of a base and a base that pairs with this base;
  • the CCA terminus of tRNA is 3 bases commonly present at the 3′ terminus of tRNA. Pairing within the tRNA means that the base pairs face each other in the cloverleaf structure of the tRNA.
  • FIG. 1 shows an example embodiment of the tRNA of the present disclosure and is a schematic diagram of a cloverleaf structure.
  • FIG. 1 shows an example of modification of the base (base number 73) adjacent to the 5′ side of the CCA terminal, modification of the combination of the third base from the 5′ end and the base (base number 70) that pairs with this base.
  • An example and a modified example of the combination of the 4th base from the 5′-end and the base (base number 69) that pairs with this base are exemplified.
  • the tRNA of the present disclosure preferably has at least one form selected from the group consisting of form (1), form (2), and form (3) below.
  • Form (1) The base adjacent to the 5' side of the CCA terminus is A or G.
  • Form (2) A combination of the third base from the 5' end and a base that pairs with this base is UA, GU or UG.
  • the third base from the 5' end is described first.
  • “UA” has U as the third base from the 5' end and A as the base that pairs with the third base in the cloverleaf structure.
  • Form (3) A combination of the 4th base from the 5' end and a base that pairs with this base is GU.
  • the fourth base from the 5' end is described first.
  • the 4th base from the 5' end is G, and the base that pairs with the 4th base in the cloverleaf structure is U.
  • Examples of embodiments of the tRNA of the present disclosure include tRNA having form (1) and form (2) and tRNA having form (1) and form (3).
  • Exemplary embodiments of tRNAs of the present disclosure include SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, A tRNA having the base sequence of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17 is included.
  • tRNA having the base sequence of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 15, or SEQ ID NO: 17 is preferable from the viewpoint of efficiently translating UAA codons into amino acids.
  • the tRNA of the present disclosure can be produced using known genetic engineering techniques based on the base sequence of wild-type tryptophan tRNA from Mycoplasma pneumoniae. For example, a tRNA gene is designed, a tRNA gene is produced by PCR (Polymerase Chain Reaction) using appropriate primers, and tRNA is transcribed from the tRNA gene.
  • PCR Polymerase Chain Reaction
  • aminoacyl-tRNA of the present disclosure is a tRNA in which an amino acid is attached to the tRNA of the present disclosure.
  • amino acid is covalently linked to the 3' end of the tRNA.
  • the tRNA of the present disclosure can be acylated at any amino acid. Therefore, the type of amino acid possessed by the aminoacyl-tRNA of the present disclosure is not limited.
  • Amino acids as used herein include natural amino acids, unnatural amino acids, modified amino acids and derivatives thereof.
  • natural amino acids refer to amino acids that generally constitute proteins, such as alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, Refers to serine, threonine, tryptophan, tyrosine and valine.
  • Natural amino acids may be natural or man-made.
  • unnatural amino acids refer to amino acids other than the 20 types of amino acids described above, including both natural and artificial products.
  • unnatural amino acids include amino acids having a chloroacetyl group (eg, chloroacetylated amino acids such as chloroacetyl lysine, chloroacetyldiaminobutyric acid).
  • unnatural amino acids include N-methylamino acids (eg, N-methylalanine, N-methylphenylalanine).
  • a modified amino acid is a labeled amino acid, which is an amino acid bound to a labeled compound.
  • a labeling compound is a substance detectable by biochemical, chemical, immunochemical or electromagnetic detection methods. Labeling compounds include dye compounds, fluorescent substances, chemiluminescent substances, bioluminescent substances, enzyme substrates, coenzymes, antigenic substances, substances that bind to specific proteins, and magnetic substances. Labeled amino acids are, for example, fluorescently labeled amino acids, photoresponsive amino acids, photoswitch amino acids, fluorescent probe amino acids, etc., when classified by function.
  • the amino acid and the labeling compound may be directly bound, or the amino acid and the labeling compound may be bound via a spacer.
  • Spacers include polyolefins such as polyethylene and polypropylene; polyethers such as polyoxyethylene, polyethylene glycol and polyvinyl alcohol; polystyrene, polyvinyl chloride, polyester, polyamide, polyimide, polyurethane and polycarbonate.
  • Derivatives of natural amino acids, unnatural amino acids or modified amino acids include, for example, hydroxy acids, mercapto acids and carboxylic acids.
  • aminoacyl-tRNAs of the present disclosure include aminoacyl-tRNAs in which one selected from the group consisting of chloroacetylated lysine, N-methylalanine, N-methylphenylalanine, and fluorescently labeled amino acids is bound to tRNA.
  • Methods for acylating the tRNA of the present disclosure with amino acids include the following methods.
  • the CA dinucleotide at the 3' end of the tRNA is omitted.
  • a CA dinucleotide is bound to the carboxyl group of the amino acid.
  • Both are ligated using RNA ligase. This method is known and its details are disclosed, for example, in WO2004/009709 and WO2007/055429.
  • the methods disclosed in these documents can be employed to produce the aminoacyl-tRNAs of the present disclosure.
  • the present disclosure provides two forms of reagents for polypeptide synthesis (ie, a first reagent for polypeptide synthesis and a second reagent for polypeptide synthesis).
  • the first reagent for polypeptide synthesis comprises a tRNA in which the tRNA for tryptophan of Mycoplasma capricolum is modified, the tRNA having CUA as an anticodon and paired with a UAG codon; A tRNA in which Mycoplasma pneumoniae tryptophan is modified, the tRNA having UUA as an anticodon and pairing with a UAA codon.
  • the above two types of tRNAs may be mixed or unmixed.
  • the first reagent for polypeptide synthesis may further contain tRNAs other than the two types of tRNAs described above.
  • the second reagent for polypeptide synthesis is an aminoacyl-tRNA obtained by modifying the tRNA for tryptophan of Mycoplasma capricolum, wherein the tRNA has CUA as an anticodon and pairs with a UAG codon, and a first unnatural amino acid is bound to the tRNA; A tRNA in which Mycoplasma pneumoniae tryptophan is modified, the tRNA having UUA as an anticodon and paired with the UAA codon, and an aminoacyl tRNA in which a second unnatural amino acid is bound to the tRNA.
  • the two types of aminoacyl-tRNA may be mixed or unmixed.
  • the second polypeptide synthesis reagent may further contain an aminoacyl-tRNA other than the above two aminoacyl-tRNAs.
  • the first non-natural amino acid and the second non-natural amino acid are different types of amino acids.
