Method for screening molecules of interest capable of forming complexes with plurality of types of target molecules, and method for producing molecules of interest including said screening method

Abstract

The present invention relates to, e.g., a method for screening molecules of interest that form complexes with a plurality of types of target molecules, the method comprising: (1) a step for preparing a linked body in which a first target molecule and a second target molecule among the plurality of types of target molecules are linked via a linking part; (2) a step (contact step) for bringing a candidate molecule or a candidate molecule-nucleic acid conjugate into contact with the linked body to form a complex; and (3) a step (cleavage step) for cleaving the linking part after step (2).

Description

A method for screening target molecules that can form complexes with multiple types of target molecules, and a method for producing target molecules including the screening method.

[0001] The present invention relates to a method for screening target molecules that can form complexes with multiple types of target molecules, and to a method for producing target molecules including the screening method.

[0002] In recent years, the search for drug targets has progressed, and there is a growing demand not only for molecules that can bind to a single target molecule, as was done in the past, but also for molecules that can form complexes with multiple types of target molecules.

[0003] As a method for obtaining such molecules, for example, Patent Document 1 discloses a method for screening candidate molecules that can form a complex with a first target molecule and a second target molecule, comprising the following steps: (1) mixing a library comprising the first target molecule linked to a first portion, the second target molecule linked to a second portion, and a plurality of test molecules, wherein the first portion and the second portion constitute part or all of a protein; and (2) recovering a complex comprising the first target molecule linked to the first portion, the second target molecule linked to the second portion, and the candidate molecules after step (1), using an affinity method mediated by a third molecule capable of detecting that the first portion and the second portion are in proximity or associated.

[0004] Furthermore, Non-Patent Documents 1 and 2 disclose a method for obtaining PROTAC molecules using DNA-encoded library (DEL) technology.

[0005] International Publication No. 2022 / 138892

[0006] ACS Chem. Biol. , 2023, 18(1), pp. 25-33 Nat. Chem. Biol. , 2024, 20(2), pp. 170-179

[0007] In a screening method for target molecules that can form complexes with multiple types of target molecules (hereinafter also referred to as "multispecific binders"), there has been a problem that when recovering the target molecules, molecules that bind to only one type of target molecule (non-target molecules; hereinafter also referred to as "monospecific binders") may also be recovered. In particular, because the ease with which binding molecules can be obtained differs depending on the target molecule, when attempting to obtain target molecules that can form complexes with multiple types of target molecules, there has been a problem that many non-target molecules that bind only to specific target molecules for which binding molecules are easily obtained may be recovered. Under conditions in which a large amount of monospecific binders are recovered, a method to reduce the recovery rate of non-target molecules is desired in order to more efficiently select target molecules.

[0008] The inventors have found that by linking multiple types of target molecules via connecting parts, then bringing them into contact with candidate molecules to form a complex, and finally cleaving the connecting parts before recovering the complex, the recovery rate of non-target molecules is reduced, resulting in more efficient acquisition of the target molecule.

[0009] Neither Patent Document 1 nor Non-Patent Documents 1 and 2 describe a method for screening target molecules that form complexes with multiple types of target molecules, in which multiple target molecules are pre-connected with connecting parts, brought into contact with candidate molecules to form complexes, and then the connecting parts are cut before the complexes are recovered, nor do they describe reducing the recovery rate of non-target molecules as a result.

[0010] This invention has been made in view of the above circumstances, and aims to provide a screening method that can more efficiently obtain target molecules that can form complexes with multiple types of target molecules. This invention also aims to provide an efficient method for producing target molecules, including the screening method.

[0011] The present invention includes, for example, the following: [1] A method for screening target molecules that form complexes with multiple types of target molecules, comprising: (1) a step of preparing a conjugate by linking a first target molecule and a second target molecule from among the multiple types of target molecules via a linking portion (linking step); (2) a step of contacting a candidate molecule or a candidate molecule-nucleic acid conjugate with the conjugate to form the complex (contact step); and (3) a step of cutting the linking portion after step (2) (cutting step). [2] A method for reducing the recovery rate of non-target molecules that bind only to a second target molecule in order to screen target molecules that form complexes with multiple types of target molecules, comprising: (1) a step of preparing a conjugate by linking a first target molecule and a second target molecule from among the multiple types of target molecules via a linking portion (linking step); (2) a step of contacting a candidate molecule or a candidate molecule-nucleic acid conjugate with the conjugate to form the complex (contact step); and (3) a step of cutting the linking portion after step (2) (cutting step). [3] The method according to [1] or [2], wherein in step (2), the multiple types of target molecules are in close proximity to each other. [4] The method according to any one of [1] to [3], wherein in step (2), the first target molecule and the second target molecule are in close proximity to each other. [5] The method according to any one of [1] to [4], further comprising (4) a step of recovering the complex after step (3) (recovery step). [6] The method according to [5], wherein step (4) is recovery via the first target molecule. [7] The method according to [5], wherein step (4) is recovery by a method utilizing affinity via the first target molecule. [8] The method according to [7], wherein the method utilizing affinity is a pull-down method. [9] The method according to [7] or [8], wherein the method utilizing affinity is a method utilizing affinity between biotin and biotin-binding protein, or affinity between antigen and antibody.

[10] The method according to any one of [7] to [9], wherein the affinity-based method is a method that utilizes the affinity between biotin and a biotin-binding protein.

[11] The method according to [9] or

[10] , wherein the first target molecule is biotinylated.

[12] The method according to any one of [9] to

[11] , wherein the biotin-binding protein is one or more selected from the group consisting of avidin, neutraavidin, and streptavidin.

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

[12] , wherein in the conjugate, the first target molecule and the second target molecule are directly linked via the linker Y which is the linking portion.

[14] The method according to

[13] , wherein the conjugate is immobilized on a solid support before contact with the candidate molecule or candidate molecule-nucleic acid conjugate in step (2).

[15] The method according to

[13] or

[14] , wherein in step (2), the first target molecule is immobilized on a solid support.

[16] The method according to any one of

[13] to

[15] , wherein in step (2), only the first target molecule among the target molecules is immobilized on a solid support.

[17] The method according to

[13] , wherein the conjugate is immobilized on a solid support after step (2) and before step (3).

[18] The method according to

[13] , wherein the conjugate is immobilized on a solid support after step (3) and before step (4).

[19] The method according to

[18] , wherein the cleavage in step (3) is performed before the first target molecule is immobilized on the solid support.

[20] The method according to

[13] , wherein the conjugate is immobilized on a solid support before the recovery of the composite in step (4).

[21] The method according to

[13] , wherein the first target molecule is immobilized on the solid support in step (4).

[22] The method according to

[13] , wherein only the first target molecule is immobilized on the solid support in step (4).

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

[12] , wherein in the linked body, the first target molecule and the second target molecule are linked via the linking portion, which is linker X - solid support - linker Z, the linker X is a linker that links the first target molecule and the solid support, and the linker Z is a linker that links the second target molecule and the solid support.

[24] The method according to any one of

[14] to

[23] , wherein the solid support is a bead, a plate, or a chip.

[25] The method according to any one of

[14] to

[24] , wherein the solid support has a biotin-binding protein on its surface.

[26] The method according to any one of

[14] to

[25] , wherein the solid support has one or more selected from the group consisting of avidin, neutraavidin, and streptavidin on its surface.

[27] Surface 1 m of the solid support. ２ per, 1 x 10 １３The method according to any one of

[14] to

[26] , wherein the target molecule is immobilized.

[28] The method according to any one of

[14] to

[27] , wherein the solid support and the first target molecule are linked by a linker X.

[29] The method according to

[28] , wherein the linker X includes the binding of biotin and a biotin-binding protein.

[30] The method according to

[29] , wherein the biotin-binding protein is one or more selected from the group consisting of avidin, neutraavidin and streptavidin.

[31] The method according to any one of

[28] to

[30] , wherein the linker X is a linker that can be cleaved using an enzyme, a linker that can be cleaved using light, or a linker that can be cleaved by competitive elution.

[32] The method according to

[31] , wherein the linker X is a linker that can be cleaved using an enzyme or a linker that can be cleaved using light.

[33] The method according to

[31] , wherein the linker X is a linker that can be cleaved using an enzyme.

[34] The method according to

[31] , wherein the linker X is a linker that can be cleaved using light.

[35] The method according to

[31] , wherein the linker X includes binding of biotin to a biotin-binding protein, binding of glutathione-S-transferase (GST) to glutathione, binding of maltose-binding protein (MBP) to amylose, binding of streptavidin or streptactin to a Strep(II)-tag, or binding of a Flag tag to an antibody that recognizes it.

