Method for screening peptide capable of binding to target molecule

Abstract

The present invention relates to a method for screening a peptide capable of binding to a target molecule, the method including: (1) a step for translating a nucleic acid library so as to obtain a peptide-nucleic acid conjugate library; (2) a step for bringing the peptide-nucleic acid conjugate library into contact with the target molecule so as to select one or a plurality of peptide-nucleic acid conjugates to be bound to the target molecule; and (3) a step for amplifying, from the selected one or plurality of peptide-nucleic acid conjugates, nucleic acids contained in the peptide-nucleic acid conjugates. The method involves suppressing the amplification in step (3) for at least one nucleic acid contained in the peptide-nucleic acid conjugates.

Description

A screening method for peptides that can bind to target molecules. 【0001】 The present invention relates to a method for screening peptides that can bind to target molecules. 【0002】 In recent years, drug discovery methods have been developed to select candidate drug substances from diverse peptide libraries. Among these methods, methods such as screening peptides from mRNA-peptide complex libraries obtained using cell-free translation systems (mRNA display library method) are attracting attention due to their diversity and ease of screening. 【0003】 As an example of the screening method described above, Patent Document 1 discloses a method for producing a specific peptide compound, which includes the step of contacting a peptide compound-nucleic acid complex library having a cyclic portion with a biomolecule and selecting a complex that has binding activity to the biomolecule. 【0004】 International Publication No. 2013 / 100132 【0005】 On the other hand, when screening peptides that can bind to a target molecule using the method described in Patent Document 1, some peptides were preferentially enriched, which sometimes hindered the acquisition of other peptides. In particular, in the initial stages of searching for hit candidates, there is a need to obtain peptides that bind to various locations on the target molecule. Therefore, the exclusive acquisition of some peptides that bind strongly to the target molecule, preventing the acquisition of other hit candidate peptides, was problematic. 【0006】 Therefore, the present invention aims to provide a method for screening other peptides that can bind to a target molecule while selectively suppressing the acquisition of specific peptides that can bind to the target molecule. 【0007】The present invention includes, for example, the following inventions: [1] A method for screening peptides that can bind to a target molecule, comprising: (1) translating a nucleic acid library to obtain a peptide-nucleic acid conjugate library; (2) contacting the peptide-nucleic acid conjugate library with the target molecule to select one or more peptide-nucleic acid conjugates that bind to the target molecule; and (3) amplifying the nucleic acid contained in the peptide-nucleic acid conjugate from the selected one or more peptide-nucleic acid conjugates, wherein the method includes suppressing the amplification of at least one nucleic acid contained in the peptide-nucleic acid conjugate in step (3). [2] The method according to [1], wherein the nucleic acid library is a DNA library. [3] The method according to [1] or [2], wherein the nucleic acid library is a cDNA library. [4] The method according to [1], wherein the nucleic acid library is an mRNA library. [5] The method according to [4], wherein step (1) includes transcribing the DNA library to obtain an mRNA library. [6] The method according to any one of [1] to [5], wherein the nucleic acid in the peptide-nucleic acid conjugate is mRNA. [7] The method according to any one of [1] to [6], wherein the nucleic acid in the peptide-nucleic acid conjugate is DNA. [8] The method according to [7], wherein the DNA is cDNA. [9] The method according to [7] or [8], wherein the DNA is single-stranded DNA.

[10] The method according to any one of [6] to [9], further comprising (2-1) a step of reverse transcribing at least one nucleic acid contained in the peptide-nucleic acid conjugate before step (3).

[11] The method according to

[10] , wherein step (2-1) is performed before step (2).

[12] The method according to

[10] or

[11] , wherein step (2-1) is performed after step (2).

[13] The method according to any one of [1] to

[12] , wherein suppressing amplification in step (3) is a reduction in the amount of DNA that serves as a template for amplification in step (3).

[14] The method according to any one of [1] to

[13] , wherein suppressing amplification in step (3) is performed by suppressing the translation of at least one nucleic acid contained in the nucleic acid library.

[15] The method according to any one of

[10] to

[14] , wherein the amplification in step (3) is suppressed by suppressing the reverse transcription of at least one nucleic acid contained in the peptide-nucleic acid conjugate in step (2-1).

[16] The method according to any one of [1] to

[15] , wherein the amplification in step (3) is suppressed by hybridizing a nucleic acid having a base sequence complementary to at least a portion of the base sequence of at least one nucleic acid contained in the nucleic acid library or at least one nucleic acid contained in the peptide-nucleic acid conjugate library with at least one nucleic acid contained in the nucleic acid library or at least one nucleic acid contained in the peptide-nucleic acid conjugate library.

[17] The method according to

[16] , wherein the nucleic acid to be hybridized is a nucleic acid containing Loc nucleic acid (LNA).

[18] The method according to

[16] or

[17] , wherein the hybridization is performed on single-stranded DNA or mRNA.

[19] The method according to

[16] , wherein the nucleic acid to be hybridized is a natural nucleic acid.

[20] The method according to

[19] , wherein the natural nucleic acid is DNA.

[21] The method according to

[19] or

[20] , wherein the hybridization is performed on single-stranded DNA.

[22] The method according to any one of

[16] to

[21] , wherein suppressing amplification in step (3) is performed by forming a structure in at least one nucleic acid contained in the nucleic acid library or at least one nucleic acid contained in the peptide-nucleic acid conjugate that inhibits the reaction of translation enzymes and / or reverse transcriptases.

[23] The method according to any one of

[16] to

[22] , wherein the hybridization is performed before step (2).

[24] The method according to any one of

[16] to

[23] , wherein the hybridization is performed before step (1).

[25] The method according to any one of

[16] to

[22] , wherein the hybridization is performed after step (2) and before step (3).

[26] The method according to any one of

[19] to

[25] , wherein suppressing amplification in step (3) is performed by treating with a nuclease that specifically recognizes the double-stranded portion formed by hybridization.

[27] The method according to

[26] , wherein the nuclease is a nuclease that degrades the double-stranded portion of DNA.

[28] The method according to

[26] or

[27] , wherein the treatment with the nuclease is performed before step (1).

[29] The method according to any one of

[26] to

[28] , wherein the treatment with the nuclease is performed after step (2) and before step (3).

[30] The method according to any one of

[16] to

[29] , wherein the nucleic acid to be hybridized is 9 bases or more.

[31] The method according to any one of

[16] to

[30] , wherein the nucleic acid to be hybridized is 12 bases or more.

[32] The method according to any one of

[16] to

[31] , wherein the nucleic acid to be hybridized is 15 bases or more.

[33] The method according to any one of

[16] to

[32] , wherein the nucleic acid to be hybridized is 18 bases or more.

[34] A method for screening peptides that can bind to a target molecule, comprising: (1) translating an mRNA library to obtain a peptide-nucleic acid conjugate library; (2) contacting the peptide-nucleic acid conjugate library with the target molecule to select one or more peptide-nucleic acid conjugates that bind to the target molecule; and (3) amplifying the nucleic acid contained in the peptide-nucleic acid conjugate from the selected one or more peptide-nucleic acid conjugates, the method comprising hybridizing a nucleic acid containing a lock nucleic acid (LNA) having a base sequence complementary to the base sequence of at least one nucleic acid contained in the nucleic acid library or at least one nucleic acid contained in the peptide-nucleic acid conjugate library with at least one of the nucleic acids contained in the nucleic acid library or at least one of the nucleic acids contained in the peptide-nucleic acid conjugate library.

[35] The method according to

[34] , wherein step (1) is transcribing a DNA library to obtain an mRNA library.

[36] The method according to

[34] or

[35] , wherein the nucleic acid in the peptide-nucleic acid conjugate is mRNA.

[37] The method according to any one of

[34] to

[36] , wherein the nucleic acid in the peptide-nucleic acid conjugate is DNA.

[38] The method according to

[37] , wherein the DNA is cDNA.

[39] The method according to

[37] or

[38] , wherein the DNA is single-stranded DNA.

[40] The method according to any one of

[34] to

[39] , further comprising (2-1) a step of reverse transcribing at least one nucleic acid contained in the peptide-nucleic acid conjugate before step (3).

[41] The method according to any one of

[34] to

[40] , wherein step (2-1) is performed before step (2).

[42] The method according to any one of

[34] to

[41] , wherein step (2-1) is performed after step (2).

[43] The method according to any one of

[34] to

[42] , wherein the hybridization is performed on single-stranded DNA or mRNA.

[44] The method according to any one of

[34] to

[43] , wherein the hybridization is performed before step (1).

[45] The method according to

[44] , wherein the hybridization suppresses the translation of at least one nucleic acid contained in the nucleic acid library and the reverse transcription of at least one nucleic acid contained in the peptide-nucleic acid conjugate.

[46] The method according to any one of

[34] to

[45] , wherein the hybridization is performed after step (1) and before step (2).

[47] The method according to any one of

[34] to

[46] , wherein the hybridization is performed after step (2) and before step (3).

[48] The method according to

[46] or

[47] , wherein the hybridization suppresses the reverse transcription of at least one nucleic acid contained in the peptide-nucleic acid conjugate.

[49] A method for screening peptides that can bind to a target molecule, comprising: (1) translating a nucleic acid library to obtain a peptide-nucleic acid conjugate library; (2) contacting the peptide-nucleic acid conjugate library with the target molecule to select one or more peptide-nucleic acid conjugates that bind to the target molecule; and (3) amplifying the nucleic acids contained in the peptide-nucleic acid conjugates from the selected one or more peptide-nucleic acid conjugates, wherein, prior to step (3), a single-stranded nucleic acid having a base sequence complementary to the base sequence of at least one DNA contained in the nucleic acid library or at least one DNA contained in the peptide-nucleic acid conjugate library is hybridized to at least one DNA contained in the nucleic acid library or at least one DNA contained in the peptide-nucleic acid conjugate library, and prior to step (3), treatment with a nuclease that specifically recognizes the double-stranded portion formed by the hybridization.

[50] The method according to

[49] , wherein the nucleic acid library is a DNA library.

[51] The method according to

[49] or

[50] , wherein the nucleic acid library is a cDNA library.

[52] The method according to

[49] , wherein the nucleic acid library is an mRNA library.

[53] The method according to

[52] , wherein step (1) comprises transcribing the DNA library to obtain an mRNA library.

[54] The method according to any one of

[49] to

[53] , wherein the nucleic acid in the peptide-nucleic acid conjugate is mRNA.

[55] The method according to any one of

[49] to

[54] , wherein the DNA is cDNA.

[56] The method according to

[55] , wherein the cDNA is single-stranded cDNA.

[57] The method according to any one of

[49] to

[56] , wherein the single-stranded nucleic acid is a single-stranded native nucleic acid.

[58] (2-1) The method according to any one of

[54] to

[57] , further comprising the step of reverse transcribing at least one nucleic acid contained in the peptide-nucleic acid conjugate before step (3).

[59] The method according to any one of the items in

[58] , wherein step (2-1) is performed before step (2).

[60] The method according to

[58] or

[59] , wherein step (2-1) is performed after step (2).

[61] The method according to any one of

[49] to

[60] , wherein the hybridization is performed before step (2).

[62] The method according to

[61] , wherein the hybridization is performed before step (1).

[63] The method according to any one of

[49] to

[62] , wherein the hybridization is performed after step (2) and before step (3).

[64] The method according to any one of

[49] to

[63] , wherein the nuclease is a nuclease that degrades the double-stranded portion of DNA.

[65] The method according to any one of

[62] to

[64] , wherein the treatment with the nuclease is performed before step (1).

[66] The method according to any one of

[63] to

[65] , wherein the treatment with the nuclease is performed after step (2) and before step (3).

[67] The method according to any one of

[34] to

[66] , wherein the nucleic acid containing the lock nucleic acid to be hybridized or the single-stranded nucleic acid is 9 nucleotides or more.

[68] The method according to any one of

[34] to

[67] , wherein the nucleic acid containing the lock nucleic acid to be hybridized or the single-stranded nucleic acid is 12 nucleotides or more.

[69] The method according to any one of

[34] to

[68] , wherein the nucleic acid containing the lock nucleic acid to be hybridized or the single-stranded nucleic acid is 15 nucleotides or more.

[70] The method according to any one of

[34] to

[69] , wherein the nucleic acid containing the lock nucleic acid to be hybridized or the single-stranded nucleic acid is 18 nucleotides or more.

[71] The method according to any one of [1] to

[70] , wherein step (2) comprises recovering one or more peptide-nucleic acid conjugates bound to the target molecule.

[72] The method according to

[71] , wherein the recovery in recovering the peptide-nucleic acid conjugate is carried out by an affinity-based method.

[73] The method according to

[72] , wherein the method utilizing the affinity is the pull-down method.

[74] The method according to any one of

[71] to

[73] , wherein step (3) comprises eluting the nucleic acid in the peptide-nucleic acid conjugate before amplifying the nucleic acid.

[75] The method according to any one of [1] to

[74] , wherein step (3) is a step of amplifying one or more cDNAs obtained from one or more selected peptide-nucleic acid conjugates.

[76] The method according to any one of [1] to

[75] , further comprising (4) a step of repeating steps (1) to (3) once or more times, using the nucleic acid amplified in step (3) as an n-th order nucleic acid library (n is an integer of 2 or more).

[77] The method according to any one of [1] to

[76] , further comprising (5) a step of identifying the peptide that can bind to the target molecule by determining the base sequence of the nucleic acid amplified in step (3).