  • the first non-natural amino acid and the second non-natural amino acid each comprise a non-natural amino acid; a natural amino acid or a modified amino acid of a non-natural amino acid; or a derivative of a natural amino acid, a non-natural amino acid or a modified amino acid.
  • the details of these amino acids are as described above.
  • a tRNA obtained by modifying the Mycoplasma capricolum tryptophan tRNA and having CUA as an anticodon and pairing with a UAG codon is referred to as "UAG codon-translated tRNA”.
  • a tRNA obtained by modifying the Mycoplasma pneumoniae tryptophan tRNA and having UUA as an anticodon and pairing with a UAA codon is referred to as "UAA codon-translated tRNA”.
  • the reagent for polypeptide synthesis of the present disclosure is a reagent that uses both UAG codon-translated tRNA and UAA codon-translated tRNA. At least two unnatural amino acids can be introduced into the polypeptide by acylating both tRNAs with separate unnatural amino acids and combining them.
  • the first polypeptide synthesis reagent is, for example, acylated with a non-natural amino acid and then added to a cell-free peptide synthesis system for use.
  • the second polypeptide synthesis reagent is used, for example, by adding it to a cell-free peptide synthesis system. According to these usage patterns, at least two types of unnatural amino acids can be introduced into a polypeptide synthesized by a cell-free peptide synthesis system. The details of the cell-free peptide synthesis system are as described later.
  • the first reagent for polypeptide synthesis may contain at least UAG codon-translated tRNA and UAA codon-translated tRNA among the elements necessary for polypeptide synthesis.
  • the second reagent for polypeptide synthesis contains UAG codon-translated tRNA bound with at least a first unnatural amino acid and UAA codon-translated tRNA bound with a second unnatural amino acid among elements necessary for polypeptide synthesis. I wish I could.
  • Reagents for polypeptide synthesis of the present disclosure may contain all the elements necessary for polypeptide synthesis.
  • Reagents for polypeptide synthesis of the disclosure may include equipment used for polypeptide synthesis.
  • a UAG codon-translated tRNA which is a variant of the Mycoplasma capricolum tryptophan tRNA, is disclosed in WO 2007/055429.
  • the method for introducing an unnatural amino acid, the method for producing a polypeptide, and the method for producing a nucleic acid display library, which will be described later. can be done.
  • the UAG codon-translated tRNA which is a variant of Mycoplasma capricolum tryptophan tRNA, is a group consisting of the following form (A), form (B) and form (C) from the viewpoint of efficiently translating the UAG codon into an amino acid. It is preferable to have at least one form selected from.
  • Form (A): GC is the combination of the 5' end and the base that pairs with this base. In the description of 2 bases, the base at the 5' end is described first. The base at the 5' end is G, and the base that pairs with the base at the 5' end in the cloverleaf structure is C.
  • Form (B): A is the fourth base from the 3' end.
  • Form (C): A, C, G or U is inserted at a position adjacent to the 5' side of the CCA terminus.
  • a variant of the tryptophan tRNA of Mycoplasma capricolum can be produced using known genetic engineering techniques based on the base sequence of the wild-type tryptophan tRNA of Mycoplasma capricolum. For example, a tRNA gene is designed, a tRNA gene is produced by PCR using appropriate primers, and a tRNA is transcribed from the tRNA gene.
  • the method of introducing unnatural amino acids of the present disclosure is a method of introducing at least two kinds of unnatural amino acids into a polypeptide, and includes expressing a polypeptide from a nucleic acid by a cell-free peptide synthesis system.
  • a method for producing a polypeptide of the present disclosure is a method for producing a polypeptide containing at least two unnatural amino acids in its amino acid sequence, and includes expressing the polypeptide from a nucleic acid by a cell-free peptide synthesis system.
  • the cell-free peptide synthesis system in the method for introducing an unnatural amino acid and the method for producing a polypeptide of the present disclosure comprises: a nucleic acid having a base sequence containing a UAG codon and a UAA codon; an aminoacyl-tRNA in which a first unnatural amino acid is bound to a UAG codon-translated tRNA, which is a variant of the Mycoplasma capricolum tryptophan tRNA; It contains an aminoacyl-tRNA in which a second unnatural amino acid is bound to a UAA codon-translated tRNA that is a variant of the Mycoplasma pneumoniae tryptophan tRNA.
  • the first unnatural amino acid and the second unnatural amino acid are different types of amino acids.
  • the first non-natural amino acid and the second non-natural amino acid each comprise a non-natural amino acid; a natural amino acid or a modified amino acid of a non-natural amino acid; or a derivative of a natural amino acid, a non-natural amino acid or a modified amino acid.
  • the details of these amino acids are as described above.
  • a cell-free peptide synthesis system is a reaction system for polypeptide synthesis that (1) translates nucleic acids or (2) transcribes and translates nucleic acids without using cells. is.
  • a cell-free peptide synthesis system consists of a template nucleic acid, ribosomes, factors and enzymes for transcription and/or translation, enzymes necessary for constructing the system, various substrates, energy sources, buffers and salts.
  • Factors and enzymes for transcription and/or translation include substances derived from prokaryotic cells such as E. coli; substances derived from eukaryotic cells such as wheat germ, animal cells and insect cells;
  • the template nucleic acid in the cell-free peptide synthesis system may be DNA or RNA. In DNA, UAG codons are replaced with TAG and UAA codons with TAA.
  • a template nucleic acid may be a single-stranded nucleic acid or a double-stranded nucleic acid.
  • a template nucleic acid may be a linear nucleic acid or a circular nucleic acid.
  • the template nucleic acid may be a nucleic acid in which a nucleotide sequence required for polypeptide synthesis is incorporated into a vector (plasmid vector, cosmid vector, etc.).
  • a template nucleic acid has a base sequence necessary for synthesizing a polypeptide by a cell-free peptide synthesis system.
  • Nucleotide sequences necessary for polypeptide synthesis include, of course, a nucleotide sequence in which codons corresponding to the amino acid sequence of a polypeptide are aligned (ie, a coding region), but also promoter sequences and ribosome binding sequences.
  • the base sequence of the template nucleic acid may or may not contain a stop codon.
  • a stop codon means a codon with no matching tRNA.
  • An example of a stop codon is the UGA codon. If the base sequence of the template nucleic acid does not contain a stop codon, an mRNA-ribosome-polypeptide conjugate can be formed.