[36] The method according to

[35] , wherein the biotin-binding protein is one or more selected from the group consisting of avidin, neutraavidin, and streptavidin.

[37] The method according to

[28] , wherein the linker X is a linker that can be cleaved using the enzyme and includes a substrate that is specifically recognized and cleaved by the enzyme.

[38] The method according to

[37] , wherein the combination of the enzyme and the substrate is a combination of a protease and a peptide containing an amino acid sequence that is specifically recognized and cleaved by the protease, or a combination of a DNA-degrading enzyme and DNA containing a base sequence that is specifically recognized and cleaved by the DNA-degrading enzyme.

[39] The method according to

[38] , wherein the protease is a TEV protease or a 3C protease.

[40] The method according to

[38] , wherein the DNA-degrading enzyme is a restriction enzyme.

[41] The method according to

[28] , wherein the linker X is a linker that can be cleaved using light and includes a structure that causes photochemical degradation.

[42] The method according to

[41] , wherein the structure that causes photochemical degradation includes a 6-nitroveratriloxycarbonyl (NVOC) structure, a coumarin structure, or a compound (peptide, etc.) that causes photochemical degradation.

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

[42] , wherein the cleavage in step (3) is performed without cleaving the bond between the first target molecule immobilized on the solid support and the solid support.

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

[43] , wherein the cleavage in step (3) is performed by one or more methods selected from the group consisting of washing, competitive elution, enzyme use, and light use.

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

[44] , wherein the cleavage in step (3) is carried out under conditions that the recovery rate of the candidate molecule-nucleic acid conjugates forming the complex is at least twice the recovery rate of the candidate molecule-nucleic acid conjugates that bind only to the second target molecule.

[46] The method according to any one of

[28] to

[45] , wherein linker Y is a different type of linker than linker X.

[47] The method according to any one of

[23] to

[46] , wherein linker Z is a different type of linker than linker X.

[48] The method according to any one of

[28] to

[47] , wherein linker X is a linker that is not cleaved under conditions in which linker Y and / or linker Z are cleaved.

[49] The method according to any one of

[13] to

[48] , wherein linker Y and linker Z are cleaved in step (3).

[50] The method according to any one of

[13] to

[49] , wherein linker Y and linker Z are linkers that can be cleaved under conditions in which the complex is not dissociated.

[51] The method according to any one of

[13] to

[50] , wherein the linker Y and / or the linker Z is a linker that can be cut by washing, a linker that can be cut by competitive elution, a linker that can be cut using an enzyme, a linker that can be cut using light, or a linker that can be cut by pH.

[52] The method according to any one of

[13] to

[50] , wherein the linker Y and / or the linker Z is a linker that can be cleaved by competitive elution, a linker that can be cleaved using an enzyme, or a linker that can be cleaved using light.

[53] The method according to any one of

[13] to

[50] , wherein the linker Y and / or the linker Z is a linker that can be cleaved by competitive elution or a linker that can be cleaved using an enzyme.

[54] The method according to any one of

[13] to

[50] , wherein the linker Y and / or the linker Z that is the link between the first target molecule and the second target molecule in step (1) includes a linkage by non-covalent bonding.

[56] The method according to

[55] , wherein the cleavage in step (3) is the cleavage of the non-covalent bond.

[57] The method according to

[55] or

[56] , wherein the non-covalent linkage includes the linkage of any one selected from the group consisting of His (histidine) consecutive His tags, HN tags consisting of His (histidine) and Asn (asparagine) consecutively, and HAT (Histidine Affinity Tag), and any one selected from the group consisting of Ni-NTA, Ni-IDA, Co-NTA, and Co-IDA.

[58] The method according to

[55] or

[56] , wherein the non-covalent linkage includes binding of biotin to a biotin-binding protein, binding of glutathione-S-transferase (GST) to glutathione, binding of maltose-binding protein (MBP) to amylose, binding of streptavidin or streptactin to a Strep(II)-tag, or binding of a Flag tag to an antibody that recognizes it.

[59] The method according to

[55] or

[56] , wherein the non-covalent linkage includes binding of a His tag to a Ni-NTA.

[60] The method according to any one of

[13] to

[59] , wherein the linker Y and / or the linker Z as a linking portion includes any of the metals nickel, cobalt, copper, and iron.

[61] The method according to any one of

[13] to

[60] , wherein the linker Y and / or the linker Z, as a connecting portion, comprises a structure in which any of nickel, cobalt, copper, and iron is immobilized using nitrilotriacetic acid (NTA) or iminodiacetic acid (IDA).

[62] The method according to any one of

[13] to

[61] , wherein the first target molecule or the second target molecule or the solid support is bonded to any of nickel, cobalt, copper, and iron using nitrilotriacetic acid (NTA) or iminodiacetic acid (IDA).

[63] The method according to any one of

[13] to

[62] , wherein the linker Y and / or the linker Z comprises a His tag consisting of His (histidine) in a continuous sequence, an HN tag consisting of His (histidine) and Asn (asparagine) in a continuous sequence, or a HAT (Histidine Affinity Tag).

[64] The method according to any one of

[13] to

[63] , wherein the first target molecule or the second target molecule or the solid support is bound to a His tag consisting of His (histidine) in a sequence, an HN tag consisting of His (histidine) and Asn (asparagine) in a sequence, or a HAT (Histidine Affinity Tag).

[65] The method according to any one of

[13] to

[64] , wherein the linker Y and / or the linker Z includes a linkage between any one selected from the group consisting of His (histidine) consecutive His tag, HN tag consisting of His (histidine) and Asn (asparagine) consecutively, and HAT (Histidine Affinity Tag), and any one selected from the group consisting of Ni-NTA, Ni-IDA, Co-NTA, and Co-IDA.

[66] The method according to any one of

[13] to

[65] , wherein the linker Y and / or the linker Z includes a His tag and a Ni-NTA.

[67] The method according to any one of

[13] to

[66] , wherein the linker Y and / or the linker Z includes a non-covalent linkage between a His tag and a Ni-NTA.

[68] The method according to

[66] or

[67] , wherein the Ni-NTA is one or more selected from the group consisting of mono-Ni-NTA, di-Ni-NTA, tetrakis-Ni-NTA and Tris-Ni-NTA.

[69] The method according to any one of

[13] to

[64] , wherein the linker Y and / or the linker Z comprises a structure selected from GST-glutathione, MBP-amylose, streptavidin or streptactin and Strep(II)-tag, and a Flag tag and an antibody that recognizes it.

[70] The method according to any one of

[13] to

[69] , wherein the competitive elution comprises competitive elution of binding of His tag and Ni-NTA, competitive elution of antigen-antibody in affinity chromatography, competitive elution of sugar in lectin chromatography, competitive elution in a biotin-avidin system, and competitive elution in a GST (glutathione S-transferase) tag system.

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

[70] , wherein the cleavage in step (3) is cleavage by competitive elution.

[72] The method according to

[71] , wherein imidazole, histidine, polyhistidine, a solution with a pH of 6 or less, or a chelating agent is used for cleavage in step (3).

[73] The method according to

[71] , wherein imidazole is used for cleavage in step (3).

[74] The method according to

[73] , wherein the concentration of imidazole is greater than 0 mM and 25 mM or less.

[75] The method according to

[73] , wherein the concentration of imidazole is 5 mM or more and 25 mM or less.

[76] The method according to

[73] , wherein the concentration of imidazole is 15 mM or more and 25 mM or less.

[77] The method according to

[73] , wherein the concentration of imidazole is 20 mM.

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

[70] , wherein the cleavage in step (3) is pH-based cleavage.

[79] The method according to

[78] , wherein a solution with a pH of 4 or less is used for cleavage in step (3).

[80] The method according to any one of

[51] to

[79] , wherein the linker that can be cleaved using the enzyme comprises a substrate that is specifically recognized and cleaved by the enzyme.

[81] The method according to

[80] , wherein the combination of the enzyme and the substrate is a combination of a protease and a peptide comprising an amino acid sequence that is specifically recognized and cleaved by the protease, or a DNA-degrading enzyme and DNA comprising a base sequence that is specifically recognized and cleaved by the DNA-degrading enzyme.

[82] The method according to

[81] , wherein the protease is a TEV protease or a 3C protease.

[83] The method according to

[81] , wherein the DNA-degrading enzyme is a restriction enzyme.