[78] A method for producing a peptide, comprising a step of screening for peptides that can bind to the target molecule by the method according to any one of [1] to

[77] .

[79] A peptide produced by a method for producing a peptide, comprising a step of screening for peptides that can bind to the target molecule by the method according to any one of [1] to

[77] . 【0008】 According to the present invention, it is possible to provide a method for screening other peptides that can bind to a target molecule while selectively suppressing the acquisition of specific peptides that can bind to the target molecule. 【0009】 The embodiments for carrying out the present invention will be described in detail below. However, the present invention is not limited to the following embodiments. 【0010】 In this specification, “one or more” means one or more numbers. When “one or more” is used in a context relating to substituents of a group, the term means a number from one up to the maximum number of substituents permitted by that group. Specifically, “one or more” could be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and / or greater numbers. 【0011】In this specification, the meaning of the terms “and / or” includes any combination of “and” and “or” as appropriate. Specifically, for example, “A, B and / or C” includes the following seven variations: (i) A, (ii) B, (iii) C, (iv) A and B, (v) A and C, (vi) B and C, (vii) A, B and C. 【0012】 [Screening Method] The screening method according to this embodiment (the screening method according to the first embodiment) is a screening method for peptides that can bind to a target molecule, comprising: (1) translating a nucleic acid library to obtain a peptide-nucleic acid conjugate library; (2) contacting the peptide-nucleic acid conjugate library with a target molecule to select one or more peptide-nucleic acid conjugates that bind to the target molecule; and (3) amplifying the nucleic acids contained in the peptide-nucleic acid conjugates from the selected one or more peptide-nucleic acid conjugates, wherein the amplification in step (3) of at least one nucleic acid contained in the peptide-nucleic acid conjugate is suppressed. 【0013】 The screening method according to this embodiment selectively suppresses the acquisition of peptides capable of binding to target molecules obtained from nucleic acids whose amplification is suppressed, by suppressing the amplification of at least one nucleic acid contained in the peptide-nucleic acid conjugate in step (3). On the other hand, peptides capable of binding to target molecules obtained from nucleic acids whose amplification is not suppressed can be screened and acquired. 【0014】 The embodiments of each process will be described in detail below. 【0015】 <Step (1)> In Step (1), the nucleic acid library is translated to obtain a peptide-nucleic acid conjugate library (acquisition step). 【0016】(Nucleic Acid Library) In this specification, "nucleic acid library" means a collection (library) of nucleic acids that encode peptides. That is, the nucleic acids contained in the nucleic acid library are nucleic acids that encode peptides that form peptide-nucleic acid conjugates. As a result, after step (3) described below, peptide-nucleic acid conjugates can be reproduced by synthesizing (translating) peptides from the amplified nucleic acids. 【0017】 The nucleic acid library may be a DNA library or an RNA library. More specifically, the nucleic acid library may be a cDNA library or an mRNA library. 【0018】 Translation is a reaction in which peptides are synthesized from mRNA. However, if the nucleic acid library is not an mRNA library, step (1) may include any means for obtaining a peptide-nucleic acid conjugate library via translation. For example, if the nucleic acid library is a DNA library, obtaining a peptide-nucleic acid conjugate library from the nucleic acid library may include transcribing the nucleic acid library (DNA library) to obtain an mRNA library. Translation can be carried out by methods well known to those skilled in the art (e.g., by using a cell-free translation system). 【0019】 (Peptide-Nucleic Acid Conjugate Library) In this specification, "peptide-nucleic acid conjugate library" means a collection (library) of conjugates (peptide-nucleic acid conjugates) in which a peptide and the nucleic acid encoding that peptide are associated. In a peptide-nucleic acid conjugate library, it is sufficient that the peptide and the nucleic acid are associated with each other, and the conjugate may be formed by the peptide and nucleic acid binding directly or via a binding site, or the conjugate may be formed without the peptide and nucleic acid binding directly or via a binding site, and it is preferable that the peptide and nucleic acid form the conjugate via a binding site. 【0020】The binding site is not limited as long as it can link the peptide and the nucleic acid, but may include, for example, the antibiotic puromycin, which is an analog of aminoacyl-tRNA. The binding site may further include spacers well known to those skilled in the art. 【0021】 The nucleic acid in the peptide-nucleic acid conjugate may contain DNA (peptide-DNA conjugate) or RNA (peptide-RNA conjugate). The DNA may be cDNA, and is preferably single-stranded DNA. The RNA may be mRNA. The nucleic acid in the peptide-nucleic acid conjugate preferably contains at least mRNA. The nucleic acid contained in the peptide-nucleic acid conjugate may be one type or multiple types. Specifically, the peptide-nucleic acid conjugate may be a peptide-DNA conjugate, a peptide-RNA conjugate, a peptide-RNA / DNA conjugate, or a peptide-mRNA / cDNA conjugate. Note that the peptide-RNA / DNA conjugate is a conjugate of a peptide with RNA and DNA. 【0022】 Furthermore, nucleic acids may have a tag sequence for identifying the peptide contained in the peptide-nucleic acid conjugate, as well as a base sequence (primer binding sequence, etc.) for amplification. The base sequence for amplification may be a base sequence common to all nucleic acids. 【0023】The peptide contained in the peptide-nucleic acid conjugate corresponds to the portion of the peptide-nucleic acid conjugate excluding the nucleic acid and the binding site. For example, in the embodiments of this application, the peptide in the peptide-nucleic acid conjugate is the portion obtained by removing the amino acid sequence (PTGTGTGKK: SEQ ID NO 65) which is the peptide linker, the binding site including puromycin, and the nucleic acid portion, and then attaching pyrrolidine to the carboxyl binding site to which the peptide linker was attached. The peptide contained in the peptide-nucleic acid conjugate can be obtained by capping the carboxyl group that was generated when the nucleic acid and binding site were removed from the peptide-nucleic acid conjugate in this way. The peptide is not particularly limited, and any compound (low molecular weight compound, medium molecular weight compound, high molecular weight compound) can be used. Preferably, the peptide is a medium molecular weight compound, and more preferably a medium molecular weight compound consisting of a cyclic peptide. 【0024】 In this specification, peptides are not particularly limited as long as the amino acid residues are linked by amide bonds or ester bonds. Peptides are preferably those in which two or more amino acid residues are linked by amide bonds. In this case, they may have ester bonds in part of the main chain, such as depsipeptides. Of the total peptides contained in the peptide-nucleic acid conjugate library, 1 × 10 ３ In the peptides described above, there are no particular restrictions on the number of amino acid residues in the peptide, but for example, it may be 5 or more, 7 or more, 8 or more, or 9 or more. Of the total peptides contained in the peptide-nucleic acid conjugate library, 1 × 10 ３ In the peptides described above, the number of amino acid residues in the peptide may also be, for example, 30 or less, 25 or less, 15 or less, or 13 or less. Of the total peptides contained in the peptide-nucleic acid conjugate library, 1 × 10 ３ In the peptides described above, the number of amino acid residues in the peptide may be, for example, 5 to 30, preferably 7 to 25, more preferably 8 to 15, and most preferably 9 to 13. The peptide may have a branched structure. 【0025】In this specification, "amino acids" include natural amino acids and non-natural amino acids. Furthermore, in this specification, "amino acid residues" include natural amino acid residues and non-natural amino acid residues. 【0026】 Natural amino acids refer to glycine (Gly), L-alanine (Ala), L-serine (Ser), L-threonine (Thr), L-valine (Val), L-leucine (Leu), L-isoleucine (Ile), L-phenylalanine (Phe), L-tyrosine (Tyr), L-tryptophan (Trp), L-histidine (His), L-glutamic acid (Glu), L-aspartic acid (Asp), L-glutamine (Gln), L-asparagine (Asn), L-cysteine ​​(Cys), L-methionine (Met), L-lysine (Lys), L-arginine (Arg), and L-proline (Pro). 【0027】 Non-natural amino acids refer to amino acids other than natural amino acids. Examples of non-natural amino acids include β-amino acids, D-type amino acids, N-substituted amino acids (excluding Pro), α,α-disubstituted amino acids, amino acids with side chains different from those of natural amino acids, and hydroxycarboxylic acids. In this specification, non-natural N-substituted amino acids refer to N-substituted amino acids other than Pro. 【0028】In this specification, any stereochemistry is permitted for the amino acids. There are no particular restrictions on the selection of the amino acid side chains, but they can be freely selected from, for example, alkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, aralkyl groups, heteroaralkyl groups, cycloalkyl groups, and spiro-bonded cycloalkyl groups, in addition to hydrogen atoms. Each of these may be substituted, and these substituents are not limited; for example, one or more substituents can be freely selected independently from any substituents including halogen atoms, O atoms, S atoms, N atoms, B atoms, Si atoms, or P atoms. Examples include substituted alkyl groups, alkoxy groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, aralkyl groups, cycloalkyl groups, etc., or oxo, aminocarbonyl, halogen atoms, etc. An amino acid according to one embodiment may be a compound having both a carboxyl group and an amino group within the same molecule (even in this case, proline, hydroxyproline, azetidine-2-carboxylic acid, etc., in which the nitrogen atom of the amino group and any atom of the side chain form a ring together, are also included as amino acids). 【0029】 Examples of halogen-derived substituents include fluoro(-F), chloro(-Cl), bromo(-Br), and iod(-I). 【0030】 Substituents derived from the oxygen atom include hydroxy (-OH), oxy (-OR), carbonyl (-C(=O)-R), and carboxyl (-CO ２ H), oxycarbonyl (-C(=O)-OR), carbonyloxy (-O-C(=O)-R), thiocarbonyl (-C(=O)-SR), carbonylthio group (-S-C(=O)-R), aminocarbonyl (-C(=O)-NHR), carbonylamino (-NH-C(=O)-R), oxycarbonylamino (-NH-C(=O)-OR), sulfonylamino (-NH-SO ２ -R), aminosulfonyl (-SO ２ -NHR), sulfamoylamino(-NH-SO) ２ -NHR), thiocarboxy(-C(=O)-SH), carboxycarbonyl(-C(=O)-CO ２ H) is one example. 【0031】 Examples of oxy (-OR) compounds include alkoxy, cycloalkoxy, alkenyloxy, alkynyloxy, aryloxy, heteroaryloxy, and aralkyloxy compounds. 【0032】 Examples of carbonyl (-C(=O)-R) include formyl (-C(=O)-H), alkylcarbonyl, cycloalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, arylcarbonyl, heteroarylcarbonyl, and aralkylcarbonyl. 【0033】 Examples of oxycarbonyl (-C(=O)-OR) include alkyloxycarbonyl, cycloalkyloxycarbonyl, alkenyloxycarbonyl, alkynyloxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, and aralkyloxycarbonyl. 【0034】 Examples of carbonyloxy (-O-C(=O)-R) include alkylcarbonyloxy, cycloalkylcarbonyloxy, alkenylcarbonyloxy, alkynylcarbonyloxy, arylcarbonyloxy, heteroarylcarbonyloxy, and aralkylcarbonyloxy. 【0035】 Examples of thiocarbonyl (-C(=O)-SR) include alkylthiocarbonyl, cycloalkylthiocarbonyl, alkenylthiocarbonyl, alkynylthiocarbonyl, arylthiocarbonyl, heteroarylthiocarbonyl, and aralkylthiocarbonyl. 【0036】 Examples of carbonylthio (-S-C(=O)-R) include alkylcarbonylthio, cycloalkylcarbonylthio, alkenylcarbonylthio, alkynylcarbonylthio, arylcarbonylthio, heteroarylcarbonylthio, and aralkylcarbonylthio. 【0037】Examples of aminocarbonyl (—C(═O)—NHR) include alkylaminocarbonyl, cycloalkylaminocarbonyl, alkenylaminocarbonyl, alkynylaminocarbonyl, arylaminocarbonyl, heteroarylaminocarbonyl, aralkylaminocarbonyl, and the like. In addition to these, compounds in which the H atom bonded to the N atom in —C(═O)—NHR is further substituted with alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl are also included. 【0038】 Examples of carbonylamino (—NH—C(═O)—R) include alkylcarbonylamino, cycloalkylcarbonylamino, alkenylcarbonylamino, alkynylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino, aralkylcarbonylamino, and the like. In addition to these, compounds in which the H atom bonded to the N atom in —NH—C(═O)—R is further substituted with alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl are also included. 【0039】 Examples of oxycarbonylamino (—NH—C(═O)—OR) include alkoxycarbonylamino, cycloalkoxycarbonylamino, alkenyloxycarbonylamino, alkynyloxycarbonylamino, aryloxycarbonylamino, heteroaryloxycarbonylamino, aralkyloxycarbonylamino, and the like. In addition to these, compounds in which the H atom bonded to the N atom in —NH—C(═O)—OR is further substituted with alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl are also included. 【0040】 Examples of sulfonylamino (—NH—SO ２ —R) include alkylsulfonylamino, cycloalkylsulfonylamino, alkenylsulfonylamino, alkynylsulfonylamino, arylsulfonylamino, heteroarylsulfonylamino, aralkylsulfonylamino, and the like. In addition to these, compounds in which the H atom bonded to the N atom in —NH—SO ２Examples include compounds in which the H atom bonded to the N atom in -R is further substituted with alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl groups. 【0041】 Aminosulfonyl (-SO ２ Examples of -NHR compounds include alkylaminosulfonyl, cycloalkylaminosulfonyl, alkenylaminosulfonyl, alkynylaminosulfonyl, arylaminosulfonyl, heteroarylaminosulfonyl, and aralkylaminosulfonyl. In addition to these, -SO ２ Examples include compounds in which the H atom bonded to the N atom in NHR is further substituted with alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl groups. 【0042】 Sulfamoylamino(-NH-SO) ２ Examples of -NH-SO include alkylsulfamoylamino, cycloalkylsulfamoylamino, alkenylsulfamoylamino, alkynylsulfamoylamino, arylsulfamoylamino, heteroarylsulfamoylamino, and aralkylsulfamoylamino. Furthermore, -NH-SO ２ The two H atoms bonded to the N atom in -NHR may be substituted with substituents independently selected from the group consisting of alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl groups, and these two substituents may form a ring. 