  • a cell-free peptide synthesis system that translates nucleic acids includes a ribosome, a translation initiation factor, a translation elongation factor, a translation termination factor, an aminoacyl-tRNA synthetase, a tRNA acylated by an aminoacyl-tRNA synthetase, and It comprises a tRNA of the present disclosure conjugated to an amino acid, and if the system is derived from E. coli, further comprises a methionyl tRNA transformylase.
  • a cell-free peptide synthesis system that transcribes and translates nucleic acids (e.g., DNA) comprises, in addition to the constituents of (1), RNA polymerase (e.g., T7 RNA polymerase) and nucleoside triphosphate, which is a substrate for RNA polymerase. Contains acid.
  • RNA polymerase e.g., T7 RNA polymerase
  • nucleoside triphosphate which is a substrate for RNA polymerase.
  • the mRNA-ribosome-polypeptide conjugate can be formed by removing the translation terminator from the constituent substances.
  • Enzymes other than factors and enzymes for transcription and/or translation include, for example, enzymes for regeneration of energy such as creatine kinase, myokinase, nucleoside diphosphate kinase (NDPK); inorganic pyrophosphatase Enzymes for decomposition of inorganic pyrophosphate generated in transcription and/or translation such as;
  • Various substrates include natural and/or unnatural amino acids, nucleotide triphosphates as energy sources, creatine phosphate, formyl folic acid, and the like.
  • Nucleotide triphosphates include ATP, GTP, CTP and UTP, ATP and GTP are used in (1), and ATP, GTP, CTP and UTP are used in (2).
  • a potassium phosphate buffer (pH 7.3) is usually used.
  • Commonly used salts include potassium glutamate, ammonium chloride, magnesium acetate, calcium chloride, putrescine, spermidine, dithiothreitol (DTT) and the like.
  • Any known cell-free peptide synthesis system can be employed for the method for introducing an unnatural amino acid and the method for producing a polypeptide of the present disclosure.
  • Examples of commercially available cell-free peptide synthesis systems include PUREfrex (Gene Frontier, "PUREfrex” is a registered trademark), Human Cell-Free Protein Expression System (Takara Bio), Rapid Translation System (Roche), and Expressway Cell-Free Expression System. (Invitrogen) and the like.
  • the UAG codon and UAA codon in the nucleotide sequence encoding the polypeptide are combined with the UAG codon-translated tRNA bound with the first unnatural amino acid and the second unnatural amino acid.
  • Each of the UAA codon-translating tRNAs to which the unnatural amino acids are attached introduces at least two unnatural amino acids into the polypeptide.
  • polypeptide embodiments include N-methylamino acids (eg, N-methylalanine or N-methylphenylalanine), amino acids with a chloroacetyl group (eg, chloroacetyldiaminobutyric acid or chloroacetylated lysine), and thiol groups.
  • N-methylamino acids eg, N-methylalanine or N-methylphenylalanine
  • amino acids with a chloroacetyl group eg, chloroacetyldiaminobutyric acid or chloroacetylated lysine
  • thiol groups eg, thiol groups
  • a UAG codon-translated tRNA bound with an N-methylamino acid and a UAA codon-translated tRNA bound with an amino acid having a chloroacetyl group (or a UAG codon-translated tRNA bound with an amino acid with a chloroacetyl group and UAA codon-translated tRNA bound with N-methyl amino acid) may be used.
  • This polypeptide is cyclized by reaction of the chloroacetyl group and the thiol group to give a cyclic N-methyl polypeptide.
  • the number of amino acid residues of the cyclic N-methyl polypeptide is, for example, 3-20.
  • a nucleic acid display library refers to a set of nucleic acid-polypeptide conjugates as a constituent unit, and a plurality of nucleic acid-polypeptide conjugates. There is no limit to the number of clones and copies of the nucleic acid-polypeptide conjugates that make up the nucleic acid display library.
  • the nucleic acid-polypeptide conjugate of the present disclosure is a nucleic acid-polypeptide conjugate in which a polypeptide containing at least two types of unnatural amino acids in its amino acid sequence and a nucleic acid having a nucleotide sequence encoding this polypeptide are linked.
  • a nucleotide sequence contains a UAG codon and a UAA codon
  • UAG codon in the nucleotide sequence encodes the first unnatural amino acid in the polypeptide
  • a UAA codon in the nucleotide sequence encodes the second unnatural amino acid in the polypeptide.
  • nucleic acid-polypeptide conjugates of the present disclosure include mRNA-polypeptide conjugates and cDNA-polypeptide conjugates.
  • a cDNA-polypeptide conjugate is the reverse transcription product of an mRNA-polypeptide conjugate.
  • UAG codons are replaced with TAG and UAA codons are replaced with TAA.
  • nucleic acid-polypeptide conjugate of the present disclosure the nucleic acid and polypeptide are linked, for example, via a puromycin linker or ribosome.
  • a method for producing a nucleic acid display library of the present disclosure includes expressing a polypeptide from a nucleic acid by a cell-free peptide synthesis system to produce a nucleic acid-polypeptide conjugate.
  • the cell-free peptide synthesis system in the method for preparing a nucleic acid display library of the present disclosure aims to introduce at least two types of unnatural amino acids into a polypeptide.
  • a nucleic acid having a base sequence containing a UAG codon and a UAA codon an aminoacyl-tRNA in which a first unnatural amino acid is bound to a UAG codon-translated tRNA, which is a variant of the Mycoplasma capricolum tryptophan tRNA
  • the cell-free peptide synthesis system may be either (1) a system that translates nucleic acids or (2) a system that transcribes and translates nucleic acids.
  • the details of the cell-free peptide synthesis system are as described above. Any known cell-free peptide synthesis system and any known nucleic acid display library preparation technique can be employed in the method for preparing the nucleic acid display library of the present disclosure.
  • the template nucleic acid in the cell-free peptide synthesis system may be DNA or RNA.
  • a template nucleic acid is, for example, a population of double-stranded DNA fragments produced by performing overlap extension PCR using random primer sets containing random sequences.
  • the random sequence is, for example, the triplet repeating sequence [NNK]m, where m is a positive integer, N is independently A, T, G or C, and K is independently T or G. Peptides of any length can be generated by setting the number of repeats of the triplet [NNK] to any number.
  • the random sequence is preferably a trimer oligonucleotide in which one type of codon is assigned to one type of amino acid, from the viewpoint of suppressing the appearance of stop codons.
  • the 3' end of the template nucleic acid has a base sequence encoding a spacer.
  • a spacer is, for example, at least one amino acid selected from glycine and serine.