[84] The method according to any one of

[51] to

[79] , wherein the linker that can be cleaved using light has a structure that undergoes photochemical degradation, and the structure comprises a 6-nitroveratriloxycarbonyl (NVOC) structure, a coumarin structure, or a compound (such as a peptide) that undergoes photochemical degradation.

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

[84] , wherein the target molecule (i) binds to a binding site that is formed only when multiple types of target molecules are in close proximity to each other, (ii) has two or more different sites that bind to each of the multiple types of target molecules, or (iii) binds to one or more types of target molecules and promotes binding to other types of target molecules.

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

[85] , wherein in step (2), the candidate molecule or the candidate molecule-nucleic acid conjugate is brought into contact with the conjugate and further into contact with target molecules other than the first target molecule and the second target molecule contained in the conjugate.

[87] The method according to

[86] , wherein there is one type of target molecule other than the first target molecule and the second target molecule.

[88] The method according to

[86] or

[87] , wherein the target molecules other than the first target molecule and the second target molecule are not immobilized on a solid support.

[89] The method according to any one of

[86] to

[88] , wherein the target molecules other than the first target molecule and the second target molecule are individual first target molecules not immobilized on a solid support.

[90] The method according to

[89] , wherein the individual first target molecule is a non-biotinized molecule.

[91] The method according to any one of

[86] to

[90] , wherein in step (2), the ratio of the concentration of target molecules other than the first target molecule and the second target molecule to the concentration of the conjugate is 1 or more.

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

[91] , wherein, in step (1), the first target molecule and the second target molecule are not linked at the conjugate, and more monospecific binder of the second target molecule is recovered than monospecific binder of the first target molecule.

[93] (5) The method according to any one of [1] to

[92] , further comprising a step of eluting the nucleic acid in the candidate molecule-nucleic acid conjugate recovered by the recovery step (elution step).

[94] The method according to

[93] , wherein the elution in the elution step is carried out by one or more methods selected from the group consisting of methods using enzymes, methods using light, methods using heat, and methods using competitive elution, which were not used in the cleavage step.

[95] The method according to

[93] , wherein the elution in the elution step is carried out by a method using enzymes.

[96] The method according to

[95] , wherein the enzyme is a TEV protease.

[97] The method according to any one of

[93] to

[96] , wherein in the elution step, the linker X between the solid support and the first target molecule is cleaved.

[98] The method according to any one of

[93] to

[97] , wherein in the elution step, the bond between the first target molecule and the candidate molecule-nucleic acid conjugate is cleaved.

[99] The method according to any one of

[93] to

[98] , wherein in the elution step, the bond between the candidate molecule and the nucleic acid is cleaved.

[100] (6) The method according to any one of [1] to

[99] , further comprising a step of amplifying the nucleic acid in the candidate molecule-nucleic acid conjugate that has formed the complex (amplification step).

[101] The method according to

[100] , wherein the amplification step is performed after the elution step.

[102] The method according to

[100] or

[101] , comprising generating a new candidate molecule-nucleic acid conjugate library from the nucleic acid amplified in the amplification step, and performing a second contact step, cleavage step, recovery step, and amplification step using the candidate molecule-nucleic acid conjugate library, wherein the first target molecule and the second target molecule are swapped and each of the second steps is performed.

[103] The method according to

[100] or

[101] , comprising generating a new candidate molecule-nucleic acid conjugate library from the nucleic acid amplified in the amplification step, and repeating the contact step, cleavage step, recovery step, and amplification step multiple times using the candidate molecule-nucleic acid conjugate library.

[104] The method according to

[100] or

[101] , wherein a new candidate molecule-nucleic acid conjugate library is generated from the nucleic acid amplified in the amplification step, and the contact step, cleavage step, recovery step, elution step and amplification step are repeated multiple times using the candidate molecule-nucleic acid conjugate library.

[105] (7) The method according to any one of [1] to

[104] , further comprising the step of identifying a target molecule from the candidate molecule-nucleic acid conjugate that has formed the complex (identification step).

[106] The method according to

[105] , wherein the identification step is performed after the elution step and / or the amplification step.

[107] The method according to

[105] or

[106] , wherein the identification step includes determining the sequence of the nucleic acid eluted in the elution step or the nucleic acid amplified in the amplification step.

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

[107] , wherein in step (2), the candidate molecule-nucleic acid conjugate is brought into contact with the conjugate to form the complex.

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

[108] , wherein the candidate molecule comprises a peptide or a protein.

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

[109] , wherein the candidate molecule is a peptide.

[111] The method according to

[110] , wherein the candidate molecule has a cyclic portion.

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

[111] , wherein the target molecule comprises a peptide, protein, sugar, lipid, or nucleic acid.

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

[112] , wherein the target molecule comprises a peptide or protein.

[114] The method according to

[113] , wherein the N-terminuses of the multiple types of target molecules are linked together via a linking portion.

[115] The method according to

[113] , wherein the C-terminuses of the multiple types of target molecules are linked together via a linking portion.

[116] The method according to

[113] , wherein the N-terminus of the first target molecule and the C-terminus of the second target molecule are linked together via the linking portion.

[117] The method according to any one of

[105] to

[116] , wherein the identification step includes comparing the recovery rate of the candidate molecule-nucleic acid conjugate (recovery rate A) when the candidate molecule-nucleic acid conjugate is in contact with the linking portion, with the recovery rate of the candidate molecule-nucleic acid conjugate (recovery rate B) when the candidate molecule-nucleic acid conjugate is in contact with only one type of target molecule selected from the multiple types of target molecules.

[118] The method according to

[117] wherein the recovery rate B includes a recovery rate B1 when the candidate molecule-nucleic acid conjugate is contacted only with a first target molecule which is one type of target molecule selected from the plurality of types of target molecules, and a recovery rate B2 when the candidate molecule-nucleic acid conjugate is contacted only with a second target molecule which is different from the first target molecule, and the identification step includes comparing recovery rate A with recovery rate B1 and comparing recovery rate A with recovery rate B2.

[119] The method according to

[117] wherein the candidate molecule is identified as the target molecule when the recovery rate A compared with recovery rate B is five times or more.

[120] The method according to

[118] wherein the candidate molecule is identified as the target molecule when the recovery rate A compared with both recovery rate B1 and recovery rate B2 is five times or more.

[121] The method according to any one of

[105] to

[120] , wherein the identification step is to compare the recovery rate of the candidate molecule-nucleic acid conjugate when the candidate molecule-nucleic acid conjugate is in contact with the conjugate (recovery rate A) with the recovery rate of the candidate molecule-nucleic acid conjugate in the absence of the target molecule (recovery rate C).

[122] The method according to

[121] , wherein the candidate molecule is identified as the target molecule when recovery rate A is five times or more compared with recovery rate C.

[123] The method according to any one of

[105] to

[122] , wherein the identification step is to compare (1) the recovery rate of the candidate molecule-nucleic acid conjugate when the candidate molecule-nucleic acid conjugate is in contact with the conjugate (recovery rate A) with (2) the recovery rate of the candidate molecule-nucleic acid conjugate when the candidate molecule-nucleic acid conjugate is in contact with a first target molecule that is immobilized on a solid support and a second target molecule that is different from the first target molecule and is not immobilized on a solid support (recovery rate D).

[124] The method according to

[123] , wherein the recovery rate D includes the recovery rate D1 of the candidate molecule-nucleic acid conjugate when the candidate molecule-nucleic acid conjugate is brought into contact with a first target molecule, which is selected from the plurality of target molecules and is immobilized on a solid support, and a second target molecule different from the first target molecule and not immobilized on a solid support, and the recovery rate D2 of the candidate molecule-nucleic acid conjugate when the candidate molecule-nucleic acid conjugate is brought into contact with the second target molecule, which is immobilized on a solid support, and the first target molecule and not immobilized on a solid support, and the identification step includes comparing the recovery rate A with the recovery rate D1 and comparing the recovery rate A with the recovery rate D2.

[125] The method according to

[123] , wherein the candidate molecule is identified as the target molecule when the recovery rate A is 5 times or more compared with the recovery rate D.

[126] The method according to

[124] , wherein the candidate molecule is identified as the target molecule when the recovery rate A is 5 times or more compared with both the recovery rate D1 and the recovery rate D2.

[127] The method according to any one of

[105] to

[126] , wherein the identification step includes comparing the recovery rate B, the recovery rate C, or the recovery rate D with respect to the recovery rate A.

[128] The method according to any one of

[105] to

[127] , wherein the identification step includes comparing the recovery rate B, the recovery rate C, and the recovery rate D with respect to the recovery rate A.