【0043】 Substituents derived from the sulfur atom include thiol (-SH), thio (-S-R), sulfinyl (-S(=O)-R), and sulfonyl (-S(O)). ２ -R), sulfo(-SO ３ H), pentafluorosulfanil (-SF ５ Examples include: 【0044】 Examples of thio(-S-R) include alkylthio, cycloalkylthio, alkenylthio, alkynylthio, arylthio, heteroarylthio, and aralkylthio. 【0045】Examples of sulfinyl (-S(=O)-R) include alkylsulfinyl, cycloalkylsulfinyl, alkenylsulfinyl, alkynylsulfinyl, arylsulfinyl, heteroarylsulfinyl, and aralkylsulfinyl. 【0046】 Sulfonyl (-S(O) ２ Examples of -R) include alkylsulfonyl, cycloalkylsulfonyl, alkenylsulfonyl, alkynylsulfonyl, arylsulfonyl, heteroarylsulfonyl, and aralkylsulfonyl. 【0047】 As substituents derived from the N atom, azide (-N ３ Also called "azide group"), cyano(-CN), primary amino(-NH) ２ ), secondary amino(-NH-R), tertiary amino(-NR(R')), amidino(-C(=NH)-NH ２ ), substitute amidino (-C (=NR)-NR'R''), guanidino (-NH-C (=NH)-NH ２ Examples include substituted guanidino (-NR-C (=NR''')-NR'R''), aminocarbonylamino (-NR-CO-NR'R''), etc. 【0048】 Examples of secondary amino acids (-NH-R) include alkylaminos, cycloalkylaminos, alkenylaminos, alkynylaminos, arylaminos, heteroarylaminos, and aralkylaminos. 【0049】 Examples of tertiary aminos (-NR(R')) include alkyl(aralkyl)aminos, and any amino group having any two substituents independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, etc., where any two substituents may form a ring. 【0050】Examples of substituted amidinos (-C(=NR)-NR'R'') include groups in which the three substituents R, R', and R'' on the N atom are independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl groups, such as alkyl(aralkyl)(aryl)amidinos. 【0051】 Examples of substituted guanidinos (-NR-C (=NR''')-NR'R'') include groups in which R, R', R'', and R'''' are independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl groups, as well as groups in which these groups form a ring. 【0052】 Examples of aminocarbonylamino (-NR-CO-NR'R'') include groups in which R, R', and R'' are independently selected from hydrogen atoms, alkyl groups, cycloalkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, and aralkyl groups, as well as groups in which these groups form a ring. 【0053】 Examples of substituents derived from the B atom include boryl (-BR(R')) and dioxyboryl (-B(OR)(OR')). These two substituents R and R' may be groups independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, etc., or groups in which these groups form a ring. Specifically, examples include cyclic boryl groups, and more specifically, pinacolate boryl groups, neopentanediolate boryl groups, catecholate boryl groups, etc. 【0054】 The amino group in the main chain of an amino acid is unsubstituted (-NH ２ ) or it may be substituted (i.e., -NHR. R represents, for example, an alkyl group, alkenyl group, alkynyl group, aryl group, heteroaryl group, aralkyl group, cycloalkyl group, etc., which may have substituents, and the carbon chain bonded to the N atom and the carbon atom at the α position may form a ring, as in proline). 【0055】In this specification, amino acid residues in which the main chain amino group is substituted are referred to as "N-substituted amino acid residues." Examples of "N-substituted amino acid residues" in this specification include N-alkyl amino acid residues and N-C １ -C ６ Alkyl amino acid residues, N-C １ -C ５ Alkyl amino acid residues, N-C １ -C ４ Alkyl amino acid residues, N-C １ -C ３ Alkyl amino acid residues, N-ethyl amino acid residues, N-methyl amino acid residues, N-C ７ -C １４ These may be aralkyl amino acid residues, N-benzyl amino acid residues, or N-phenethyl amino acid residues. 【0056】 Specifically, the substituent on the nitrogen atom of an N-substituted amino acid residue in this specification (the R in -NHR mentioned above) is an alkyl group (preferably C １ -C ６ Alkyl alkyl group, comfortable C １ -C ４ Alkyl alkyl group, comfortable C １ -C ３ Alkyl group, more preferably ethyl group or methyl group), C ７ -C １４ Examples include aralkyl groups, benzyl groups, and phenethyl groups. Substituents on the nitrogen atom of N-substituted amino acids include, for example, C １ -C ６ It may be an alkyl group, preferably C １ -C ３ It is an alkyl group, more preferably an ethyl group or a methyl group, and most preferably a methyl group. (That is, as the N-substituted amino acid, N-methylamino acid is most preferred). 【0057】In this specification, "amino acid" includes all of its corresponding isotopes. An isotope of an "amino acid" is one in which at least one atom is replaced by an atom with the same atomic number (number of protons) but a different mass number (sum of protons and neutrons). Examples of isotopes included in "amino acids" in this specification include hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine atoms, respectively. ２ H, ３ H, １３ C, １４ C, １５ N, １７ O, １８ O, ３２ P, ３５ S, １８ F, ３６ It contains chlorine, etc. 【0058】 Of all the peptides contained in the peptide-nucleic acid conjugate library, 1 × 10 ３ In the peptides described above, the number of non-natural amino acid residues contained in the peptide may be, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more. Also, of the total peptides contained in the peptide-nucleic acid conjugate library, 1 × 10 ３ In the peptides described above, the number of non-natural amino acid residues contained in the peptide may be, for example, 30 or less, 25 or less, 20 or less, 15 or less, 14 or less, 13 or less, or 12 or less. Of the total peptides contained in the peptide-nucleic acid conjugate library, 1 × 10 ３ In the peptides described above, examples of the range of non-natural amino acid residues contained in the peptides include 1 to 30, 2 to 25, 3 to 20, 4 to 15, 5 to 14, 6 to 13, 7 to 12, or 11. Of the total peptides contained in the peptide-nucleic acid conjugate library, 1 × 10 ３ In the peptides described above, the range of the number of non-natural amino acid residues contained in the peptide is, for example, 1 to 13, preferably 3 to 12, more preferably 4 to 11, and most preferably 5 to 10. 【0059】Of all the peptides contained in the peptide-nucleic acid conjugate library, 1 × 10 ３ In the peptides described above, the number of N-substituted amino acid residues in the peptide may be, for example, 1 or more, 2 or more, 3 or more, 4 or more, or 5 or more. Of the total peptides contained in the peptide-nucleic acid conjugate library, 1 × 10 ３ In the peptides described above, the number of N-substituted amino acid residues contained in the peptide is, for example, 10 or less, 9 or less, preferably 8 or less, more preferably 7 or less, and most preferably 6 or less. Of the total peptides contained in the peptide-nucleic acid conjugate library, 1 × 10 ３ In the peptides described above, examples of the range of the number of N-substituted amino acid residues contained in the peptides include 1 to 10, 1 to 8, 1 to 7, and 2 to 6. Of the total peptides contained in the peptide-nucleic acid conjugate library, 1 × 10 ３ In the peptides described above, the number of N-substituted amino acid residues contained in the peptide is, for example, 1 to 13, preferably 3 to 12, more preferably 4 to 11, and most preferably 5 to 10. 【0060】 The peptide may be a cyclic peptide. In this specification, "cyclic peptide" is not particularly limited as long as it has a cyclic portion composed of five or more amino acid residues. Of the total peptides contained in the peptide-nucleic acid conjugate library, 1 × 10 ３ In the peptides described above, the number of amino acid residues constituting the cyclic portion of the cyclic peptide may be, for example, 5 to 15, 6 to 15, 6 to 14, 7 to 14, 8 to 14, 7 to 13, 7 to 12, 8 to 12, 8 to 11, 9 to 11, 10, or 11. Of the total peptides contained in the peptide-nucleic acid conjugate library, 1 × 10 ３In the peptides described above, the number of amino acid residues constituting the cyclic portion may be, for example, 5 to 15, preferably 9 to 15, more preferably 10 to 14, and most preferably 11 to 13. Having the number of amino acid residues constituting the cyclic portion within this range increases the efficiency of obtaining peptides with high membrane permeability and metabolic stability. The cyclic portion is preferably formed via covalent bonds such as amide bonds, carbon-carbon bond formation reactions, S-S bonds, thioether bonds, and triazole bonds. Cyclization can take any form, including cyclization via carbon-nitrogen bonds such as amide bonds, cyclization via carbon-oxygen bonds such as ester bonds and ether bonds, cyclization via carbon-sulfur bonds such as thioether bonds, cyclization via carbon-carbon bonds, or cyclization by heterocycle construction. Of these, cyclization via covalent bonds such as amide bonds and carbon-carbon bonds is preferred, and cyclization via amide bonds between the carboxyl group of the side chain and the amino group of the main chain is more preferred. The positions of the carboxyl group and amino group used in cyclization can be on the main chain or on the side chain, and are not particularly limited as long as they are in a position where cyclization is possible. 【0061】 A cyclic peptide may have a linear portion in addition to the cyclic portion. The specific configuration of the number of amino acid residues of a cyclic peptide is the same as the specific configuration of the number of amino acid residues of the peptide described above. When a cyclic peptide has a linear portion, it is preferable that the total number of amino acid residues of the cyclic and linear portions fall within the same range. Furthermore, when a cyclic peptide has a linear portion, of all peptides contained in the peptide-nucleic acid conjugate library, 1 × 10⁻¹⁶ ３ In the peptides described above, the number of amino acid residues constituting the cyclic portion may be, for example, 5 to 15, 6 to 15, 6 to 14, 7 to 14, 8 to 14, 7 to 13, 7 to 12, 8 to 11, 9 to 11, 10, or 11, and the number of amino acid residues constituting the linear portion may be, for example, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, or 1 to 3. When the cyclic peptide has a linear portion, of all peptides contained in the peptide-nucleic acid conjugate library, 1 × 10 ３In the peptides described above, the number of amino acid residues constituting the cyclic portion may be, for example, 5 to 15, preferably 9 to 15, more preferably 10 to 14, and most preferably 11 to 13. The number of amino acid residues constituting the linear portion may be, for example, 1 to 8, preferably 1 to 6, more preferably 1 to 4, and most preferably 1 to 3. Having the number of amino acid residues constituting the linear portion within the above ranges increases the efficiency of obtaining target molecules with high membrane permeability and metabolic stability. 【0062】 The peptide-nucleic acid conjugate library may be an mRNA display library or a cDNA display library, and is preferably an mRNA display library. 【0063】 The peptides contained in the peptide-nucleic acid conjugate library are 1 × 10⁻¹⁶ ３ The diversity of the peptides in the library may be 5 × 10⁻⁶. This can improve the screening efficiency of the target molecule. ３ The above is 1 x 10 ４ The above 5 x 10 ４ The above is 1 x 10 ５ The above 5 x 10 ５ The above is 1 x 10 ６ The above 5 x 10 ６ The above is 1 x 10 ７ The above 5 x 10 ７ The above is 1 x 10 ８ The above 5 x 10 ８ The above is 1 x 10 ９ The above 5 x 10 ９ The above is 1 x 10 １０ The above 5 x 10 １０ The above, or 1 x 10 １１ The above is sufficient. The diversity of peptides contained in the peptide-nucleic acid conjugate library is, for example, 1 × 10⁻⁶. ３ The above is sufficient, preferably 1 × 10 ５ More than 1 x 10 ７ In summary, the most preferred is 1 x 10 ９ That concludes the explanation. Peptide diversity is synonymous with the number of different types of peptides. 【0064】Among all the peptides contained in the peptide-nucleic acid conjugate library, 1 × 10 ３ or more peptides, the molecular weight of the peptide may be, for example, 300 g / mol or more, 350 g / mol or more, 400 g / mol or more, 450 g / mol or more, or 500 g / mol or more, and may be 5,000 g / mol or less, 4,500 g / mol or less, 4,000 g / mol or less, 3,500 g / mol or less, 3,000 g / mol or less, 2,500 g / mol or less, or 2,000 g / mol or less. Among all the peptides contained in the peptide-nucleic acid conjugate library, 1 × 10 ３ or more peptides, the molecular weight of the peptide may be, for example, 300 g / mol or more and 5,000 g / mol or less, preferably 350 g / mol or more and 4,500 g / mol or less, more preferably 400 g / mol or more and 4,000 g / mol or less, and most preferably 450 g / mol or more and 3,500 g / mol or less. The molecular weight in this specification means the sum of the atomic weights of the atoms constituting the compound molecule (unit: "g / mol"), and is obtained by calculating the sum of the atomic weights of the atoms included in the molecular structural formula (unit "g / mol"). 【0065】(Method for preparing a peptide-nucleic acid conjugate library) A peptide-nucleic acid conjugate library can be prepared by methods well known to those skilled in the art, for example, by the method described in International Publication No. 2013 / 100132. For example, an mRNA display library can be prepared as follows, although not limited to this. First, a DNA library with a desired base sequence placed downstream of a promoter is chemically synthesized, and this is used as a template to produce double-stranded DNA by a primer extension reaction. Next, this is used as a template to transcribe into mRNA using RNA polymerase. A linker (spacer) with an aminoacyl-tRNA analog such as the antibiotic puromycin is attached to the 3' end of this mRNA. This is added to a known cell-free translation system and incubated to translate the mRNA, and the mRNA and the peptide encoded therein are linked via the linker containing puromycin, etc. In this way, an mRNA display library consisting of complexes of mRNA and its products can be constructed. Furthermore, by contacting the mRNA display library with a desired immobilized target molecule and washing away peptide-nucleic acid conjugates that do not bind to the target molecule, peptide-nucleic acid conjugates that do bind to the target molecule can be concentrated (panning). By synthesizing cDNA from the mRNA contained in the selected peptide-nucleic acid conjugates, performing PCR amplification, and analyzing the base sequence, the amino acid sequence of the bound peptide can be determined. 【0066】 In this specification, of the total peptides contained in the peptide-nucleic acid conjugate library, 1 × 10 ３ To determine whether the above peptides fall within a specific value range, we calculate the value of each peptide actually contained in the peptide-nucleic acid conjugate library, and the number of values ​​is 1 × 10⁻⁶. ３ This can be determined by checking whether or not the result is greater than or equal to the above. 