  • An example embodiment of a method for generating a nucleic acid display library of the present disclosure includes generating mRNA-polypeptide conjugates and reverse transcribing the mRNA of the mRNA-polypeptide conjugates to generate cDNA-polypeptide conjugates. including. According to this embodiment, since the nucleic acid of the nucleic acid-polypeptide conjugate is DNA, a chemically more stable nucleic acid display library can be obtained.
  • the nucleic acid to be translated (usually mRNA) and the translation product may be ligated via a puromycin linker, ligation via a ribosome, or the like.
  • the ligation between the nucleic acid to be translated and the translation product is preferably ligation via a puromycin linker from the viewpoints of facilitating the formation of an appropriate higher-order structure of the translation product and facilitating evaluation of the function of the translation product. Therefore, the nucleic acid to be translated in the cell-free peptide synthesis system is preferably a nucleic acid with a puromycin linker added to its 3' end.
  • a linker for adding puromycin to a nucleic acid As a linker for adding puromycin to a nucleic acid, a 2'-O-methylated nucleic acid linker or a nucleic acid linker having an ultraviolet crosslinkable compound at the 5' end is preferable from the viewpoint of suppressing dissociation of the nucleic acid and puromycin. .
  • the method for preparing a nucleic acid display library of the present disclosure uses UAG codons and UAA codons for amino acid translation while preserving the codons for 20 types of natural amino acids. You can diversify.
  • the screening method of the present disclosure comprises preparing a nucleic acid display library by the method of preparing a nucleic acid display library of the present disclosure, selecting nucleic acid-polypeptide conjugates having a desired activity from the nucleic acid display library, identifying the base sequence of the nucleic acid of the peptide conjugate.
  • Target activity is, for example, binding to a target substance.
  • a target substance is a term that includes all chemical substances that exhibit physiological activity, and includes compounds, groups, molecules, proteins, nucleic acids, lipids, carbohydrates, complexes thereof, and the like.
  • Target substances are, for example, receptors, transcription factors, enzymes, coenzymes, regulatory factors, antibodies, antigens, DNA, RNA, fragments thereof, complexes thereof, modification groups thereof.
  • An example of an embodiment of the screening method of the present disclosure includes contacting and incubating a nucleic acid display library and a target substance (eg, target protein).
  • a target substance eg, target protein
  • the nucleic acid display library and the target substance are brought into contact with each other in a buffer solution, and incubated while adjusting the pH and temperature of the buffer solution and the contact time.
  • the target substance may be immobilized on a solid-phase carrier, and the nucleic acid display library may be brought into contact with the immobilized target substance.
  • the solid phase carrier is not limited as long as it can immobilize the target substance, and includes microtiter plates, substrates, beads, magnetic beads, nitrocellulose membranes, nylon membranes, PVDF membranes and the like. Target substances are immobilized on these solid phase carriers by known techniques.
  • nucleic acid-polypeptide conjugate bound to the target substance eg, target protein
  • the nucleic acid base sequence of the extracted nucleic acid-polypeptide conjugate is identified. Nucleotide sequence identification can be performed using a nucleic acid amplification system and a sequencer.
  • a nucleic acid amplification system refers to a system that uses a nucleic acid as a template to amplify nucleic acid.
  • the nucleic acid amplification reaction of the nucleic acid amplification system is polymerase chain reaction (PCR), ligase chain reaction (LCR), TMA (transcription mediated amplification), NASBA (nucleic acid sequence-based amplification), etc. It's okay.
  • sequencer is a term that includes first-generation sequencers (capillary sequencers), second-generation sequencers (next-generation sequencers), third-generation sequencers, fourth-generation sequencers, and sequencers to be developed in the future.
  • the sequencer may be a capillary sequencer, a next-generation sequencer, or any other sequencer.
  • a next-generation sequencer is preferable from the viewpoints of speed of analysis, a large number of samples that can be processed at one time, and the like.
  • a next generation sequencer refers to a sequencer classified as opposed to a capillary sequencer (called a first generation sequencer) using the Sanger method.
  • next-generation sequencers are sequencers based on the principle of determining base sequences by capturing fluorescence or luminescence associated with complementary strand synthesis by DNA polymerase or complementary strand binding by DNA ligase.
  • Specific examples include MiSeq (Illumina Inc., MiSeq is a registered trademark), HiSeq2000 (Illumina Inc., HiSeq is a registered trademark), Roche454 (Roche Inc.), and the like.
  • a polypeptide having a desired activity, its encoding base sequence, and its amino acid sequence can be found among polypeptides into which at least two types of unnatural amino acids have been introduced.
  • tRNA and the like of the present disclosure will be described more specifically below with specific examples. Materials, processing procedures, and the like shown in the following specific examples can be changed as appropriate without departing from the gist of the present disclosure. The scope of the tRNA, etc. of the present disclosure should not be construed to be limited by the specific examples shown below.
  • tRNAs for tryptophan of Mycoplasma capricolum, Mycoplasma pneumoniae, Staphylococcus aureus, and Shewanella oneidensis were modified, respectively, and tRNAs of SEQ ID NOs: 1 to 4 were designed as tRNAs having UUA in the anticodon.
  • tRNA gene Two oligonucleotides were chemically synthesized as primers based on the nucleotide sequences of SEQ ID NOS: 1-4.
  • the forward primer contains the T7 promoter and the 1st to 51st base sequences of tRNA.
  • the reverse primer contains the nucleotide sequence from 36th to 74th (up to the first C of the CCA end) of tRNA. PCR was performed using these primers, and the PCR product was purified to obtain the tRNA gene. This gene is called tRNA-CA gene.
  • the PCR reaction solution contains KOD Dash Buffer, 0.2 mM dNTP, 2.5 units KOD Dash, and 0.2 nmol of each primer in 100 ⁇ L.
  • MinElute PCR Purification Kit (Qiagen) was used for purification of PCR products.
  • tRNA-CA Preparation of tRNA -
  • the tRNA-CA gene was transcribed and the transcript was purified to obtain tRNA. This tRNA is called tRNA-CA.