[129] The method according to any one of

[105] to

[128] , wherein the candidate molecule is identified as the target molecule when the recovery rate A is 5 times or more when compared with any of the recovery rates B, C, or D.

[130] The method according to any one of

[105] to

[129] , wherein the identification step includes comparing the recovery rate D with respect to the recovery rate B.

[131] The method according to any one of

[105] to

[130] , wherein the candidate molecule is identified as the target molecule when the recovery rate D is 5 times or more when compared with the recovery rate B.

[132] The method according to any of [1] to

[131] , performed in vitro.

[133] The method according to any of [1] to

[132] , without using fluorescence by GFP or luminescence by luciferase.

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

[133] , wherein the candidate molecule is a library.

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

[134] , wherein the candidate molecule-nucleic acid conjugate is a library.

[136] The method according to

[134] or

[135] , wherein the library is a display library.

[137] The method according to any one of

[134] to

[136] , wherein the library is a mRNA display library.

[138] The method according to

[134] or

[135] , wherein the library is a DNA-encoded library.

[139] The candidate molecule contained in the library is 1×10. ３ The method according to any one of

[134] to

[138] , having a diversity of more than.

[140] The candidate molecule contained in the library is 1×10 ５ The method according to any one of

[134] to

[138] , having a diversity of more than.

[141] The candidate molecule contained in the library is 1×10 ７ The method according to any one of

[134] to

[138] , having a diversity of more than.

[142] The candidate molecule contained in the library is 1×10 ９ The method according to any one of

[134] to

[138] , having a diversity of more than.

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

[142] , wherein the nucleic acid is a nucleic acid encoding a candidate molecule that forms the nucleic acid and the candidate molecule-nucleic acid conjugate.

[144] The method according to

[143] , wherein the nucleic acid is DNA.

[145] The method according to

[143] , wherein the nucleic acid is mRNA.

[146] Among the candidate molecules contained in the library, in 1×10 ３ The method according to any one of

[134] to

[145] , wherein in the above candidate molecules, the molecular weight of the candidate molecule is 300 g / mol or more and 5,000 g / mol or less.

[147] Among the candidate molecules contained in the library, in 1×10 ３ The method according to any one of

[134] to

[146] , wherein in the above candidate molecules, the number of amino acid residues contained in the candidate molecule is 5 or more and 30 or less.

[148] Among the candidate molecules contained in the library, in 1×10 ３The method according to any one of

[134] to

[147] , wherein the candidate molecule contains one or more unnatural amino acid residues.

[149] Among the candidate molecules included in the library, 1 × 10 ３ The method according to any one of

[134] to

[148] , wherein the candidate molecule contains one or more N-substituted amino acid residues.

[150] The substituent on the nitrogen atom of the N-substituted amino acid residue is C １ -C ６ The method according to

[149] , wherein the alkyl group is selected.

[151] Among the candidate molecules included in the library, 1 × 10 ３ The method according to any one of

[134] to

[150] , wherein the number of amino acid residues constituting the cyclic portion in the above candidate molecules is 5 or more and 15 or less.

[152] Among the candidate molecules included in the library, 1 × 10 ３ The method according to any one of

[134] to

[151] , wherein the ClogP of the candidate molecule is 4 or more and 25 or less.

[153] Among the candidate molecules included in the library, 1 × 10 ３The method according to any one of

[134] to

[152] , wherein the candidate molecule has a ClogP / amino acid residue count of 1.0 or more.

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

[153] , wherein there are two or more types of multiple target molecules and no more than five types.

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

[153] , wherein there are two types of multiple target molecules.

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

[155] , wherein the dissociation constant between at least two target molecules selected from the multiple target molecules exceeds 10 μM.

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

[155] , wherein the dissociation constants between all of the multiple target molecules exceed 10 μM.

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

[157] , wherein the target molecule is a protein.

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

[158] , wherein the target molecule is a molecule present in the human body.

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

[159] , wherein the target molecule is a molecule that does not contain a split protein.

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

[160] , wherein the target molecule is a molecule that does not contain either GFP or its fragments.

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

[161] , wherein the target molecule is a molecule that does not contain either luciferase or its fragments.

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

[162] , wherein the target molecule is a molecule that does not contain either a SNAP tag or its fragments.

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

[163] , wherein the target molecule is a molecule that does not contain either ubiquitin or its fragments.

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

[164] , wherein the target molecule comprises one or more selected from the group consisting of FK506-binding protein (FKBP), FKBP-rapamycin-binding protein (FRB), cyclophyllin A (CyA), and calcineurin (Cn).

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

[165] , comprising FKBP as the target molecule.

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

[166] , comprising CyA as the target molecule.

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

[167] , comprising FKBP and FRB as the target molecules.

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

[168] , wherein the target molecules are CyA and Cn.

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

[169] , wherein the target molecules are FKBP and FRB.

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

[170] , wherein the target molecules are CyA and Cn.

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

[171] , wherein the target molecules are FKBP and Cn.

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

[172] , wherein the candidate molecule and the nucleic acid form a conjugate via a binding site.

[174] The method according to

[173] , wherein the binding site is puromycin.

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

[174] , wherein the dissociation constant between at least two target molecules selected from the plurality of target molecules in the presence of 10 μM of the target molecule is 10 μM or less.

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

[174] , wherein the dissociation constants of the multiple types of target molecules in the presence of 10 μM of the target molecule are all 10 μM or less.

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

[176] , wherein the target molecule is a candidate molecule when the recovery rate A is 5 times or more compared to the recovery rate B.

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

[177] , wherein the target molecule is a candidate molecule when the recovery rate A is 5 times or more compared to the recovery rate C.

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

[178] , wherein the target molecule is a candidate molecule when the recovery rate A is 5 times or more compared to the recovery rate D.

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

[179] , wherein the target molecule is a molecule that can form a complex with the multiple types of target molecules.

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

[180] , wherein the value of the dissociation constant between the multiple types of target molecules in the presence of 10 μM of the target molecule is smaller than the value of the dissociation constant between the multiple types of target molecules in the absence of the target molecule.

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

[181] , wherein the value of the dissociation constant between the multiple types of target molecules in the presence of 10 μM of the target molecule is less than one-fifth of the value of the dissociation constant between the multiple types of target molecules in the absence of the target molecule.

[183] ​​The method according to any one of [1] to

[182] , wherein the value of the dissociation constant between the multiple types of target molecules in the presence of 10 μM of the target molecule is 30 μM or less.

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

[183] , wherein the value of the dissociation constant between the multiple types of target molecules in the presence of 10 μM of the target molecule is 30 μM or less, and is less than one-fifth of the value of the dissociation constant between the multiple types of target molecules in the absence of the target molecule.

[185] A method for producing a peptide, comprising the method according to any one of [1] to

[184] .

[186] A method for producing a peptide, comprising the following steps: (i) a step of screening the target molecule by the method according to any one of [1] to

[184] ; and (ii) a step of producing a peptide based on the amino acid sequence of the target molecule selected in (i).

[187] A method for identifying or selecting a candidate molecule having desired properties, comprising the steps of carrying out the method of any one of [1] to

[184] , wherein a candidate molecule-nucleic acid conjugate minilibrary is used as the candidate molecule or the candidate molecule-nucleic acid conjugate, and at least one step selected from the contact step, the cleavage step, and the recovery step is carried out under a plurality of conditions with mutually different parameters, and the amount of each candidate molecule-nucleic acid conjugate recovered under each of the plurality of conditions is quantified by analyzing the nucleic acid sequence bound thereto, and the recovery rates under each condition are compared to identify or select a candidate molecule having desired properties.

[188] The candidate molecule-nucleic acid conjugate minilibrary is 2 or more, 1 × 10. ８The method according to

[187] , having the following diversity:

[189] The method according to

[187] or

[188] , wherein the candidate molecule-nucleic acid conjugate minilibrary comprises one or more candidate molecules enriched by carrying out the method according to any one of [1] to

[184] at least once.

[190] The method according to

[187] or

[188] , wherein the candidate molecule-nucleic acid conjugate minilibrary comprises at least one candidate molecule selected from the group consisting of a specific parent sequence and a child sequence in which one or more amino acid residues of the parent sequence are substituted with other amino acid residues.

[191] The method according to any one of

[187] to

[190] , wherein the plurality of different conditions are conditions in which the concentration of the competing substance used in the cleavage step is different.

[192] The method according to

[191] , wherein the competing substance is imidazole.

[193] The method according to any one of

[187] to

[190] , wherein the plurality of different conditions are conditions in which the parameters in the contact step are different.