【0067】 However, the total number of peptides contained in the peptide-nucleic acid conjugate library is 1 × 10⁻¹⁶. ３ If the above conditions are met, we assume all types of peptides that could theoretically be included in the peptide-nucleic acid conjugate library, and of those, 1 × 10３ If more than one type of peptide-nucleic acid conjugate falls within a specific value range, then of all peptides contained in the peptide-nucleic acid conjugate library, 1 × 10 ３ Assume that the above peptides fall within a specific range of values. 【0068】 <Step (2)> In step (2), the peptide-nucleic acid conjugate library is brought into contact with the target molecule to select one or more peptide-nucleic acid conjugates that bind to the target molecule (contact step). In step (2), by bringing the peptide-nucleic acid conjugate library into contact with the target molecule, one or more peptide-nucleic acid conjugates that bind to the target molecule can be separated from one or more peptide-nucleic acid conjugates that do not bind to the target molecule. Therefore, one or more peptide-nucleic acid conjugates that bind to the target molecule can be selected. 【0069】 (Target Molecules) The target molecules may be one or more types, and are not limited to any one type. For example, they may be two or more types, three or more types, four or more types, five or more types, six or more types, seven or more types, eight or more types, nine or more types, or ten or more types, and may be 10 or fewer types, nine or fewer types, eight or fewer types, seven or fewer types, six or fewer types, five or fewer types, four or fewer types, three or fewer types, or two or fewer types. For example, the target molecules may be one or more types and five or fewer types, preferably one or more types and four or fewer types, more preferably one or more types and three or fewer types, even more preferably one or more types and two or fewer types, and most preferably one type. 【0070】 Specific examples of target molecules are not particularly limited as long as they can be bound to peptide-nucleic acid conjugates, but examples include proteins, sugars (oligosaccharides, polysaccharides, etc.), lipids, and nucleic acids (DNA, RNA, etc.), with proteins being preferred. 【0071】 When the target molecule is a protein, examples of target molecules include glutathione S-transferase (GST), extracellular signal-regulated kinase (ERK), FK506-binding protein (FKBP), FKBP-rapamycin-binding protein (FRB), cyclophyllin, and calcineurin (Cn). 【0072】The target molecule may be immobilized on a solid-phase carrier. There is no particular limitation on the shape of the solid-phase carrier, and for example, it may be in the shape of beads, plates, chips, or the like. The solid-phase carrier may have a molecule that can be used for affinity binding on its surface. Specifically, for example, the solid-phase carrier may have one or more selected from the group consisting of avidin, neutravidin, and streptavidin on its surface. 【0073】 When the target molecule is immobilized on the solid-phase carrier, the target molecule is present on the surface of the solid-phase carrier at 1 m ２ per area, 1×10 １３ or more, 5×10 １３ or more, 1×10 １４ or more, or 5×10 １４ or more may be immobilized. 【0074】 (Method for carrying out step (2)) The means for contacting the peptide-nucleic acid conjugate library with the target molecule is not particularly limited, and for example, it may include mixing the peptide-nucleic acid conjugate library and the target molecule. 【0075】 As a specific example of carrying out step (2), a method of adding the peptide-nucleic acid conjugate library to a liquid containing the target molecule, mixing, and then incubating can be mentioned. The conditions (incubation time, temperature during incubation, etc.) when contacting the peptide-nucleic acid conjugate library with the target molecule can be appropriately selected according to the type of the target molecule, the type of the peptide-nucleic acid conjugate library, and the like. 【0076】 Step (2) may include recovering one or more peptide-nucleic acid conjugates that bind to the target molecule. The recovery of the peptide-nucleic acid conjugate can be carried out by a method well known to those skilled in the art. For example, the peptide-nucleic acid conjugate can be recovered by a method using affinity such as the pull-down method. 【0077】Methods utilizing affinity include, for example, methods utilizing affinity between biotin and biotin-binding proteins, affinity between His tags and Ni-NTA, or affinity between antigens and antibodies, with the preferred method utilizing affinity between biotin and biotin-binding proteins. 【0078】 For example, by biotin modification of a target molecule and mixing it with a solid support (e.g., beads) to which biotin-binding proteins are attached, affinity binding between biotin and biotin-binding proteins can be established. By recovering the beads by centrifugation or other means, peptide-nucleic acid conjugates that have formed a complex with the target molecule can be recovered along with the target molecule. 【0079】 Examples of biotin-binding proteins used herein include avidin, neutraavidin, and streptavidin. 【0080】 <Step (2-1)> If the nucleic acid in the peptide-nucleic acid conjugate contains mRNA, the screening method according to this embodiment may further include a step of reverse transcribing at least one nucleic acid contained in the peptide-nucleic acid conjugate before step (3). In step (2-1), a peptide-mRNA / cDNA conjugate is generated from the peptide-mRNA conjugate by reverse transcribing at least one nucleic acid contained in the peptide-nucleic acid conjugate. If the nucleic acid in the peptide-nucleic acid conjugate contains mRNA, step (2-1) can be included to make step (3) easier to carry out. Reverse transcription can be carried out by methods well known to those skilled in the art. 【0081】 Step (2-1) may be performed before step (3), for example, during step (1), before step (2), or after step (2). 【0082】 <Step (3)> In step (3), the nucleic acid contained in one or more selected peptide-nucleic acid conjugates is amplified (amplification step). By performing step (3), a peptide that can bind to a target molecule can be obtained from the amplified nucleic acid. 【0083】 The nucleic acid contained in the peptide-nucleic acid conjugate may be a nucleic acid contained in a nucleic acid library, or a nucleic acid having a sequence complementary to the nucleic acid contained in a nucleic acid library. 【0084】 Nucleic acid amplification can be carried out, for example, by PCR using a nucleic acid contained in one or more selected peptide-nucleic acid conjugates as a template and a primer that binds to a sequence in the nucleic acid for amplification. 【0085】 Step (3) may further include eluting nucleic acids from the peptide-nucleic acid conjugates in order to amplify the nucleic acids contained in the selected peptide-nucleic acid conjugates. 【0086】 In other words, step (3) may be a step of amplifying one or more nucleic acids obtained from one or more selected peptide-nucleic acid conjugates, a step of amplifying one or more DNAs obtained from one or more selected peptide-nucleic acid conjugates, or a step of amplifying one or more cDNAs obtained from one or more selected peptide-nucleic acid conjugates. 【0087】 More specifically, step (3) may be a step of obtaining nucleic acid from the recovered peptide-nucleic acid conjugate and amplifying the nucleic acid by PCR or the like, a step of obtaining DNA from the recovered peptide-nucleic acid conjugate and amplifying the DNA by PCR or the like, or a step of obtaining cDNA from the recovered peptide-nucleic acid conjugate and amplifying the cDNA by PCR or the like. 【0088】 In eluting nucleic acids from recovered peptide-nucleic acid conjugates, the nucleic acids may be eluted in the form of the nucleic acid itself, the peptide-nucleic acid conjugate, or a complex of the target molecule and the peptide-nucleic acid conjugate. 【0089】In eluting nucleic acids from a peptide-nucleic acid conjugate, the bond between the peptide and the nucleic acid may be cleaved, the bond between the target molecule and the peptide-nucleic acid conjugate may be cleaved, and / or the bond between the solid support and the target molecule may be cleaved. By cleaving the bond between the peptide and the nucleic acid, the nucleic acid can be eluted in the form of the nucleic acid itself. By cleaving the bond between the target molecule and the peptide-nucleic acid conjugate, the nucleic acid can be eluted in the form of the peptide-nucleic acid conjugate. By cleaving the bond between the solid support and the target molecule, the nucleic acid can be eluted in the form of a complex of the target molecule and the peptide-nucleic acid conjugate. 【0090】 The bond can be cleaved by one or more methods selected from the group consisting of, for example, methods using enzymes, methods using light, or methods using heat. Preferably, the bond can be cleaved by methods using enzymes. 【0091】 Enzymatic cleavage of bonds can be performed, for example, by including a substrate that is specifically recognized and cleaved by the enzyme at the binding site between a peptide and a nucleic acid, the binding site between a target molecule and a peptide-nucleic acid conjugate, or the binding site between a solid support and a target molecule. By acting the enzyme on the complex containing the substrate, the bond is cleaved and the nucleic acid is eluted. Specific examples of enzyme-substrate combinations include, for example, a combination of a protease such as TEV protease or 3C protease and a peptide containing an amino acid sequence specifically recognized and cleaved by the protease, or a combination of a DNA-degrading enzyme such as a restriction enzyme and DNA containing a base sequence specifically recognized and cleaved by the DNA-degrading enzyme. 【0092】 Binding cleavage using light can be achieved, for example, by incorporating a structure that induces photochemical degradation at the binding site between the peptide and nucleic acid, the binding site between the target molecule and the peptide-nucleic acid conjugate, or the binding site between the solid support and the target molecule. By irradiating the complex containing such a structure with light of an appropriate wavelength, the bond is cleaved and the nucleic acid is eluted. Specific examples of structures that induce photochemical degradation include, for example, the 6-nitroveratriloxycarbonyl (NVOC) structure and the coumarin structure. 【0093】 The cleavage of bonds using heat can be achieved, for example, by using affinity-based bonding (e.g., the binding of biotin to a biotin-binding protein) for the binding of a peptide to a nucleic acid, a target molecule to a peptide-nucleic acid conjugate, or a solid support to a target molecule. By applying heat (e.g., 95°C for 10 minutes) to the complex having such a bond, the bond is cleaved and the nucleic acid is eluted. 【0094】 <Suppressing amplification in step (3)> The screening method according to this embodiment includes suppressing the amplification of at least one nucleic acid contained in the peptide-nucleic acid conjugate in step (3). By selectively suppressing the amplification in step (3), it is possible to selectively suppress the acquisition of peptides encoded by the nucleic acid whose amplification has been suppressed, among the peptides that can bind to the target molecule. 【0095】 In the amplification suppression in step (3) of the nucleic acid, at least one nucleic acid included in the peptide-nucleic acid conjugate that suppresses amplification can be arbitrarily selected. For example, it may be a nucleic acid that can be identified from peptides that are publicly known to be able to bind to a target molecule through databases, etc. Alternatively, it may be a nucleic acid encoding a peptide that has been identified as being able to bind to a target molecule as a result of performing steps (1) to (3) at least once without suppressing the amplification of the nucleic acid in the present invention. 【0096】 At least one nucleic acid contained in the peptide-nucleic acid conjugate that suppresses amplification may be some of the nucleic acids contained in multiple types of peptide-nucleic acid conjugates, for example, it may be 2 or more, 3 or more, 4 or more, or 5 or more nucleic acids, or it may be 10 or less, 9 or less, 8 or less, 7 or less, or 6 or less nucleic acids. 【0097】The amplification suppression in step (3) of the nucleic acid may be, for example, by reducing the amount of DNA that serves as the amplification template in step (3). Reducing the amount of DNA that serves as the amplification template in step (3) may be done, for example, by suppressing the translation of at least one nucleic acid contained in the nucleic acid library. Reducing the amount of DNA that serves as the amplification template in step (3) may also be done, for example, by degrading at least one nucleic acid contained in the nucleic acid library. In these cases, the amount of peptide-nucleic acid conjugates obtained from at least one nucleic acid contained in the nucleic acid library decreases, so the amount of such peptide-nucleic acid conjugates selected in step (2) also decreases, and the amount of DNA that serves as the amplification template in step (3) decreases. This makes it possible to suppress the amplification in step (3) of the nucleic acid. 【0098】 Furthermore, if the screening method according to this embodiment includes step (2-1) and the nucleic acid in the peptide-nucleic acid conjugate contains mRNA but not DNA, reducing the amount of DNA that serves as a template for amplification in step (3) may be done, for example, by suppressing the reverse transcription of at least one nucleic acid contained in the peptide-nucleic acid conjugate in step (2-1). In this case, the formation of a peptide-nucleic acid conjugate containing DNA is suppressed, so the amount of DNA that serves as a template for amplification in step (3) is reduced. This makes it possible to suppress the amplification of nucleic acids in step (3). 【0099】 The suppression of translation of at least one nucleic acid in the nucleic acid library can be confirmed, for example, by calculating the ratio of the number of cDNA molecules in the peptide-nucleic acid conjugate that are recovered after binding to the target molecule to the number of cDNA molecules in the peptide-nucleic acid conjugate. The specific calculation method may be as described in the examples. The suppression of translation of at least one nucleic acid in the nucleic acid library suppresses the formation of peptide-nucleic acid conjugates, and the binding ability of these peptide-nucleic acid conjugates to the target molecule is weakened, thus reducing the ratio. 