  • 100 ⁇ L of the reaction solution for transcription was 40 mM Tris-HCl (pH 8.0), 20 mM MgCl 2 , 5 mM DTT, 4 mM NTP, 20 mM GMP, 2 mM Spermidine, 10 ⁇ g/mL BSA, 40 units Ribonuclease inhibitor, 0.5 units Inorganic, Pyrophosphate 7.
  • RNA Polymerase containing 6 ⁇ g tRNA-CA gene. The reaction solution was allowed to react at 37° C. for 12 hours, and tRNA-CA was purified using RNEasy Minelute Cleanup Kit (Qiagen).
  • tRNA-BODIPYFL-aminophenylalanine conjugated to tRNA.
  • This tRNA is called fluorescent-labeled amino acid-tRNA.
  • 5 ⁇ Ligation Buffer 275 mM Hepes-Na (pH 7.5), 75 mM MgCl 2 , 16.5 mM DTT, 5 mM ATP
  • 4 ⁇ L 200 ⁇ M tRNA-CA 2.5 ⁇ L
  • DNA containing a base sequence (SEQ ID NO: 21: ATGTGCAAATAAAACCGCGGAGCAAAACATGAGCGATTATAAAGATGATGATGATAAG) encoding a polypeptide consisting of 20 amino acids (SEQ ID NO: 20: MCKXKPRSKNMSDYKDDDDK, X is an unnatural amino acid) was prepared.
  • This polypeptide and DNA are referred to as polypeptide (1) and DNA (1).
  • Polypeptide (1) contains a FLAG tag, and the 13th to 20th amino acid sequences are FLAG tags.
  • DNA (1) contains all the base sequences required for expression of polypeptide (1) by PUREfrex2.0.
  • DNA (1) was transcribed and the transcript was purified to obtain mRNA. Transcription from DNA (1) was performed in the same manner as the above transcription from the tRNA-CA gene, except that 20 mM GMP was not added to the reaction solution. Using fluorescence-labeled amino acid-tRNA and PUREfrex2.0 (Gene Frontier), polypeptide (1) was translated from mRNA, which was the transcript and purified product of DNA (1). 10 ⁇ L of the reaction solution contains 5 ⁇ L of PUREfrex 2.0 Solution I, 0.5 ⁇ L of Solution II, 1 ⁇ L of Solution III, 1 ⁇ L of 16 ⁇ M mRNA, 1 ⁇ L of fluorescence-labeled amino acid-tRNA solution, and 1.5 ⁇ L of water. The translation reaction was carried out at 25°C for 1 hour.
  • Example 2 A tRNA for tryptophan of Mycoplasma pneumoniae was modified to design tRNAs of SEQ ID NOS: 5 to 9 as tRNAs having UUA in the anticodon.
  • tRNAs of SEQ ID NOs: 5 to 9 the same methods as in Example 1 were used to perform tRNA gene preparation, tRNA preparation, tRNA aminoacylation, introduction of fluorescent-labeled amino acids into polypeptides, and introduction efficiency evaluation.
  • Table 3 shows the relative values with SEQ ID NO: 2 as the reference value of 1.00.
  • the tRNAs of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9 had a high introduction efficiency of fluorescent-labeled amino acids. This indicates the superiority of Mycoplasma pneumoniae tryptophan tRNAs in which the combination of the third base from the 5' end and the base paired with this base is UA, GU or UG.
  • Example 3 A tRNA for tryptophan of Mycoplasma pneumoniae was modified to design tRNAs of SEQ ID NOS: 10 to 14 as tRNAs having UUA in the anticodon.
  • tRNAs of SEQ ID NOS: 10 to 14 the same methods as in Example 1 were used to perform tRNA gene preparation, tRNA preparation, tRNA aminoacylation, introduction of fluorescently labeled amino acids into polypeptides, and introduction efficiency evaluation.
  • Table 4 shows relative values with SEQ ID NO: 2 as the reference value of 1.00.
  • the tRNA of SEQ ID NO: 13 had a high introduction efficiency of fluorescently labeled amino acids. This indicates the superiority of the tRNA for Mycoplasma pneumoniae for tryptophan in which the combination of the 4th base from the 5' end and the base paired with this base is GU.
  • Example 4 A tRNA for tryptophan of Mycoplasma pneumoniae was modified to design tRNAs of SEQ ID NOS: 15 to 17 as tRNAs having UUA in the anticodon.
  • tRNAs of SEQ ID NOS: 15 to 17 the same methods as in Example 1 were used to perform tRNA gene preparation, tRNA preparation, tRNA aminoacylation, introduction of fluorescently labeled amino acids into polypeptides, and introduction efficiency evaluation.
  • Table 5 shows relative values with SEQ ID NO: 2 as the reference value of 1.00.
  • the tRNA of SEQ ID NO: 17 had a high introduction efficiency of the fluorescent-labeled amino acid. From this, in the tRNA for tryptophan of Mycoplasma pneumoniae, the combination of the 4th base from the 5' end and the base that pairs with this base is GU, and the base adjacent to the 5' side of the CCA terminus is G. The superiority of certain tRNAs was demonstrated.
  • Example 5 Formamide was added to the tRNAs of SEQ ID NOS: 2 and 5 to 17, heat-treated (95°C, 3 minutes), and mixed with 10x Loading Buffer (TaKaRa, 9157). Electrophoresis was performed on an acrylamide gel containing urea (SuperSepTMRNA, 15%, 17well (Wako, 194-15881)), followed by staining with a staining reagent (Gel Red Nucleic Acid Gel Stain (Biotium, 41003)) for detection.
  • FIG. 2 shows the electrophoresis gel after staining.
  • the numbers on the gel in FIG. 2 are the sequence numbers.
  • DNAs (a) to (d) were prepared by substituting the base sequence (SEQ ID NO: 21) encoding polypeptide (1) in DNA (1).
  • DNAs (a) to (d) each have a base sequence obtained by substituting SEQ ID NO: 21 with the following substitutions.
  • DNA (a) DNA obtained by substituting TAG for the 4th codon and TAA for the 11th codon in SEQ ID NO:21.
  • DNA (b) DNA obtained by substituting TAG for the 4th and 5th codons and TAA for the 11th codon in SEQ ID NO:21.
  • DNA (c) DNA obtained by substituting TAG for the 4th and 6th codons and TAA for the 11th codon in SEQ ID NO:21.
  • DNA (d) DNA obtained by substituting TAG for the 4th and 7th codons and TAA for the 11th codon in SEQ ID NO:21.
  • the tRNA of SEQ ID NO: 19 was prepared as UAG codon-translated tRNA, which is a variant of Mycoplasma capricolum tryptophan tRNA, and N-methylphenylalanine or BODIPYFL-aminophenylalanine was bound to this tRNA.
  • the tRNA of SEQ ID NO: 2 was prepared as a UAA codon-translated tRNA, which is a modified tRNA for tryptophan of Mycoplasma pneumoniae, and N-methylphenylalanine or BODIPYFL-aminophenylalanine was bound to this tRNA.
  • FIG. 3 shows an electrophoretic gel.
  • the gel on the left side of FIG. 3 is the electrophoresis gel of the sample before purification, and the gel on the right side of FIG. 3 is the electrophoresis gel of the sample after purification using the FLAG tag.
  • Numbers on the gel in FIG. 3 indicate the following polypeptides.
  • Example 7 The same experiment as in Example 6 was performed with the addition of binding puromycin to the 3' end of the mRNA to be translated. UAG codon-translated tRNA and UAA codon-translated tRNA were bound with N-methylphenylalanine, N-methylalanine, or BODIPYFL-aminophenylalanine.
  • FIG. 4 shows an electrophoretic gel.
  • (11) A polypeptide translated from DNA (a) using BODIPYFL-aminophenylalanine-linked UAG codon-translated tRNA and N-methylalanine-linked UAA codon-translated tRNA.
  • the lower thick band is BODIPYFL-aminophenylalanine
  • the slightly upper band is the polypeptide
  • the thin band around 98 kDa is the mRNA-polypeptide conjugate.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
PCT/JP2022/035791 2021-09-24 2022-09-26 ｔＲＮＡ、アミノアシルｔＲＮＡ、ポリペプチド合成用試薬、非天然アミノ酸の導入方法、ポリペプチドの作製方法、核酸ディスプレイライブラリの作製方法、核酸－ポリペプチド連結体及びスクリーニング方法 Ceased WO2023048291A1 (ja)