[194] The method according to

[193] , wherein the parameter is at least one selected from the group consisting of the concentration of a first target molecule, the concentration of a second target molecule, pH, salt concentration, and temperature.

[0012] The present invention provides a screening method that can more efficiently obtain target molecules capable of forming complexes with multiple types of target molecules. The present invention also provides an efficient method for producing target molecules, including the screening method.

[0013] 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.

[0014] 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.

[0015] In this specification, the range indicated by "~" includes the values ​​at both ends of the range. For example, "A~B" means a range where A is greater than or equal to B and B is less than or equal to B.

[0016] In this specification, the term "approximately" when used in combination with a number means a range of +10% and -10% of that number.

[0017] In this invention, the meaning of the terms "and / or" includes any combination in which "and" and "or" are appropriately combined. 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.

[0018] [Screening Method] The screening method according to the present invention relates to a method for screening target molecules that form complexes with multiple types of target molecules.

[0019] The screening method includes (1) a step of preparing a conjugate by linking a first target molecule and a second target molecule from among the plurality of target molecules via a connecting portion (linking step), (2) a step of contacting a candidate molecule or a candidate molecule-nucleic acid conjugate with the conjugate to form a complex (contact step), and (3) a step of cutting the connecting portion after step (2) (cutting step).

[0020] The screening method may further include (4) a step of recovering the composite (recovery step), in addition to the linking step, contact step and cutting step.

[0021] Furthermore, when a candidate molecule-nucleic acid conjugate is used in step (2) (contact step), the screening method may further include (5) a step of eluting the nucleic acid in the candidate molecule-nucleic acid conjugate recovered in the recovery step (elution step), (6) a step of amplifying the nucleic acid in the candidate molecule-nucleic acid conjugate that has formed the complex (amplification step), and / or (7) a step of identifying the target molecule from the candidate molecule-nucleic acid conjugate that has formed the complex (identification step).

[0022] [Method for reducing the recovery rate of non-target molecules] The method for reducing the recovery rate of non-target molecules according to the present invention relates to a method for reducing the recovery rate of non-target molecules that bind only to a second target molecule in order to screen for target molecules that form complexes with multiple types of target molecules.

[0023] The method for reducing the recovery rate of the non-target molecules includes (1) a step of preparing a conjugate by linking a first target molecule and a second target molecule from among the multiple types of target molecules via a connecting portion (linking step), (2) a step of contacting a candidate molecule or a candidate molecule-nucleic acid conjugate with the conjugate to form a complex (contact step), and (3) a step of cutting the connecting portion after step (2) (cutting step).

[0024] The method for reducing the recovery rate of the non-target molecules may further include (4) a step of recovering the composite (recovery step), in addition to the linking step, contact step and cleavage step.

[0025] Furthermore, when a candidate molecule-nucleic acid conjugate is used in step (2) (contact step), the screening method may further include (5) a step of eluting the nucleic acid in the candidate molecule-nucleic acid conjugate recovered in the recovery step (elution step), (6) a step of amplifying the nucleic acid in the candidate molecule-nucleic acid conjugate that has formed the complex (amplification step), and / or (7) a step of identifying the target molecule from the candidate molecule-nucleic acid conjugate that has formed the complex (identification step).

[0026] [Step (1): Linking Step] In Step (1) (linking step), a linked body is prepared by linking a first target molecule and a second target molecule from among several types of target molecules via a linking portion. The first target molecule and the second target molecule may be the same or different, but it is preferable that they be different.

[0027] The linked structure, in which a first target molecule and a second target molecule are linked via a linking portion, is not particularly limited, but examples include (1) a linked structure in which the first target molecule and the second target molecule are directly linked via a linker (let's call it linker Y), and (2) a linked structure in which the first target molecule and a solid support are linked via a linker (let's call it linker X), and the solid support and the second target molecule are further linked via a linker (let's call it linker Z). In embodiment (1), linker Y constitutes the linking portion. In embodiment (2), the linking portion consists of "linker X - solid support - linker Z".

[0028] A conjugate in which a first target molecule and a second target molecule are linked via a connecting portion may further contain a third target molecule, different from the first and second target molecules, also linked via a connecting portion. Similarly, a fourth target molecule, different from the first, second, and third target molecules, a fifth target molecule, and so on, may further contain connecting portions. In the same way, all of the multiple target molecules may be linked via connecting portions.

[0029] If the target molecule is a peptide or protein, in the conjugate, the N-terminuses of multiple types of target molecules may be linked to each other via a linking portion, the C-terminuses of multiple types of target molecules may be linked to each other via a linking portion, and the N-terminuses of some of the multiple types of target molecules may be linked to the C-terminuses of other target molecules via a linking portion. Furthermore, the N-terminus of the first target molecule and the N-terminus of the second target molecule may be linked via the linking portion, the C-terminus of the first target molecule and the C-terminus of the second target molecule may be linked via the linking portion, and the C-terminus of the first target molecule and the N-terminus of the second target molecule may be linked via the linking portion.

[0030] The linker included in the connecting portion may be, for example, a linker that can be cleaved by washing, a linker that can be cleaved using an enzyme, a linker that can be cleaved using light, a linker that can be cleaved by competitive elution, or a linker that can be cleaved by pH. Furthermore, the linker included in the connecting portion may be a linker that can be cleaved under conditions that do not dissociate the complex.

[0031] The linker that can be cut by the washing process may also be a linker that can be cut by a mild physical action such as stirring or inverting the solution containing the composite.

[0032] A linker that can be cleaved using an enzyme may contain a substrate that is specifically recognized and cleaved by the enzyme. The linker is cleaved by acting an enzyme on a linker containing the substrate. Specific examples of enzyme-substrate combinations include, for example, a combination of a protease and a peptide containing an amino acid sequence that is specifically recognized and cleaved by the protease, and a combination of a DNA-degrading enzyme and DNA containing a base sequence that is specifically recognized and cleaved by the DNA-degrading enzyme. Examples of proteases include TEV proteases and 3C proteases. Examples of DNA-degrading enzymes include restriction enzymes.

[0033] A linker that can be cleaved using light may contain a structure that undergoes photochemical decomposition. The linker is cleaved by irradiating the linker containing such a structure with light of an appropriate wavelength. Specific examples of structures that undergo photochemical decomposition include, for example, the 6-nitroveratriloxycarbonyl (NVOC) structure, the coumarin structure, and other compounds (peptides, etc.) that undergo photochemical decomposition.

[0034] Linkers that can be cleaved by competitive elution may include those that utilize affinity-based binding.

[0035] Specific examples of affinity-based binding include the binding of biotin to biotin-binding proteins, glutathione-S-transferase (GST) to glutathione, maltose-binding protein (MBP) to amylose, streptavidin or streptactin to Strep(II)-tag, Flag tag to an antibody that recognizes it, and metal to a histidine-containing peptide.

[0036] In the binding of biotin to a biotin-binding protein, the biotin-binding protein may be one or more selected from the group consisting of, for example, avidin, neutraavidin, and streptavidin.

[0037] In the binding of a metal to a histidine-containing peptide, the metal may be one or more selected from the group consisting of, for example, nickel, cobalt, copper, and iron. The histidine-containing peptide may be, for example, a His tag consisting of His (histidine) in sequence, an HN tag consisting of His (histidine) and Asn (asparagine) in sequence, or a HAT (Histidine Affinity Tag). The binding of the metal to the histidine-containing peptide may further include a matrix for immobilizing the metal on a target molecule or solid support. Examples of the matrix include nitrilotriacetic acid (NTA) or iminodiacetic acid (IDA). A specific example of the binding of a metal to a histidine-containing peptide is, for example, the binding of Ni-NTA to a His tag (non-covalent bond). Examples of Ni-NTAs include one or more selected from the group consisting of mono-Ni-NTA, di-Ni-NTA, tetrakis-Ni-NTA, and Tris-Ni-NTA (J. Am. Chem. Soc., 2005, vol. 127, pp. 10205-10215).

[0038] By bringing a solution containing a competing substance into contact with the linker containing the bond in question, one of the bonds is replaced by the competing substance, thereby cleaving the linker. Examples of competing substances include substances that can specifically bind to one of the molecules in the affinity-based bond. The competing substance may also be the molecule itself that is included in the affinity-based bond. Specific examples of competing substances include, for example, when the affinity-based bond is between a metal and a peptide containing histidine, imidazole, histidine, polyhistidine, a solution with a pH of 6 or less, and a chelating agent (e.g., ethylenediaminetetraacetic acid (EDTA)). In the case of a bond between GST and glutathione, glutathione is an example.