【0100】Suppressing the translation of at least one nucleic acid contained in the nucleic acid library may, for example, reduce the percentage calculated by the above method by 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more compared to the case where no processing other than the processing necessary for nucleic acid translation was performed. 【0101】 The degradation of at least one nucleic acid in the nucleic acid library can be confirmed, for example, by calculating the ratio of the number of cDNA molecules in the peptide-nucleic acid conjugate before binding to the target molecule to the number of cDNA molecules in the peptide-nucleic acid conjugate recovered after binding to the target molecule. A specific comparison method may be as described in the examples. If at least one nucleic acid in the nucleic acid library is degraded, the number of cDNA molecules in the peptide-nucleic acid conjugate recovered after binding to the target molecule will decrease. 【0102】 Degrading at least one nucleic acid in the nucleic acid library may, for example, reduce the number of cDNA molecules in the peptide-nucleic acid conjugate recovered after binding to the target molecule by 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more compared to the number of cDNA molecules in the peptide-nucleic acid conjugate before binding to the target molecule. 【0103】 The suppression of reverse transcription of at least one nucleic acid contained in the peptide-nucleic acid conjugate can be confirmed, for example, by calculating the ratio of the number of cDNA molecules contained in the peptide-nucleic acid conjugate before binding to the target molecule to the number of cDNA molecules contained in the peptide-nucleic acid conjugate after binding to the target molecule and recovery. The specific comparison method may be as described in the examples. If the reverse transcription of at least one nucleic acid contained in the peptide-nucleic acid conjugate is suppressed, the number of cDNA molecules contained in the peptide-nucleic acid conjugate after binding to the target molecule and recovery will decrease. 【0104】Suppressing the reverse transcription of at least one nucleic acid contained in the peptide-nucleic acid conjugate may, for example, be achieved by reducing the number of cDNA molecules contained in the peptide-nucleic acid conjugate that has been bound to a target molecule and recovered by 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more compared to the number of cDNA molecules contained in the peptide-nucleic acid conjugate before binding to the target molecule. 【0105】 In suppressing amplification in nucleic acid process (3), "at least one nucleic acid contained in the nucleic acid library or at least one nucleic acid contained in the peptide-nucleic acid conjugate" is, in other words, a nucleic acid used as the target for translation and / or reverse transcription. 【0106】 Suppressing amplification in step (3) may be done by hybridizing a nucleic acid having a base sequence complementary to at least one nucleic acid in the nucleic acid library or at least one nucleic acid in the peptide-nucleic acid conjugate library (a nucleic acid used as the target of translation and / or reverse transcription) with at least one nucleic acid in the nucleic acid library or at least one nucleic acid in the peptide-nucleic acid conjugate library (a nucleic acid used as the target of translation and / or reverse transcription). By hybridizing the nucleic acid, a double-stranded structure of the nucleic acid can be formed, and a structure that inhibits the reaction of translating enzymes and / or reverse transcriptases can be formed. That is, in this case, suppressing amplification in step (3), suppressing the translation of at least one nucleic acid in the nucleic acid library, and suppressing the reverse transcription of the peptide-nucleic acid conjugate in step (2-1), may include, for example, forming a structure in at least one nucleic acid in the nucleic acid library or at least one nucleic acid in the peptide-nucleic acid conjugate that inhibits the reaction of translating enzymes and / or reverse transcriptases. 【0107】Examples of structures that inhibit the reaction of translation enzymes and / or reverse transcriptases include double-stranded nucleic acid structures and debasicated nucleic acid structures. If suppressing amplification in step (3) involves forming a structure that inhibits the reaction of translation enzymes and / or reverse transcriptases in at least one nucleic acid contained in the nucleic acid library or at least one nucleic acid contained in the peptide-nucleic acid conjugate, then suppressing amplification in step (3) may include debasing in addition to nucleic acid hybridization. 【0108】 The nucleic acid to be hybridized may be any nucleic acid having a single-stranded portion, and may be a natural nucleic acid or a non-natural nucleic acid (artificial nucleic acid), but a single-stranded nucleic acid is preferred. In this specification, "natural nucleic acid" means a nucleic acid (oligonucleotide) consisting of naturally occurring nucleotides. In this specification, "non-natural nucleic acid" means a nucleic acid (oligonucleotide) that contains at least a portion of chemically modified nucleotides that do not exist in nature. "Chemically modified nucleotide" means a nucleotide in which any or all of the bases, sugars, and phosphate groups constituting the nucleotide have been chemically modified. 【0109】 Specific examples of chemically modified nucleotides include, for example, LNA (Locked Nucleic Acid), PNA (Peptide Nucleic Acid), BNA (Bridged Nucleic Acid), and photocrosslinkable artificial nucleic acids. 【0110】 In non-natural nucleic acids, the number of chemically modified nucleotides relative to the number of naturally occurring nucleotides, i.e., the content of chemically modified nucleotides in non-natural nucleic acids, may be, for example, 1% or more, 5% or more, 10% or more, 15% or more, or 20% or more, and may be 100% or less, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, or 30% or less. 【0111】If the nucleic acid to be hybridized is a non-natural nucleic acid, it is preferable that the non-natural nucleic acid to be hybridized is a nucleic acid containing LNA. The nucleic acid to be hybridized is preferably a single-stranded non-natural nucleic acid, preferably a single-stranded nucleic acid containing LNA, and more preferably a single-stranded nucleic acid consisting of LNA. LNA is a cross-linked non-natural nucleic acid in which the oxygen atom at the 2' position and the carbon atom at the 4' position of RNA are cross-linked with methylene, and ribose is immobilized. If the nucleic acid to be hybridized is a nucleic acid containing LNA, the hybridization may be performed on single-stranded DNA or mRNA. 【0112】 When the nucleic acid to be hybridized is a natural nucleic acid, it is preferable that the natural nucleic acid to be hybridized is a single-stranded natural nucleic acid. It is preferable that the natural nucleic acid to be hybridized is DNA. In this case, the hybridization may be performed on single-stranded DNA or mRNA. 【0113】 The nucleic acids used for hybridization may, for example, consist of 9 or more bases, 12 or more bases, or 15 or more bases, and may also consist of 100 or fewer bases, 90 or fewer bases, 80 or fewer bases, 70 or fewer bases, 60 or fewer bases, 50 or fewer bases, 40 or fewer bases, or 35 or fewer bases. 【0114】 The above hybridization may be performed before step (3), after step (2), before step (2), after step (1), before step (1), after step (1) and before step (2), or after step (2) and before step (3), or any combination thereof. If the above hybridization is performed before step (1), the translation of at least one nucleic acid contained in the nucleic acid library can be suppressed. If the above hybridization is performed after step (1) and before step (2), or after step (2) and before step (3), the reverse transcription of the peptide-nucleic acid conjugate in step (2-1) can be suppressed. 【0115】The amplification inhibition in step (3) of the nucleic acid may include treatment with a nuclease that specifically recognizes the double-stranded portion formed by hybridization. The nuclease recognizes the double-stranded portion and degrades at least one nucleic acid contained in the nucleic acid library or at least one nucleic acid contained in the peptide-nucleic acid conjugate library (a nucleic acid used as the target for translation and / or reverse transcription). The nuclease may be a nuclease that degrades the DNA of the double-stranded portion. 【0116】 If the amplification inhibition in step (3) of the nucleic acid involves treatment with the above-mentioned nuclease, then at least one nucleic acid contained in the nucleic acid library or at least one nucleic acid contained in the peptide-nucleic acid conjugate library is preferably DNA, and more preferably cDNA. 【0117】 If the amplification inhibition in step (3) of the nucleic acid process includes treatment with the above-mentioned nuclease, the nucleic acid to be hybridized may be a natural nucleic acid or a non-natural nucleic acid, but it is preferable that it be a natural nucleic acid. 【0118】 If the screening method according to this embodiment includes step (4) described later, a single-stranded cDNA library may be obtained from the peptide-nucleic acid conjugate library by performing step (3), and a single-stranded nucleic acid having a base sequence complementary to at least a portion of the base sequence of at least one single-stranded cDNA contained in the single-stranded cDNA library may be hybridized. 【0119】 The above nuclease may specifically be, for example, dsDNasedsRNase, duplex specific nuclease, RNaseH, etc. 【0120】The treatment with the above nuclease can be performed at any time before step (3), after hybridizing a nucleic acid having a base sequence complementary to at least a portion of the base sequence of at least one nucleic acid in the nucleic acid library or at least one nucleic acid in the peptide-nucleic acid conjugate library with at least one nucleic acid in the nucleic acid library or at least one nucleic acid in the peptide-nucleic acid conjugate library. Specifically, the treatment with the above nuclease may be performed, for example, before step (1), or after step (2) and before step (3), but it is preferable to perform it before step (1). 【0121】 <Step (4)> The screening method according to this embodiment may further include a step (4) in which the nucleic acids amplified in step (3) are used as an n-th order nucleic acid library (where n is an integer of 2 or more), and steps (1) to (3) are repeated once or multiple times. 【0122】 By repeating steps (1) to (3) once or more times, a library containing the target molecule can be obtained. The number of repetitions may be one or more, two or more, three or more, four or more, or five or more, and specifically, for example, it may be one, two, three, four, or five times. 【0123】 <Step (5)> The screening method according to this embodiment may further include a step (identification step) of identifying the peptide that can bind to the target molecule by determining the base sequence of the nucleic acid amplified in step (3). Step (5) may include, after determining the base sequence of the nucleic acid amplified in step (3), identifying the amino acid sequence of the peptide that can bind to the target molecule based on the said base sequence. Determining the base sequence of the amplified nucleic acid and identifying the amino acid sequence of the peptide that can bind to the target molecule can be carried out by methods well known to those skilled in the art. 【0124】A screening method according to another embodiment (a screening method according to the second embodiment) is a screening method for peptides that can bind to a target molecule, comprising: (1) translating an mRNA library to obtain a peptide-nucleic acid conjugate library; (2) contacting the peptide-nucleic acid conjugate library with a target molecule to select one or more peptide-nucleic acid conjugates that bind to the target molecule; and (3) amplifying the nucleic acids contained in the peptide-nucleic acid conjugates from the selected one or more peptide-nucleic acid conjugates, and comprising hybridizing a nucleic acid containing a lock nucleic acid (LNA) having a base sequence complementary to the base sequence of at least one nucleic acid contained in the nucleic acid library or at least one nucleic acid contained in the peptide-nucleic acid conjugate library with at least one nucleic acid contained in the nucleic acid library or at least one nucleic acid contained in the peptide-nucleic acid conjugate library. 【0125】 As a specific embodiment of the screening method according to the second embodiment, the specific embodiment of the screening method according to the first embodiment can be applied. 【0126】 Furthermore, a screening method according to another embodiment (a screening method according to a third embodiment) is a screening method for peptides that can bind to a target molecule, comprising: (1) translating a nucleic acid library to obtain a peptide-nucleic acid conjugate library; (2) contacting the peptide-nucleic acid conjugate library with a target molecule to select one or more peptide-nucleic acid conjugates that bind to the target molecule; and (3) amplifying the nucleic acid contained in the peptide-nucleic acid conjugate from the selected one or more peptide-nucleic acid conjugates, wherein, prior to step (3), a single-stranded nucleic acid having a base sequence complementary to the base sequence of at least one DNA contained in the nucleic acid library or at least one DNA contained in the peptide-nucleic acid conjugate library is hybridized to at least one DNA contained in the nucleic acid library or at least one DNA contained in the peptide-nucleic acid conjugate library, and prior to step (3), treatment with a nuclease that specifically recognizes the double-stranded portion formed by hybridization. 【0127】 As a specific embodiment of the screening method according to the third embodiment, a specific embodiment of the screening method according to the first embodiment can be applied. 【0128】 [Method for producing peptides] The method for producing peptides according to this embodiment includes a step of screening peptides that can bind to a target molecule using the screening method according to this embodiment described above. This makes it possible to produce peptides that can bind to a target molecule. 