Priority Applications (4)

Application Number Priority Date Filing Date Title
CN202280063245.1A CN118019845A (zh) 2021-09-24 2022-09-26 tRNA、氨酰基tRNA、多肽合成用试剂、非天然氨基酸的导入方法、多肽的制作方法、核酸展示文库的制作方法、核酸-多肽连接体及筛选方法
JP2023549778A JPWO2023048291A1 (https=) 2021-09-24 2022-09-26
EP22873042.0A EP4389890A4 (en) 2021-09-24 2022-09-26 ARNT, AMINOACYL-ARNT, POLYPEPTIDE SYNTHESIS REAGENT, PROCESS FOR INCORPORATION OF NON-NATURAL AMINO ACIDS, PROCESS FOR PRODUCING POLYPEPTIDES, PROCESS FOR PRODUCING NUCLEIC ACID DISPLAY BANK LAYERS, NUCLEIC ACID/POLYPEPTIDE CONJUGATE, AND SCREENING PROCESS
US18/611,981 US20240229015A1 (en) 2021-09-24 2024-03-21 tRNA, AMINOACYL tRNA, REAGENT FOR POLYPEPTIDE SYNTHESIS, INTRODUCTION METHOD OF UNNATURAL AMINO ACID, PRODUCTION METHOD OF POLYPEPTIDE, PRODUCTION METHOD OF NUCLEIC ACID DISPLAY LIBRARY, NUCLEIC ACID-POLYPEPTIDE CONJUGATE, AND SCREENING METHOD