[0039] Competitive elution may be at least one selected from the group consisting of His tags (consecutive Histidine units), HN tags (consecutive Histidine and Asparagine units), and HAT (Histidine Affinity Tag), and the binding of any one selected from the group consisting of Ni-NTA, Ni-IDA, Co-NTA, and Co-IDA, particularly competitive elution of His tags and Ni-NTA binding, competitive antigen-antibody elution in affinity chromatography, competitive sugar elution in lectin chromatography, competitive elution in biotin-avidin systems, and competitive elution in GST (glutathione S-transferase) tag systems.

[0040] A linker that can be cleaved by pH may include a bond that utilizes affinity. Specific examples of a linker that can be cleaved by pH are the same as those of a linker that can be cleaved using competitive elution. The linker is cleaved by contacting the linker containing the bond with a solution (e.g., a buffer solution) at a pH that causes the affinity bond to dissociate. Examples of such pH values ​​include 6 or less, 5 or less, or 4 or less, when the affinity bond is between a metal and a histidine-containing peptide.

[0041] The method of linking a first target molecule and a second target molecule with the linker described above can be carried out by methods known in the art. Specifically, for example, a linked product can be obtained by adding Ni-NTA to the first target molecule and His-tag to the second target molecule, and then mixing the two. The same applies to the method of linking the first target molecule or the second target molecule with a solid support with the linker described above.

[0042] Furthermore, in step (1), if the first target molecule and the second target molecule are not linked at the connecting portion, the process may be carried out in a system in which more monospecific binder of the second target molecule is recovered than monospecific binder of the first target molecule.

[0043] In the above embodiment (2), there are no particular restrictions on the shape of the solid support constituting the connecting portion, but it may be in the shape of, for example, beads (e.g., magnetic beads), plates, or chips. The solid support may have molecules on its surface that can be used for affinity binding. Specifically, for example, the solid support may have a biotin-binding protein on its surface, or it may have one or more selected from the group consisting of avidin, neutraavidin, and streptavidin on its surface.

[0044] In the above embodiment (2), the surface 1 m of the solid support ２ For example, around here, 1 x 10 １３ The above 5 x 10 １３ The above is 1 x 10 １４ The above, or 5 x 10 １４ The target molecules mentioned above may be immobilized. Also, for example, 1 × 10 １３ Preferably 5 x 10 １３ More than 1 x 10 １４ In summary, the most preferred is 5 x 10 １４ The target molecules described above may be immobilized. This increases the frequency with which multiple types of target molecules come into close proximity to each other. Preferably, the first target molecule and the second target molecule are immobilized on the solid support in roughly equal mole amounts, preferably equimolar amounts.

[0045] In the above embodiment (2), the surface 1 m of the solid support ２ For example, 1 x 10 １３ The above 5 x 10 １３ The above is 1 x 10 １４ The above, or 5 x 10 １４ The process may include a step of bringing the target molecules into contact with the solid support. For example, 1 × 10 １３ Preferably 5 x 10 １３ More than 1 x 10 １４ In summary, the most preferred is 5 x 10 １４ The process may include a step of bringing the target molecules into contact with the solid support. This increases the frequency with which multiple types of target molecules come into close proximity to each other. Preferably, the target molecules brought into contact with the solid support contain the first target molecule and the second target molecule in roughly equal mole amounts, preferably equimolar amounts.

[0046] In embodiment (1) above, the conjugate in which the first target molecule and the second target molecule are directly linked via a linker Y may be immobilized on a solid support. The specific embodiment of the solid support is as described above. Immobilization to the solid support may be, for example, by immobilizing the first target molecule contained in the conjugate and the solid support via a linker (referred to as linker X, as in embodiment (2)).

[0047] In the above embodiment (1), the timing for immobilizing the first target molecule on the solid support may be, for example, before contact with the candidate molecule or candidate molecule-nucleic acid conjugate in step (2) (contact step), before recovery of the complex in step (4) (recovery step), after step (2) (contact step) and before step (3) (cleavage step), or after step (3) (cleavage step) and before step (4) (recovery step).

[0048] In the above embodiment (2), the first target molecule and the second target molecule are linked to the solid support, which is the linking part, in step (1) (linking step), and are therefore immobilized on the solid support before contact with the candidate molecule or candidate molecule-nucleic acid conjugate in step (2) (contact step).

[0049] Furthermore, linkers Y and Z may be linkers capable of cleaving the complex under conditions that do not cause it to dissociate. The conditions that do not cause the complex to dissociate are not particularly limited, as long as the bond between the candidate molecule and the target molecule is not cleaved after the candidate molecule or candidate molecule-nucleic acid conjugate is brought into contact with the conjugate.

[0050] (Target Molecules) In one embodiment, the multiple types of target molecules may be any number or type, and are not limited to any particular type. For example, they may be two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more, and may be ten or fewer, nine or fewer, eight or fewer, seven or fewer, six or fewer, five or fewer, four or fewer, three or fewer, or two or fewer. The multiple types of target molecules may be, for example, two or more and five or fewer, preferably two or more and four or fewer, more preferably two or more and three or fewer, and most preferably two. Having the types of target molecules within the above range makes it easier to obtain the target molecule.

[0051] In one embodiment, the dissociation constants of at least two (two types) of target molecules selected from a plurality of target molecules may exceed 10 μM, and preferably, the dissociation constants of all of the plurality of target molecules exceed 10 μM. The screening method according to the present invention includes a step of contacting a candidate molecule or a candidate molecule-nucleic acid conjugate with a conjugate formed by linking a first target molecule and a second target molecule via a linking portion. Thus, it is possible to select target molecules that can form complexes even with a plurality of target molecules that have large dissociation constants.

[0052] The dissociation constants of at least two (two types) target molecules selected from the aforementioned multiple types of target molecules may be, for example, greater than 10 μM, 15 μM or more, 20 μM or more, 25 μM or more, 30 μM or more, 35 μM or more, 40 μM or more, 45 μM or more, or 50 μM or more. The dissociation constants of at least two (two types) target molecules selected from the aforementioned multiple types of target molecules may be, for example, greater than 10 μM, preferably 20 μM or more, more preferably 30 μM or more, and most preferably 50 μM or more. According to the present invention, target molecules that can form complexes with multiple types of target molecules having dissociation constants within the above range can be obtained more efficiently.

[0053] The target molecules are not particularly limited, but examples include peptides, proteins, sugars (oligosaccharides, polysaccharides, etc.), lipids, and nucleic acids (DNA, RNA, etc.). Peptides or proteins are preferred as target molecules.

[0054] Furthermore, the target molecule is preferably a molecule present in the human body. Furthermore, the target molecule is preferably a molecule that does not contain split proteins. Furthermore, the target molecule is preferably a molecule that does not contain GFP or its fragments. Furthermore, the target molecule is preferably a molecule that does not contain luciferase or its fragments. Furthermore, the target molecule is preferably a molecule that does not contain SNAP tags or their fragments. Furthermore, the target molecule is preferably a molecule that does not contain ubiquitin or its fragments.

[0055] The target molecule may include one or more selected from the group consisting of FK506-binding protein (FKB), FKB-rapamycin-binding protein (FRB), cyclophyllin, and calcineurin (Cn).

[0056] The target molecule may contain FKBP. Preferred FKBPs include FKBP12, FKBP12.6, FKBP13, FKBP19, FKBP22, FKBP23, FKBP25, FKBP36, FKBP38, FKBP51, FKBP52, FKBP60, and FKBP65.

[0057] The target molecule may contain cyclophylline. Preferred cyclophyllines include cyclophylline A (CyA), cyclophylline B, cyclophylline C, cyclophylline D, cyclophylline E, cyclophylline F, cyclophylline G, cyclophylline H, cyclophylline I, cyclophylline J, cyclophylline 40, NKTR, PPWD1, PPIL1, PPIL2, PPIL4, PPIL5, RANBP2, and SDCCAG-10.

[0058] The target molecule may, for example, include FKBP and FRB, or it may be FKBP and FRB.

[0059] The target molecule may, for example, contain cyclophylline and Cn, or it may contain cyclophylline and Cn.

[0060] The target molecule may, for example, contain FKPB and Cn, or it may contain FKBP and Cn.

[0061] The cyclophylline may be, for example, CyA.

[0062] [Step (2): Contact Step] In Step (2) (Contact Step), the candidate molecule or the candidate molecule-nucleic acid conjugate is brought into contact with the conjugate.