【0129】 The peptide production method according to this embodiment includes an acquisition step (step (1)), a contact step (step (2)), and an amplification step (step (3)). Furthermore, a reverse transcription step may be included before the amplification step as needed, the contact step may include a recovery step, the amplification step may include an elution step, and an identification step may be included after the amplification step. Additionally, a step (translation step) may be included in which the nucleic acid amplified in the amplification step is used as a template for transcription and translation to obtain a peptide capable of binding to a target molecule. 【0130】 [Peptides] The peptides according to this embodiment are produced by a peptide production method that includes a step of screening peptides that can bind to a target molecule using the screening method according to this embodiment described above. 【0131】 The following describes preferred specific embodiments of the present invention as examples, but the present invention is not limited thereto. 【0132】 [Example 1: Pull-down assay] In Example 1, the following (1) and (2) were confirmed by inhibiting the translation and reverse transcription reactions during the preparation of mRNA display peptides (mRNA / cDNA-peptide conjugates) using LNA-antisense oligo (LNA-ASO). (1) In the above translation reaction, the amount of mRNA display peptide bound to the target molecule is reduced because the mRNA display peptide is not correctly generated from mRNA. (2) In the above reverse transcription reaction, the cDNA encoding the mRNA display peptide is not amplified because the generation of cDNA from mRNA is inhibited. 【0133】Specifically, an LNA-ASO was designed to hybridize with the mRNA encoding the target peptide. This LNA-ASO was added to the mRNA during the preparation of the mRNA display peptide, allowing it to hybridize. Translation and reverse transcription reactions were then performed to prepare the mRNA display peptide. A pull-down assay was performed on the prepared mRNA display peptide against the target protein (corresponding to the target molecule). The number of cDNA molecules in the prepared mRNA display peptide solution after the reverse transcription reaction and in the mRNA display peptide solution bound to the target protein was measured to verify the translation inhibitory effect of the LNA-ASO on the target peptide. The translation inhibitory effect on the target peptide was verified by comparing the ratio of cDNA molecules of the mRNA display peptide recovered bound to the target protein to the number of cDNA molecules of the added mRNA display peptide. The reverse transcription inhibitory effect on the target peptide was verified by measuring and comparing the number of cDNA molecules in the mRNA display peptide solution after the reverse transcription reaction. It is easy to imagine that if translation is inhibited and peptides are not properly generated, their binding ability to target proteins will decrease. Therefore, the decrease in the proportion of cDNA molecules mentioned above is thought to suggest an inhibitory effect on translation. The number of cDNA molecules was measured by qPCR. 【0134】 <Preparation of Double-Stranded DNA> Double-stranded DNA encoding a peptide compound library was prepared. Using synthetic oligoDNA (SEQ ID NOs. 1-6) as a template, PCR was performed using synthetic oligoDNA (SEQ ID NOs. 7) and synthetic oligoDNA (SEQ ID NOs. 8) with ExTaq (Takara). After denaturation at 95°C for 2 minutes, double-stranded DNA (SEQ ID NOs. 9-14) was prepared by repeating a cycle of 95°C for 10 seconds, 57°C for 20 seconds, and 72°C for 30 seconds 25 times. 【0135】Sequence ID 1 TAAGGAGATATAAAAATAGGGTAACCTGAGGACTGTGCATATAGCCCGACCGGCACCGGC Sequence ID 2 TAAGGAGATATAAAAATGATTCATACTGCTTGCGAATGCTCTTTCGGTAAGCCCGACCGGCACCGGC Sequence ID 3 TAAGGAGATATAAAAATAGCCGAACTTGTTCTCGTGCGTGAATTTGTGCCAGTAGCCCGACCGGCACCGGC Sequence ID 4 TAAGGAGATATAAAAATAGGGTCCCGCGAACAGGTTCGAATGAACCATTAAGCCCGACCGGCACCGGC Sequence ID 5 TAAGGAGATATAAAATAGCATTCCGGGGTGAGTGCCATACTAGTTTCTAAGCCGCACCGGCACCGGCACCGGC Sequence No. 6 TAAGGAGATATAAAATAGTTTTTCCGGAGTGCACTAACCACAGCCGTGTTAAGCCGCACCGGCACCGGC Sequence No. 7 GTAATACGACTACACTATAGGGGTTAACTTTAAAAGGAGATATAAAATAG Sequence No. 8 TTTTTTTTGCGCGGGCGCGCGGGC Sequence No. 9 GTAATACGACTCACTATAGGGTTAACTTTAATAAGGAGATATAAAATATGGGTAACCTGAGGACTGTGCATAGTGTGTAGCCGACCGGCACCGGCACCGGCAAAAAAAA SEQ ID NO: 10 GTAATACGACTCACTATAGGGTTAACTTTAATAAGGAGATATAAATATGATTCATACTGCTTGCGATGCTCTTCGGTAGCCGACCGGCACCGGCACCGGCAAAAAAAA SEQ ID NO: 11 GTAATACGACTCACTATAGGGTTAACTTTAATAAGGAGATATAAATATGCCGAACTTGTCTTGCGGTGATTTGCAGTAGCCGACCGGCACCGGCACCGGCAAAAAAAA SEQ ID NO: 12 GTAATACGACTCACTATAGGGTTAACTTTAATAAGGAGATATAAATATGGGTCCGCCGAACAGGTCGATGAACCATTAGCCGACCGGCACCGGCACCGGCAAAAAAAA SEQ ID NO: 13GTAATACGACTCACTATAGGGTTAACTTTAATAAGGAGATATAAATATGCATTCGGGTGAGTGCCATACTAGTTCTTAGCCGACCGGCACCGGCACCGGCAAAAAAAA SEQ ID NO: 14 GTAATACGACTCACTATAGGGTTAACTTTAATAAGGAGATATAAATATGTTTTCGGAGTGCACTAACCAGCCGTTGTAGCCGACCGGCACCGGCACCGGCAAAAAAAA 【0136】 <Preparation of mRNA-Puromycin Linker Ligation Products> Using a DNA library (SEQ ID NOs. 9-14) prepared by PCR as a template, the following mRNAs (SEQ ID NOs. 15-20) were prepared by in vitro transcription and purified using the RNeasy mini kit (Qiagen). 13 μM mRNA was mixed with 257.5 μM puromycin linker (Sigma) (sequence A), T4 RNA ligase reaction buffer (50 mM HEPES-KOH (pH 8.3), 10 mM MgCl) ２ Adding 2.67 mM dithiothreitol, 0.667 mM ATP), 10% PEG8000, 2 units / μl T4 RNA ligase (Newengl and Bio Lab.), the ligation reaction was carried out at 37°C for 60 minutes, and then purified using the RNeasy Mini Kit (Qiagen). Sequence IDs 15-20 were ligated on a 30 μl scale, purified, and mRNA-puromycin linker ligation products (mRNA-Pu) were prepared. 【0137】Array code 15 GGGUUAACUUUAAUAAGGAGAAUAUAAUAUAUGGGGUAACCUGUGCAAUAUAGUGUGUAGCGCACCGGCACCGGCACCGGCAAAAAAAA Array code 16 GGGUUAACUUUAAUAAGGAGAAUAUAUAAUAUCAAUAUUCUCUGGAAUUCUCUCUGGGGUAGCGCACCGGCACCGGCACCGGCAAAAAAAA Array code 17 GGGUUAACUUUAAUAAGGAGAAUAUAAUAUAUGCCGAACUUGUCUUGCGGGUGAAUUUGCCAGUAGCCGCACCGGCACCGGCACCGGCAAAAAAAAA Array 18 GGGUUAACUUUAAUAAGGAGAAUAUAUAAUAGGGGUCCGCCGCAACAGGUUCGAAUAUAACCCAAUAUAGCCGCACCGGCACCGGCACAAAAAAA Array 19 GGGUUAACUUUAAUAAGGAGAAUAUAAUAUAUGCAAUUCGGGUGAGUGCCAAUACUAGUUCUAGCGCACCGGCACCGGCACCGGCAAAAAAAAA Sequence ID 20 GGGUUAACUUUAAUAAGGAGAAUAUAUAUAUUCGGGAGUGCCAACUAACCAGCGCGCACCGGCACCGGCAAAAAAAAA Puromycin Linker Sequence A [P]dTdTdT[Spacer18 (hexaethylene glycol)][Spacer18][Spacer18][Spacer18]dCdC[puromycin] ([P]: 5' phosphorylation) 【0138】<Preparation of Biotinylated Target Proteins> Glutathione S-transferase (GST), Extracellular signal-regulated kinase (ERK), FRB, and FKBP12 were used as target proteins. These proteins were prepared by expression in Escherichia coli. Following the method described in WO2022 / 138892, GST-HisTEVAvi (SEQ ID NO: 63) was modified with a His tag, a TEV protease cleavage tag, and a biotinylated enzyme recognition tag to the C-terminus to prepare GST-HisTEVAvi (SEQ ID NO: 63) to produce GST-HisTEVAvi (SEQ ID NO: 64) to produce ERK-HisTEVAvi to produce ERK-HisTEV-Bio. For FRB and FKBP12, FLAG-Avi-bio-TEV-FKBP-FRB-FLAG(N-to-C) was prepared according to WO2024214825. 【0139】Sequence ID 63 MSPILGYWKIKGLVQPTRLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVK LTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEM LKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYL KSSKYIAWPLQGWQATFGGGGDHPPKSDGGSSHHHHHHSSGENLYFQGSGGGLNDIFEAQKIEWHE Sequence ID 64 MAAAAAAAQGGGGGEPRRTEGVGPVGVPGEVEMVKGQPFDVGPRYTQLQYIGEGAYGMVSSAYDHVRKTRVAIKKISPFEHQTYCQRTLRREIQILLRFRHENVIGIRDILRASTLEAMRDVYIVQDLMETDLYKLLKSQQLSNDHHICYFLYQILRGLKYIHSANVLHRDLKPSNLLINTTCCDLKICDFGLARIAADPEHDHTGFLTEYVATR WYRAPEIMLNSKGYTKSIDIWSVGCILAEMLSNRPIFPGKHYLDQLNHILGILGSPSQEDLNCIINMKARNYLQSLPSKTKVAWAKLFPKSDSKALDLLDRMLT FNPNKRITVEEALAHPYLEQYYDPTDEPVAEEPFTFAMELDDLPKERLKELIFQETARFQPGVLEAPGGSSHHHHHHSGENLYFQGSGGGLNDIFEAQKIEWHE 【0140】<Synthesis of acylated tRNA for translation> The acylated tRNA used for translation in Example 1 was prepared in accordance with the methods described in WO2018 / 143145 and WO2018 / 225864. A mixture of elongator aminoacylated tRNAs was prepared using 19 amino acids: Asp(SMe), Phe(3-Cl), Hph(3-Cl), MeAla(3-Pyr), MeGly, MeHnl(7-F2), MeHph, MeSer(nPr), MeSer(tBuOH), Nle, Pro(4-pip-4-F2), Pic(2), Ser(3-F-5-Me-Pyr), Ser(NtBu-Aca), Ser(Ph-2-Cl), Ser(iPen), cisPro(4-pip-4-F2), D-MeSer, and nBuGly. The final concentrations of each acylated tRNA in the translation solution ranged from 10 μM to 20 μM. The pCpA amino acids protected with Pnaz were processed after phenol extraction without deprotection. The initiator aminoacylated tRNA was the same compound as Acbz-MeCys(StBu)-tRNAfMetCAU described in WO2018 / 225864, and was added to the translation solution to a final concentration of 25 μM. The abbreviations of the amino acids exemplified herein and their relationship to their structures are shown in Table 1 below. 【0141】 【0142】<Translation solution used in pull-down assay> The translation solution used in the pull-down assay contains the following substances: 1 mM GTP, 1 mM ATP, 20 mM creatine phosphate, 50 mM HEPES-KOH pH 7.6, 100 mM potassium acetate, 6 mM magnesium acetate, 2 mM spermidine, 1 mM dithiothreitol, 1 mg / ml E. coli MRE600 (RNase negative) derived tRNA (Roche Inc.) (Non-patent literature: Yokogawa T, Kitamura Y, Nakamura D, Ohno S, Nishikawa K. 2010. Nucleic acids research) (Partially tRNA removed by the method described in 38:e89), 4 μg / ml creatine kinase, 6.72 units / mL myokinase, 2 units / mL inorganic pyrophosphatase, 1.1 μg / mL nucleoside diphosphate kinase, 0.26 μM EF-G, 2.7 μM IF1, 0.4 μM IF2, 1.5 μM IF3, 40 μM EF-Tu, 60 μM EF-Ts, 1 μM EF- P-Lys, 1.2 μM ribosome, 1.37 μM AlaRS, 1 μM GlyRS, 0.04 μM IleRS, 0.5 μM mutant PheRS (WO2016 / 148044), 0.16 μM ProRS, 1 μM mutant SerRS (WO2016 / 148044), 0.09 μM ThrRS, 1 μM mutant ValRS (WO2016 / 148044), 0.11 μM LysRS, 3 μM in In vitro transcription of E. coli tRNA Ala1B, 250 μM glycine, 10 μM isoleucine, 250 μM proline, 250 μM threonine, 250 μM lysine, 5 mM N-methylvaline, 5 mM N-methylserine, 2.5 mM N-methylalanine, 5 mM N-methylphenylalanine, Elongator aminoacyl-tRNA mixture, 25 μM Initiator aminoacyl-tRNA, 0.5 μM Penicillin G Amida (PGA), 0.4 units / μL RNasin® Ribonuclease Inhibitor (PROMEGA). 【0143】<Pull-down assay procedure> Using the mRNA-Pu and translation solution described above, an mRNA / cDNA-peptide conjugate solution was prepared in accordance with Patent Document 1 (WO2013 / 100132) and a pull-down assay was performed. Specifically, regarding the LNA-ASO addition conditions, each LNA-ASO (SEQ ID NOs. 21-26) was added to mRNA-Pu and heated at 95°C for 2 minutes, then cooled to 4°C before adding the translation solution and performing translation. To this reaction solution, 30 mM TCEP TEA solution (pH 7.0), 30 mM Tris-HCl solution (pH 8.3), 20 mM KOH, and 12.5 mM EDTA were mixed and then heated at 37°C for 1 hour and 30 minutes to advance the peptide cyclization reaction. Furthermore, 12.5 mM glutathione, 175 mM TCEP TEA solution (pH 7.0), and 0.8 M VA-044 were added, and the mixture was heated at 42°C for 2 hours and 30 minutes to promote the desulfurization reaction of the peptide. After this, the mRNA-peptide conjugate was purified. The reverse transcription reaction of the mRNA-peptide conjugate was also carried out in the same manner after adding LNA-ASO. Each of the LNA-ASOs used was designed as a sequence complementary to the mRNA sequence encoding a specific four amino acid portion of the target peptide. Sequence ID 21 targets nBuG:S3F5MePyr:T:MeHnl7F2 of Sequence ID 15, Sequence ID 22 targets Pic2:cisP4pip4F2:MeA:MeHph of Sequence ID 16, Sequence ID 23 targets SPh2Cl:SNtBuAca:Pic2:G of Sequence ID 17, Sequence ID 24 targets P:Nle:S3F5MePyr:MeG of Sequence ID 18, Sequence ID 25 targets G:dMeS:Pic2:Hph3Cl of Sequence ID 19, and Sequence ID 26 targets the mRNA sequence encoding MeG:dMeS:Pic2:T of Sequence ID 20. 【0144】Proteins linked with GST, ERK, or FKBP12 and FRB via a linker have a TEV protease recognition sequence introduced between them and biotin; therefore, the target peptide was eluted from the biotinylated target protein using TEV protease. After the target peptide interacted with the biotinylated protein, it was collected using streptavidin-immobilized magnetic beads, washed, and then the beads were treated with TEV elution solution (50 mM Tris-HCl pH 8.0, 0.5 mM EDTA, 1 mM DTT, 0.1 U / μL AcTEV protease (Thermo Fisher Scientific, product number 12575015)) and reacted. After the reaction, the supernatant was collected. The amount of cDNA in this supernatant and the amount of cDNA in the prepared mRNA / cDNA-peptide conjugate solution after the reverse transcription reaction were measured by qPCR. For qPCR, Fw primer (SEQ ID NO: 62) and Rv primer (SEQ ID NO: 8) were used as primers. The qPCR reaction mixture consisted of 1x Ex Taq buffer, 0.2 mM dNTPs, 0.5 μM Fw primer, 0.5 μM Rv primer, 50,000-fold diluted SYBR Green I (Lonza, catalog #50513), and 0.02 U / μL Ex Taq polymerase (TAKARA). The mixture was heated at 95°C for 2 minutes, followed by 40 cycles of heating at 95°C for 10 seconds, 57°C for 20 seconds, and 72°C for 30 seconds. 【0145】 SEQ ID NO: 21 CACAGTCCTCCAG (Nucleic acids at positions 3, 5, 7, 8, 10, and 11 from the 5' end are LNA) SEQ ID NO: 22 AAGAGCATCGCA (Nucleic acids at positions 2, 3, 6, 8, 9, and 11 from the 5' end are LNA) SEQ ID NO: 23 ACCGCAAGACAA (Nucleic acids at positions 2, 3, 6, 8, 10, and 11 from the 5' end are LNA) SEQ ID NO: 24 CGACCTGTTCGGG (Nucleic acids at positions 2, 4, 6, 8, 10, and 11 from the 5' end are LNA) SEQ ID NO: 25 ATGGCAACTCACC (Nucleic acids at positions 3, 4, 5, 7, 9, and 11 from the 5' end are LNA) SEQ ID NO: 26 AGTGCACTCCGA (Nucleic acids at positions 2, 4, 5, 7, 9, and 11 from the 5' end are LNA) SEQ ID NO: 62 GTAATACGACTCACTATAGGGTTAACTTTTAATAAGGGAG 【0146】 <Results> The recovery rate of mRNA / cDNA-peptide conjugate clones was evaluated from the qPCR results. The recovery rate (%) of mRNA / cDNA-peptide conjugate clones was calculated using the following formula. The results are shown in Table 2. Input cDNA amount refers to the number of cDNA molecules of the prepared mRNA / cDNA-peptide conjugate before being subjected to the pull-down assay. Output cDNA amount refers to the number of cDNA molecules of the mRNA / cDNA-peptide conjugate bound to the target protein and recovered after pull-down. (Formula): Recovery rate (%) of mRNA / cDNA-peptide conjugate clones = {Output cDNA amount (number of molecules) of mRNA / cDNA-peptide conjugate clones / Input cDNA amount (number of molecules) of mRNA / cDNA-peptide conjugate clones} × 100 【0147】 【0148】 As shown in Table 2, the input cDNA amount, output cDNA amount, and recovery rate of mRNA / cDNA-peptide conjugate clones containing the target mRNA were significantly reduced when LNA-ASO was used (#2, #7, #12, #16, #18, and #21). The reduced recovery rate of mRNA / cDNA-peptide conjugate clones suggests that inhibiting the translation process by LNA-ASO prevents the proper generation of mRNA display peptides from mRNA, resulting in a decrease in the amount of peptides bound to the target molecule. Furthermore, the reduced input cDNA amount of mRNA / cDNA-peptide conjugate clones suggests that inhibiting the reverse transcription process by LNA-ASO inhibits cDNA generation from mRNA, preventing the amplification of cDNA encoding the mRNA display peptide. 