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2021-156183 2021-09-24
JP2021156183 2021-09-24

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US18/611,981 Continuation US20240229015A1 (en) 2021-09-24 2024-03-21 tRNA, AMINOACYL tRNA, REAGENT FOR POLYPEPTIDE SYNTHESIS, INTRODUCTION METHOD OF UNNATURAL AMINO ACID, PRODUCTION METHOD OF POLYPEPTIDE, PRODUCTION METHOD OF NUCLEIC ACID DISPLAY LIBRARY, NUCLEIC ACID-POLYPEPTIDE CONJUGATE, AND SCREENING METHOD

Publications (1)

Publication Number Publication Date
WO2023048291A1 true WO2023048291A1 (ja) 2023-03-30

Family

ID=85720849

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2022/035791 Ceased WO2023048291A1 (ja) 2021-09-24 2022-09-26 ｔＲＮＡ、アミノアシルｔＲＮＡ、ポリペプチド合成用試薬、非天然アミノ酸の導入方法、ポリペプチドの作製方法、核酸ディスプレイライブラリの作製方法、核酸－ポリペプチド連結体及びスクリーニング方法

Country Status (5)

Country Link
US (1) US20240229015A1 (https=)
EP (1) EP4389890A4 (https=)
JP (1) JPWO2023048291A1 (https=)
CN (1) CN118019845A (https=)
WO (1) WO2023048291A1 (https=)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024214800A1 (ja) * 2023-04-11 2024-10-17 富士フイルム株式会社 生理活性評価用のポリペプチド溶液を調製する方法、及びポリペプチドの生理活性を評価する方法

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004009709A1 (ja) 2002-07-18 2004-01-29 Protein Express Co., Ltd. 非天然型標識化アミノ酸および該アミノ酸-ｔRNA結合体の作製方法
WO2007055429A1 (ja) 2005-11-14 2007-05-18 Japan Science And Technology Agency 非天然アミノ酸をタンパク質に導入するためのtRNA変異体
JP2008193911A (ja) * 2007-02-08 2008-08-28 Protein Express:Kk タンパク質のｎ末端を特異的に修飾する方法
JP2012058092A (ja) 2010-09-09 2012-03-22 Univ Of Tokyo Ｎ−メチルアミノ酸およびその他の特殊アミノ酸を含む特殊ペプチド化合物ライブラリーの翻訳構築と活性種探索法
WO2018174078A1 (ja) 2017-03-21 2018-09-27 富士フイルム株式会社 ペプチド化合物およびその製造方法、スクリーニング用組成物、並びにペプチド化合物の選択方法
WO2021026506A2 (en) * 2019-08-08 2021-02-11 Brickbio, Inc. Aminoacyl-trna synthetases and cell lines for site-specific incorporation of unnatural amino acids
JP2021156183A (ja) 2020-03-25 2021-10-07 トヨタ自動車株式会社 内燃機関の制御装置