[0063] (Candidate Molecules and Candidate Molecules-Nucleic Acid Conjugates) Candidate molecules are not particularly limited, and any compound (low molecular weight compounds, medium molecular weight compounds, high molecular weight compounds) can be used. Preferably, candidate molecules are medium molecular weight compounds such as peptides, high molecular weight compounds such as proteins, more preferably medium molecular weight compounds such as peptides, and even more preferably cyclic peptides.

[0064] A candidate molecule-nucleic acid conjugate is a conjugate in which a candidate molecule and a nucleic acid as a tag are associated (candidate molecule-nucleic acid conjugate). In a candidate molecule-nucleic acid conjugate, it is sufficient that there is a one-to-one correspondence between the type of candidate molecule and the type of nucleic acid. The conjugate may be formed by the candidate molecule and the nucleic acid binding directly or via a binding site, or the conjugate may be formed without the candidate molecule and the nucleic acid binding directly or via a binding site. In one non-limiting embodiment, it is preferable that the candidate molecule and the nucleic acid form the conjugate via a binding site.

[0065] The binding site is not limited as long as it can link the candidate molecule with the nucleic acid, but may include, for example, the antibiotic puromycin, an analog of aminoacyl-tRNA. The binding site may further include spacers well known to those skilled in the art.

[0066] The nucleic acid may be a nucleic acid that encodes a candidate molecule that forms a candidate molecule-nucleic acid conjugate with the nucleic acid. This allows for easy reproduction of the candidate molecule-nucleic acid conjugate by amplifying the nucleic acid from the recovered candidate molecule-nucleic acid conjugate and then synthesizing (translating) a peptide from the amplified nucleic acid.

[0067] The nucleic acid may be DNA or mRNA. Furthermore, the nucleic acid may have a tag sequence for identifying candidate molecules contained in the conjugate, as well as a sequence for amplification (such as a primer-binding sequence). The sequence for amplification may be a sequence common to all nucleic acids.

[0068] 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. The number of amino acid residues in a peptide is not particularly limited, but may be, for example, five or more, seven or more, eight or more, or nine or more. The number of amino acid residues in a peptide may also be, for example, 30 or less, 25 or less, 15 or less, or 13 or less. The number of amino acid residues in a peptide may be, for example, 5 to 30, preferably 7 to 25, more preferably 8 to 15, most preferably 9 to 13. Having the number of amino acid residues in the peptide within the above range increases the efficiency of obtaining target molecules with high membrane permeability and metabolic stability. Peptides may have a branched structure.

[0069] 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.

[0070] 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).

[0071] 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.

[0072] 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).

[0073] Examples of halogen-derived substituents include fluoro(-F), chloro(-Cl), bromo(-Br), and iod(-I).

[0074] Substituents derived from the oxygen atom include hydroxy (-OH), oxy (-OR), carbonyl (-C(=O)-R), and carboxyl (-CO ２H), 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.

[0075] Examples of oxy (-OR) compounds include alkoxy, cycloalkoxy, alkenyloxy, alkynyloxy, aryloxy, heteroaryloxy, and aralkyloxy compounds.

[0076] Examples of carbonyl (-C(=O)-R) include formyl (-C(=O)-H), alkylcarbonyl, cycloalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, arylcarbonyl, heteroarylcarbonyl, and aralkylcarbonyl.

[0077] Examples of oxycarbonyl (-C(=O)-OR) include alkyloxycarbonyl, cycloalkyloxycarbonyl, alkenyloxycarbonyl, alkynyloxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, and aralkyloxycarbonyl.

[0078] Examples of carbonyloxy (-O-C(=O)-R) include alkylcarbonyloxy, cycloalkylcarbonyloxy, alkenylcarbonyloxy, alkynylcarbonyloxy, arylcarbonyloxy, heteroarylcarbonyloxy, and aralkylcarbonyloxy.

[0079] Examples of thiocarbonyl (-C(=O)-SR) include alkylthiocarbonyl, cycloalkylthiocarbonyl, alkenylthiocarbonyl, alkynylthiocarbonyl, arylthiocarbonyl, heteroarylthiocarbonyl, and aralkylthiocarbonyl.

[0080] Examples of carbonylthio (-S-C(=O)-R) include alkylcarbonylthio, cycloalkylcarbonylthio, alkenylcarbonylthio, alkynylcarbonylthio, arylcarbonylthio, heteroarylcarbonylthio, and aralkylcarbonylthio.

[0081] Examples of aminocarbonyl (-C(=O)-NHR) include alkylaminocarbonyl, cycloalkylaminocarbonyl, alkenylaminocarbonyl, alkynylaminocarbonyl, arylaminocarbonyl, heteroarylaminocarbonyl, and aralkylaminocarbonyl. 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 compounds are also included.

[0082] Examples of carbonylamino (-NH-C(=O)-R) include alkylcarbonylamino, cycloalkylcarbonylamino, alkenylcarbonylamino, alkynylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino, and aralkylcarbonylamino. 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 compounds are also included.

[0083] Examples of oxycarbonylaminos (-NH-C(=O)-OR) include alkoxycarbonylaminos, cycloalkoxycarbonylaminos, alkenyloxycarbonylaminos, alkynyloxycarbonylaminos, aryloxycarbonylaminos, heteroaryloxycarbonylaminos, and aralkyloxycarbonylaminos. 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 groups are also included.

[0084] Sulfonylamino(-NH-SO) ２ Examples of -R) include alkylsulfonylaminos, cycloalkylsulfonylaminos, alkenylsulfonylaminos, alkynylsulfonylaminos, arylsulfonylaminos, heteroarylsulfonylaminos, and aralkylsulfonylaminos. In addition to these, -NH-SO ２ Examples 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.

[0085] 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.

[0086] Sulfamoylamino(-NH-SO) ２Examples 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.

[0087] 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:

[0088] Examples of thio(-S-R) include alkylthio, cycloalkylthio, alkenylthio, alkynylthio, arylthio, heteroarylthio, and aralkylthio.

[0089] Examples of sulfinyl (-S(=O)-R) include alkylsulfinyl, cycloalkylsulfinyl, alkenylsulfinyl, alkynylsulfinyl, arylsulfinyl, heteroarylsulfinyl, and aralkylsulfinyl.

[0090] Sulfonyl (-S(O) ２ Examples of -R) include alkylsulfonyl, cycloalkylsulfonyl, alkenylsulfonyl, alkynylsulfonyl, arylsulfonyl, heteroarylsulfonyl, and aralkylsulfonyl.

[0091] 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)-NH２ ), 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.

[0092] Examples of secondary amino acids (-NH-R) include alkylaminos, cycloalkylaminos, alkenylaminos, alkynylaminos, arylaminos, heteroarylaminos, and aralkylaminos.

[0093] 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.

[0094] 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.

[0095] 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.

[0096] 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.

[0097] 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.

[0098] 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).

[0099] 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.

[0100] 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 C１ -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).

[0101] 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.

[0102] The peptide may be a cyclic peptide. In this specification, "cyclic peptide" is not particularly limited as long as it is a peptide having a cyclic portion composed of five or more amino acid residues. The number of amino acid residues constituting the cyclic portion of a 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. 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 the above range increases the efficiency of obtaining target molecules 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, such as cyclization via carbon-nitrogen bonds like amide bonds, cyclization via carbon-oxygen bonds like ester bonds and ether bonds, cyclization via carbon-sulfur bonds like 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 a carboxyl group in the side chain and an amino group in 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.

[0103] A cyclic peptide may have a linear portion in addition to a cyclic portion. The specific configuration of the number of amino acid residues in a cyclic peptide is the same as the specific configuration of the number of amino acid residues in the peptides described above. When a cyclic peptide has a linear portion, it is preferable that the total number of amino acid residues in the cyclic and linear portions fall within the same range. Furthermore, when a cyclic peptide has a linear portion, 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 a cyclic peptide has a linear portion, 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.

[0104] The molecular weight of the candidate molecule 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. The molecular weight of the candidate molecule 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. In this specification, molecular weight refers to 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 structure formula (unit: "g / mol"). Having the molecular weight of a candidate molecule within the above range increases the efficiency of obtaining the target molecule.

[0105] The ClogP of a candidate molecule may be, for example, between 4 and 25. ClogP is a computer-calculated partition coefficient and can be determined according to the principles described in "CLOGP Reference Manual DailyLight Version 4.9 (Release Date: August 1, 2011, https: / / www.daylight.com / dayhtml / doc / clogp / )". As an example of how to calculate ClogP, see DailyLight Chemical Information Systems, Inc. One possible method is to use Daylight Version 4.95 (release date: August 1, 2011, ClogP algorithm version 5.4, database version 28, https: / / www.daylight.com / dayhtml / doc / release_notes / index.html) for the calculation.