【0149】[Example 2: Mini-library assay] As in Example 1, the inhibitory effect of LNA-ASO on translation and reverse transcription reactions was confirmed in a mixed solution containing multiple mRNAs encoding mRNA display peptides. Specifically, in a solution containing mRNA-Pu encoding multiple types of mRNA display peptides, one type of LNA-ASO or a mixture of two types of LNA-ASO was used to hybridize an LNA-ASO complementary to the target mRNA sequence, similar to Example 1. This was used to verify whether the translation and reverse transcription reactions were inhibited, reducing the recovery rate and cDNA amount of the target mRNA display peptide and decreasing its relative abundance in the mixed solution. 【0150】 <Preparation of Double-Stranded DNA> Double-stranded DNA encoding the peptide compound library was prepared in the same manner as in Example 1. Using synthetic oligoDNA (SEQ ID NOs. 27-31) as a template, PCR was performed using synthetic oligoDNA (SEQ ID NOs. 7) and synthetic oligoDNA (SEQ ID NOs. 8) with ExTaq (Takara). After denaturation at 95°C for 2 minutes, the cycle of 95°C for 10 seconds, 57°C for 20 seconds, and 72°C for 30 seconds was repeated 25 times to prepare double-stranded DNA (SEQ ID NOs. 32-36). 【0151】Sequence ID 27 TAAGGAGATATAAAAATGATTCATAGTATTGTGGTATTCTGTGGTAGCGCACCGGCACCGGC Sequence ID 28 TAAGGAGATATAAAAATGTGGCGGAGAGAGTTTTTTAGGGTGATAGTGTGTTAGCGCACCGGCACCGGC Sequence ID 29 TAAGGAGATATAAAAATGTGCGGGAGCGGTTTAGGGGGTTAGAGTGTGTTAGCGCACCGGCACCGGC Sequence ID 30 TAAGGAGATATAAAAATGTTCGTTCGTCATCTGTGGCATATTAGGTGAGCACCGGCACCGGC Sequence ID 31 TAAGGAGATATAAAAATGTGGGGTTTTGTCTGTGCCTACTGGGGGTAGCGCACCGCACCGC Sequence ID 32 GTAATACGACTCACTATAGGGGGTATAAAAAGGAGATATAAAAATGATTCATATAGTATTTAGTGGGGTATTCTGTGCGCACCGCACCGCACCGCACCGCAAAAAA Sequence ID 33 GTAATACGACTCACTATAGGGGTTAAACTTTTAAAGGAGAATATAAATGTGCGAGAAGTTTTTAAGGGTCGAATGAGTGTGTTAGCGCACCGCGCACCGCGCACCGCCAAAAAAA Sequence ID 34 GTAATACGACTCACTATAGGGGTTAAACTTTTAAAGGAGAATATAAAGGCGCGAGCGCGTTAAAGGGGTTAAGGCGCACCGCACCGCGCCAAAAAAA Sequence ID 35 GTAATACGACTCACTATAGGGTTAACTTTAATAAGGAGATATAAATATGTCTGGTCGTCATCTGTGCCATATTAGGTAGCCGACCGGCACCGGCACCGGCAAAAAAAA SEQ ID NO: 36 GTAATACGACTCACTATAGGGTTAACTTTAATAAGGAGATATAAATATGTGGGTTTTGCTGTGCTCTACTGGTGGTTAGCCGACCGGCACCGGCACCGGCAAAAAAAA 【0152】<Preparation of mRNA-Puromycin Linker Ligation Products> Using the DNA library (SEQ ID NOs. 32-36) prepared by PCR as in Example 1, the following mRNAs (SEQ ID NOs. 37-41) were prepared by in vitro transcription using the PCR-prepared DNA library (SEQ ID NOs. 37-41) and purified using the RNeasy mini kit (Qiagen). 13 μM mRNA was mixed with 257.5 μM puromycin linker (Sigma) (Sequence A), T4 RNA ligase reaction buffer (50 mM HEPES-KOH (pH 8.3), 10 mM MgCl). ２ 2.67 mM dithiothreitol, 0.667 mM ATP), 10% PEG8000, 2 units / μl T4 RNA ligase (New England Bio Lab.) were added, and the ligation reaction was carried out at 37°C for 60 minutes, followed by purification using the RNeasy Mini Kit (Qiagen). Sequence IDs 37-41 were purified by ligation with puromycin linker on a 30 μl scale to prepare mRNA-puromycin linker ligation products (mRNA-Pu). 【0153】Array code 37 GGGUUAACUUUAAAGGAGAAUAUAAUAUAAUUCAUAGUGUGUAUAGUUCUCGGCACCGGCACCGGCACCGGCAAAAAAAA Array code 38 GGGUUAACUUUAAAGGAGAAUAUAAUAUAUAGUGGCGAGAAUGAGUGUUUAGUUCGAAUGAGUGUGUAGCGCACCGGCACCGGCACCGGCAAAAAAAA Array code 39 GGGUUAACUUUAAUAAGGAGAAUAUAAUAUAGCGGGGAGCGGGUUUAAGGGGUUAAGCGGCACCGGCACCGGCACCGGCAAAAAAAAAA Sequence ID 40 GGGUUAACUUUAAUAAGGAGAAUAUAUAUAUAGUCUGUCGCUCAUUCUGUCCCAUAUAUAAGGGGCACCGGCACCGGCACCGGCAAAAAAAAAA Sequence ID 41 GGGUUAACUUUAAUAAGGAGAUAUAAAAUUGGGGUUUUGCUGUGCUCUACUGGUGGUUAGCCGACCGGCACCGGCACCGGCAAAAAAAA 【0154】 <Preparation of Biotinylated Target Proteins> The proteins GST-HisTEVBio, ERK-HisTEVBio, and FLAG-Avi-bio-TEV-FKBP-FRB-FLAG(N-to-C) prepared in Example 1 were used. 【0155】 <Translation solution used for pull-down assay> A translation solution prepared in the same manner as in Example 1 was used. 【0156】<Pull-down assay procedure> For the prepared mRNA-Pu, mRNA-Pu mixture 1, which contained mRNA-Pu corresponding to SEQ ID NOs. 15, 16, and 37; mRNA-Pu mixture 2, which contained mRNA-Pu corresponding to SEQ ID NOs. 17, 18, 38, and 39; and mRNA-Pu mixture 3, which contained mRNA-Pu corresponding to SEQ ID NOs. 19, 20, 40, and 41 were used thereafter. Using the mRNA-Pu mixture and translation solution, mRNA / cDNA-peptide conjugate solutions were prepared in accordance with Patent Document 1 (WO2013 / 100132) and a pull-down assay was performed. Regarding the LNA-ASO addition conditions, LNA-ASO (SEQ ID NOs. 21-25, 42) was added to the mRNA-Pu mixture according to Table 3 below, heated at 95°C for 2 minutes, cooled to 4°C, and then the translation solution was added to perform translation. Furthermore, for the reverse transcription reaction of the peptide-mRNA conjugate, LNA-ASO was added before the reverse transcription reaction was carried out. Sequence ID No. 42 ATGGCACAGATG (the nucleic acids at positions 3, 4, 5, 7, 9, and 11 from the 5' end are LNA) 【0157】 Since a TEV protease recognition sequence was introduced between GST or ERK and biotin, the target peptide was eluted from the biotinylated target protein using TEV protease. After the peptide interacted with the biotinylated protein, it was collected using streptavidin-immobilized magnetic beads, washed, and then TEV elution solution (50 mM Tris-HCl pH 8.0, 0.5 mM EDTA, 1 mM DTT, 0.1 U / μL AcTEV protease (Thermo Fisher Scientific, product number 12575015)) was added to the beads and the reaction was allowed to proceed. After the reaction, the supernatant was collected and the cDNA in this supernatant was amplified by PCR. The base sequence analysis of this DNA pool was performed, and the relative abundance of each peptide sequence was calculated. The results are shown in Tables 4-6. 【0158】 【0159】 【0160】 【0161】 【0162】 <Results> As shown in Tables 4-6, under conditions where an LNA-ASO with a complementary base sequence was added, the relative abundance of mRNA encoding the target peptide decreased (SEQ ID NO: 15 in Condition 2, SEQ ID NOs: 15 and 16 in Condition 3, SEQ ID NO: 17 in Condition 6, SEQ ID NOs: 17 and 18 in Condition 7, SEQ ID NO: 19 in Condition 10, and SEQ ID NOs: 19 and 39 in Condition 11). 【0163】 [Example 3: Selective Degradation of Dominant Clones Using dsDNase Enzyme Reaction] When preparing mRNA display peptides, we confirmed that nucleic acid sequences (DNA sequences) encoding specific peptide sequences in the library can be selectively degraded by the enzyme. Specifically, the DNA encoding the peptide was prepared from template DNA by PCR, and the mRNA sequence was prepared from this DNA by a transcription reaction. After creating double-stranded mRNA / cDNA nucleic acids by reverse transcription, single-stranded cDNA was obtained using RNaseH. To this single-stranded cDNA, DNA with a complementary base sequence (driveroligo) was added and hybridized, and dsDNase was added and the reaction was carried out. Since dsDNase selectively degrades double-stranded DNA, we confirmed whether the target single-stranded cDNA could be selectively degraded by designing and using driveroligo to hybridize with the target single-stranded cDNA. 【0164】 <Preparation of double-stranded DNA> Sequence IDs 9-14, 35, and 36 were prepared from template DNA by PCR, as in Example 1. 【0165】 <Preparation of mRNA> Sequence IDs 15-20, 40, and 41 were prepared by transcription reaction from Sequence IDs 9-14, 35, and 36, in the same manner as in Example 1. 【0166】 <Preparation of single-stranded cDNA sequence> The prepared mRNA was subjected to a reverse transcription reaction to prepare mRNA / cDNA. 5 μM mRNA was mixed with 5 μM Rv primer (SEQ ID NO: 8), 50 mM TrisHCl (pH 8.3), 75 mM KCl, and 3 mM MgCl. ２0.5 mM dGTP, 0.5 mM dATP, 0.5 mM dCTP, 0.5 mM dTTP, and 8 U / μL M-MLV Reverse Transcriptase (H-) (Promega, M368B) were added, and the mixture was made up with Nuclear-Free Water (NFW). The solution was incubated in 20 μL at 42°C for 60 minutes to perform the reverse transcription reaction. Subsequently, RNaseH (Thermofisher, EN0201) was added to this mRNA / cDNA. After inactivating RNaseH by incubation at 37°C for 30 minutes and heating at 65°C for 15 minutes, single-stranded cDNA (SEQ ID NOs. 43-50) was obtained. 【0167】SEQ ID NO: 43 TTTTTTTGCCGTGFCGTGLCGTCGLTTAGAAAGLATCGAAGLGTATGATCATATTTATAATCCTTTATTAAATTAAD T T T 【0168】<Confirmation of Selective Degradation by dsDNase> According to Table 7, single-stranded cDNA sequences equivalent to 5 μM (SEQ ID NOs. 43-50) were prepared. Ultrapure water or driveroligo (SEQ ID NOs. 51-58) and 10x reaction buffer (Thermofisher, EN0771) were added, and the mixture was made up with ultrapure water. The mixture was heated in a thermal cycle at 95°C for 3 minutes and then cooled to 4°C. 0.20 U / μL of dsDNase (Thermofisher, EN0771) was added to this solution, and the mixture was heated at 37°C for 10 minutes, followed by heating at 55°C for 5 minutes to inactivate the dsDNase and obtain a reaction solution. The amount of cDNA contained in this reaction solution was quantified by qPCR. Fw primer (SEQ ID NOs. 62) and Rv primer (SEQ ID NOs. 8) were used as qPCR primers. The qPCR reaction mixture consisted of 1x Ex Taq buffer, 0.2 mM dNTPs, 0.5 μM Fw primer, 0.5 μM Rv primer, 50,000-fold diluted SYBR Green I (Lonza, catalog #50513), and 0.02 U / μL Ex Taq polymerase (TAKARA). The mixture was heated at 95°C for 2 minutes, followed by 40 cycles of heating at 95°C for 10 seconds, 57°C for 20 seconds, and 72°C for 30 seconds. 【0169】 【0170】 Sequence ID 51 ATGGGTAACCTGAGACTGTGCATATAGGTGTAG Sequence ID 52 ATGATTCATACTGCTTGCGAATGCTCTCTCGGGTAG Sequence ID 53 ATGCCCGAACTGTGTTCTTGCGGGTGAATTTGCAG Sequence ID 54 ATGGGTCCGCGAACAGGTTCGAATGAACCATTAG Sequence ID 55 ATGCATTCGCGGGTGAGTGCCATACTATAGTTTCTAG Sequence ID 56 ATGTTTTCGCGGGAGTGCACTAACCAGCCGTGTAG Sequence ID 57 ATGTCTGGTCTCCATCTGTGCCATATATAGGTAG Sequence ID 58 ATGTGGGTTTTGCTGTGCTCCTACTGGTGGTTAG 【0171】 【0172】 <Results> Table 8 shows the molecular weight of the target single-stranded cDNA under each condition quantified by qPCR. As shown in Table 8, the amount of cDNA decreased and the target sequence was selectively degraded by dsDNase only when driveroligo, which has a complementary base sequence to each single-stranded cDNA and is designed to hybridize, was used (Table 8 #2, 5, 8, 11, 14, 17, 20, 23). 【0173】 [Example 4: Selective Degradation of Dominant Clones in a Library Using dsDNase Enzyme Reaction] When preparing mRNA display peptides, it was confirmed that nucleic acid sequences encoding specific peptide sequences in a library could be selectively degraded by the enzyme in a mixture containing multiple mRNAs encoding mRNA display peptides. Specifically, in a solution containing mRNA-Pu encoding multiple types of mRNA display peptides, one type of driveroligo or a mixture of two types of driveroligo was used to hybridize the driveroligo corresponding to the target mRNA sequence, as in Example 3, and dsDNase was added to react. This confirmed whether the target cDNA sequence could be selectively degraded. 【0174】 <Preparation of double-stranded DNA> Sequence IDs 32-34 were prepared from template DNA by PCR, in the same manner as in Example 1. 【0175】 <mRNA preparation> Sequence IDs 37-39 were prepared from Sequence IDs 32-34 by transcription reaction, similar to Example 1. 【0176】 <Preparation of single-stranded cDNA sequence> The prepared mRNA was subjected to a reverse transcription reaction to prepare mRNA / cDNA. 5 μM mRNA was mixed with 5 μM Rv primer (SEQ ID NO: 8), 50 mM TrisHCl (pH 8.3), 75 mM KCl, and 3 mM MgCl. ２0.5 mM dGTP, 0.5 mM dATP, 0.5 mM dCTP, 0.5 mM dTTP, and 8 U / μL M-MLV Reverse Transcriptase (H-) (Promega, M368B) were added, and the mixture was made up with nucleotide-free water. The solution was incubated in 20 μL at 42°C for 60 minutes to perform the reverse transcription reaction. Subsequently, RNaseH (Thermofisher, EN0201) was added to this mRNA / cDNA. After inactivating RNaseH by incubation at 37°C for 30 minutes and heating at 65°C for 15 minutes, single-stranded cDNA sequences (SEQ ID NOs. 59-61) were obtained. 【0177】 SEQ ID NO: 59 TTTTTTTTGCCGGTGCCGGTGCCGGTCGGCTACCACAGAATAACCACTAATACTATGAATCATATTTATATCTCCTTATTAAAGTTAACCC SEQ ID NO: 60 TTTTTTTTGCCGGTGCCGGTGCCGGTCGGCTACACACTCATCGACCTAAAACTCTCGCACATATTTATATCTCCTTATTAAAGTTAACCC SEQ ID NO: 61 TTTTTTTTGCCGGTGCCGGTGCCGGTCGGCTACACACTCATACCCCCTAAACCGCTCCCGCATATTTATATCTCCTTATTAAAGTTAACCC 【0178】<Confirmation of selective degradation by dsDNA> For the prepared single-stranded cDNA, single-stranded cDNA mixture 1, which was a mixture of single-stranded cDNAs corresponding to SEQ ID NOs. 43, 44, and 59; single-stranded cDNA mixture 2, which was a mixture of single-stranded cDNAs corresponding to SEQ ID NOs. 45, 46, 60, and 61; and single-stranded cDNA mixture 3, which was a mixture of single-stranded cDNAs corresponding to SEQ ID NOs. 47, 48, 49, and 50 were used thereafter. To the prepared single-stranded cDNA mixture according to Table 9, ultrapure water or driveroligo (SEQ ID NOs. 51-54, 55, 57), 10x reaction buffer (Thermofisher, EN0771) was added, and the mixture was made up with ultrapure water. The mixture was heated in a thermal cycle at 95°C for 3 minutes and then cooled to 4°C. To this solution, 0.20 U / μL of dsDNase (Thermofisher, EN0771) was added and heated at 37°C for 10 minutes, then heated at 55°C for 5 minutes to inactivate the dsDNase and obtain a reaction solution. The amount of cDNA contained in this reaction solution was quantified by qPCR. Fw primer (SEQ ID NO: 62) and Rv primer (SEQ ID NO: 8) were used as qPCR primers. The qPCR reaction mixture consisted of 1x Ex Taq buffer, 0.2 mM dNTPs, 0.5 μM Fw primer, 0.5 μM Rv primer, 50,000-fold diluted SYBR Green I (Lonza, catalog #50513), and 0.02 U / μL Ex Taq polymerase (TAKARA). The mixture was heated at 95°C for 2 minutes, followed by 40 cycles of heating at 95°C for 10 seconds, 57°C for 20 seconds, and 72°C for 30 seconds. 【0179】 【0180】 【0181】 【0182】 【0183】<Results> Tables 10-12 show the molecular weight of the target single-stranded cDNA quantified by qPCR under each condition. As shown in Tables 10-12, under the conditions in which driveroligo was added, the abundance of the target single-stranded cDNA decreased (SEQ ID NO: 43 in Condition 2, SEQ ID NOs: 43 and 44 in Condition 3, SEQ ID NO: 45 in Condition 5, SEQ ID NOs: 45 and 46 in Condition 6, SEQ ID NO: 47 in Condition 8, and SEQ ID NOs: 47 and 49 in Condition 9). These results suggest that it is possible to suppress the acquisition of target DNA by selectively degrading it in the library using dsDNase and reducing its abundance.