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR102930465B1 (ko) * 2017-11-02 2026-02-25 더 위스타 인스티튜트 오브 아나토미 앤드 바이올로지 ACE-tRNA를 이용한 유전 재할당을 통한 정지 코돈의 구조 방법

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004009709A1 (ja) 2002-07-18 2004-01-29 Protein Express Co., Ltd. 非天然型標識化アミノ酸および該アミノ酸-ｔRNA結合体の作製方法
WO2007055429A1 (ja) 2005-11-14 2007-05-18 Japan Science And Technology Agency 非天然アミノ酸をタンパク質に導入するためのtRNA変異体
JP2008193911A (ja) * 2007-02-08 2008-08-28 Protein Express:Kk タンパク質のｎ末端を特異的に修飾する方法
JP2012058092A (ja) 2010-09-09 2012-03-22 Univ Of Tokyo Ｎ−メチルアミノ酸およびその他の特殊アミノ酸を含む特殊ペプチド化合物ライブラリーの翻訳構築と活性種探索法
WO2018174078A1 (ja) 2017-03-21 2018-09-27 富士フイルム株式会社 ペプチド化合物およびその製造方法、スクリーニング用組成物、並びにペプチド化合物の選択方法
WO2021026506A2 (en) * 2019-08-08 2021-02-11 Brickbio, Inc. Aminoacyl-trna synthetases and cell lines for site-specific incorporation of unnatural amino acids
JP2021156183A (ja) 2020-03-25 2021-10-07 トヨタ自動車株式会社 内燃機関の制御装置

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
NUCLEIC ACIDS RES, vol. 21, no. 21, 25 October 1993 (1993-10-25), pages 4967 - 4974
See also references of EP4389890A4
SIMONEAU PHILIPPE, LI CHENG-MING, LOECHEL STEVE, WENZE RAINER, HERRMANN RICHARD, HU PING-CHUAN: "Codon reading scheme in Mycoplasma pneumoniae revealed by the analysis of the complete set of tRNA genes", NUCLEIC ACIDS RESEARCH, OXFORD UNIVERSITY PRESS, GB, vol. 21, no. 21, 25 October 1993 (1993-10-25), GB , pages 4967 - 4974, XP093054265, ISSN: 0305-1048, DOI: 10.1093/nar/21.21.4967 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024214800A1 (ja) * 2023-04-11 2024-10-17 富士フイルム株式会社 生理活性評価用のポリペプチド溶液を調製する方法、及びポリペプチドの生理活性を評価する方法

Also Published As

Publication number Publication date
EP4389890A1 (en) 2024-06-26
US20240229015A1 (en) 2024-07-11
EP4389890A4 (en) 2025-12-31
JPWO2023048291A1 (https=) 2023-03-30
CN118019845A (zh) 2024-05-10

Similar Documents

Publication Publication Date Title
JP7825552B2 (ja) 非天然ポリペプチドのインビボ合成のための組成物および方法
US20180171321A1 (en) Platform for a non-natural amino acid incorporation into proteins
WO2014119600A1 (ja) Ｆｌｅｘｉｂｌｅ Ｄｉｓｐｌａｙ法
US20240200070A1 (en) Expanding the chemical substrates for genetic code reprogramming
WO2012026566A1 (ja) 新規人工翻訳合成系
US11001868B2 (en) Cell-free protein expression using double-stranded concatameric DNA
US20240247253A1 (en) Production method of polypeptide, tag, expression vector, evaluation method of polypeptide, production method of nucleic acid display library, and screening method
JP2025165960A (ja) 遺伝暗号の再プログラミングのため、長鎖炭素と環状アミノ酸が包含されるよう化学的基質を拡張する
US20240229015A1 (en) tRNA, AMINOACYL tRNA, REAGENT FOR POLYPEPTIDE SYNTHESIS, INTRODUCTION METHOD OF UNNATURAL AMINO ACID, PRODUCTION METHOD OF POLYPEPTIDE, PRODUCTION METHOD OF NUCLEIC ACID DISPLAY LIBRARY, NUCLEIC ACID-POLYPEPTIDE CONJUGATE, AND SCREENING METHOD
US20240124910A1 (en) Ribosome-mediated polymerization of novel chemistries
US20220002719A1 (en) Oligonucleotide-mediated sense codon reassignment
JP2017216961A (ja) 非天然アミノ酸含有ペプチドライブラリ
JP7774799B2 (ja) タンパク質の製造方法
WO2007055429A1 (ja) 非天然アミノ酸をタンパク質に導入するためのtRNA変異体
JP7777904B2 (ja) リンカー
JP2012139197A (ja) ｍＲＮＡ／ｃＤＮＡ−タンパク質連結体作製用リンカーとそれを用いたヌクレオチド−タンパク質連結体の精製方法
HK40087096A (zh) 将用於遗传密码重编程的化学底物扩展至包括长链碳和环状氨基酸
KR20250151188A (ko) 비천연 아미노산 도입 단백질의 효율적인 합성을 위한 유전체 가공 균주 및 세포 파쇄액 제작 방법
Suga et al. In vitro synthetic biology of the genetic code: its development and applications

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22873042

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 202280063245.1

Country of ref document: CN

Ref document number: 2023549778

Country of ref document: JP

WWE Wipo information: entry into national phase

Ref document number: 2022873042

Country of ref document: EP

ENP Entry into the national phase

Ref document number: 2022873042

Country of ref document: EP

Effective date: 20240321

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 11202401982Q

Country of ref document: SG