Claims

1. A method for screening peptides that can bind to a target molecule, comprising: (1) translating a nucleic acid library to obtain a peptide-nucleic acid conjugate library; (2) contacting the peptide-nucleic acid conjugate library with the target molecule to select one or more peptide-nucleic acid conjugates that bind to the target molecule; and (3) amplifying the nucleic acids contained in the peptide-nucleic acid conjugates from the selected one or more peptide-nucleic acid conjugates, wherein the method includes suppressing the amplification of at least one nucleic acid contained in the peptide-nucleic acid conjugate in step (3).

2. The method according to claim 1, wherein the nucleic acid in the peptide-nucleic acid conjugate comprises mRNA, and (2-1) the method further comprises the step of reverse transcribing at least one nucleic acid contained in the peptide-nucleic acid conjugate before step (3).

3. The method according to claim 1 or 2, wherein the amplification in step (3) is suppressed by hybridizing a nucleic acid having a base sequence complementary to at least a portion of the base sequence of at least one nucleic acid contained in the nucleic acid library or at least one nucleic acid contained in the peptide-nucleic acid conjugate library with at least one nucleic acid contained in the nucleic acid library or at least one nucleic acid contained in the peptide-nucleic acid conjugate library.

4. The method according to claim 3, wherein the nucleic acid to be hybridized is a nucleic acid containing locking nucleic acid (LNA).

5. The method according to claim 4, wherein the hybridization is performed on single-stranded DNA or mRNA.

6. The method according to claim 4, wherein suppressing amplification in step (3) comprises forming a structure in at least one nucleic acid contained in the nucleic acid library or at least one nucleic acid contained in the peptide-nucleic acid conjugate that inhibits the reaction of translation enzymes and / or reverse transcriptases.

7. The method according to claim 3, wherein the nucleic acid to be hybridized is a natural nucleic acid, and suppressing the amplification in step (3) includes treatment with a nuclease that specifically recognizes the double-stranded portion formed by hybridization.

8. The method according to claim 7, wherein the nuclease is a nuclease that degrades the double-stranded portion of DNA.

9. The method according to claim 7, wherein the treatment with the nuclease is performed after step (2) and before step (3).

10. The method according to claim 3, wherein the nucleic acid to be hybridized is 9 bases or more.

11. The method according to claim 3, wherein the nucleic acid to be hybridized is 12 bases or more.

12. The method according to claim 1 or 2, wherein step (2) comprises recovering one or more peptide-nucleic acid conjugates bound to the target molecule.

13. The method according to claim 12, wherein step (3) comprises eluting the nucleic acid in the peptide-nucleic acid conjugate before amplifying the nucleic acid.

14. (4) The method according to claim 1 or 2, further comprising the step of repeating steps (1) to (3) once or more times, using the nucleic acid amplified in step (3) as an n-th order nucleic acid library (where n is an integer of 2 or more).

15. A method for producing a peptide, comprising the step of screening peptides that can bind to the target molecule by the method described in claim 1 or 2.

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