[0106] Furthermore, the principles described in the CLOGP Reference Manual Daily Version 4.9 (release date: August 1, 2011, https: / / www.dailylight.com / dailyhtml / doc / clogp / ) are as described in paragraph 0101 of the specification of the international application under the Patent Cooperation Treaty: application number PCT / JP2024 / 014861.

[0107] The ClogP of a candidate molecule may be, for example, 24 or less, 23 or less, 22 or less, 21 or less, or 20 or less. The lower limit of the ClogP of a candidate molecule may be, for example, 5 or more, 6 or more, 7 or more, 8 or more, or 10 or more. The range of the ClogP of a candidate molecule may be, for example, 5 to 24, 6 to 23, 7 to 22, 8 to 21, 9 to 20, 10 to 20, or 11 to 18. The range of the ClogP of a candidate molecule may be, for example, 4 to 25, preferably 6 to 23, more preferably 8 to 21, and most preferably 9 to 20.

[0108] If the candidate molecule is a peptide, the peptide's ClogP / amino acid residue ratio may be, for example, 1.0 or higher. "Amino acid residue ratio" refers to the total number of amino acid residues that make up the peptide. For example, a cyclic peptide consisting of a cyclic portion with 10 amino acid residues and a linear portion with 1 amino acid residue has 11 amino acid residues. The ClogP / amino acid residue ratio is calculated by dividing the peptide's ClogP by the number of amino acid residues contained in the peptide. For example, if the peptide's ClogP is 14.0 and the peptide contains 7 amino acid residues, the ClogP / amino acid residue ratio of the peptide is calculated to be 2.0.

[0109] When the candidate molecule is a peptide, the ClogP / amino acid residue number of the peptide may be, for example, 1.1 or more, 1.2 or more. The upper limit of the ClogP / amino acid residue number of the peptide may be, for example, 1.8 or less, 1.7 or less, 1.6 or less, 1.5 or less. The range of the ClogP / amino acid residue number of the peptide is, for example, 1.0 or more and 1.8 or less, preferably 1.0 or more and 1.7 or less, more preferably 1.1 or more and 1.6 or less, and most preferably 1.1 or more and 1.5 or less.

[0110] The candidate molecule used in step (2) (contact step) may be only one type of candidate molecule, or may be a library of candidate molecules (also referred to as "candidate molecule library" in this specification). Similarly, the candidate molecule-nucleic acid conjugate used in step (2) (contact step) may be only one type of candidate molecule-nucleic acid conjugate, or may be a library of candidate molecule-nucleic acid conjugates (also referred to as "candidate molecule-nucleic acid conjugate library" in this specification). The candidate molecule library is a collection of multiple types of candidate molecules. Similarly, the candidate molecule-nucleic acid conjugate library is a collection of multiple types of candidate molecule-nucleic acid conjugates.

[0111] The library may be a display library such as a phage display library, ribosome display library, bacterial display library, yeast display library, mRNA display library, and DNA display library, or a DNA encoded library (DNA encoded library). The library is preferably an mRNA display library or a DNA encoded library, and more preferably an mRNA display library.

[0112] The candidate molecules contained in the library may have a diversity of 1×10 ３ or more. Thereby, the screening efficiency of the target molecule can be improved. The diversity of the candidate molecules contained in the library is 5×10 ３ or more, 1×10 ４ or more, 5×10 ４ or more, 1×10 ５ or more, 5×10 ５ or more, 1×10６ or more, 5 × 10 ６ or more, 1 × 10 ７ or more, 5 × 10 ７ or more, 1 × 10 ８ or more, 5 × 10 ８ or more, 1 × 10 ９ or more, 5 × 10 ９ or more, 1 × 10 １０ or more, 5 × 10 １０ or more, or 1 × 10 １１ or more may be included. The diversity of candidate molecules included in the library may be, for example, 1 × 10 ３ or more, preferably 1 × 10 ５ or more, more preferably 1 × 10 ７ or more, most preferably 1 × 10 ９ or more. The diversity of candidate molecules is synonymous with the number of types of candidate molecules. When the diversity of candidate molecules included in the library is within the above range, the efficiency of obtaining the target molecule is further enhanced.

[0113] Note that in this specification, "candidate molecules included in the library" means, in the case of a candidate molecule-nucleic acid conjugate library, the part other than the nucleic acid and the binding part in the candidate molecule-nucleic acid conjugate (the part of the candidate molecule).

[0114] The library can be prepared by methods well known to those skilled in the art. For example, an mRNA display library can be prepared as follows, although it is not limited to this method. First, a DNA library with the desired base sequence positioned downstream of the promoter is chemically synthesized, and this is used as a template to create 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 by it are linked via the linker containing puromycin, etc. In this way, an mRNA display library can be constructed consisting of complexes of mRNA and its corresponding products. Furthermore, by contacting the mRNA display library with a desired immobilized target molecule and washing away complexes that do not bind to the target molecule, the complexes that bind to the target molecule can be enriched (panning). By synthesizing cDNA from the mRNA contained in the selected complexes, performing PCR amplification, and analyzing the base sequence, the amino acid sequence of the bound peptide can be determined.

[0115] Of the candidate molecules included in the library, 1 × 10 ３ In the above candidate molecules, the molecular weight of each candidate molecule 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. Of the candidate molecules included in the library, 1 × 10 ３In the above candidate molecules, the molecular weight of the candidate molecule 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.

[0116] Of the candidate molecules included in the library, 1 × 10 ３ In the above candidate molecules, the ClogP value of each candidate molecule may be, for example, between 4 and 25. Of the candidate molecules included in the library, 1 × 10 ３ In the above candidate molecules, the ClogP of each candidate molecule may be, for example, 24 or less, 23 or less, 22 or less, 21 or less, or 20 or less. Of the candidate molecules included in the library, 1 × 10 ３ In the above candidate molecules, the lower limit of ClogP for each candidate molecule may be, for example, 5 or more, 6 or more, 7 or more, 8 or more, or 10 or more. Of the candidate molecules included in the library, 1 × 10 ３ In the above candidate molecules, the range of ClogP for each candidate molecule may be, for example, 5 to 24, 6 to 23, 7 to 22, 8 to 21, 9 to 20, 10 to 20, and 11 to 18. Of the candidate molecules included in the library, 1 × 10 ３ In the above candidate molecules, the range of ClogP for each candidate molecule may be, for example, 4 to 25, preferably 6 to 23, more preferably 8 to 21, and most preferably 9 to 20.

[0117] If the candidate molecule is a peptide, then from the candidate molecules included in the library, 1 × 10 ３ In the above candidate molecules, there are no particular restrictions on the number of amino acid residues in each candidate molecule, but for example, it may be 5 or more, 7 or more, 8 or more, or 9 or more. Of the candidate molecules included in the library, 1 × 10 ３ In the above candidate molecules, the number of amino acid residues in each candidate molecule may also be, for example, 30 or less, 25 or less, 15 or less, or 13 or less. Of the candidate molecules included in the library, 1 × 10 ３In the above candidate molecules, the number of amino acid residues in each candidate molecule may be, for example, 5 to 30, preferably 7 to 25, more preferably 8 to 15, and most preferably 9 to 13.

[0118] If the candidate molecule is a peptide, then from the candidate molecules included in the library, 1 × 10 ３ In the above candidate molecules, the number of non-natural amino acid residues contained in each candidate molecule 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, among the candidate molecules included in the library, 1 × 10 ３ In the above candidate molecules, the number of non-natural amino acid residues contained in each candidate molecule 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 candidate molecules included in the library, 1 × 10 ３ Among the candidate molecules described above, examples of the range of the number of non-natural amino acid residues contained in each candidate molecule 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 candidate molecules included in the library, 1 × 10 ３ In the above candidate molecules, the range of the number of non-natural amino acid residues contained in each candidate molecule is, for example, 1 to 13, preferably 3 to 12, more preferably 4 to 11, and most preferably 5 to 10.

[0119] If the candidate molecule is a peptide, then from the candidate molecules included in the library, 1 × 10 ３ In the above candidate molecules, the number of N-substituted amino acid residues in each candidate molecule may be, for example, 1 or more, 2 or more, 3 or more, 4 or more, or 5 or more. Of the candidate molecules included in the library, 1 × 10 ３ In the above candidate molecules, the number of N-substituted amino acid residues contained in each candidate molecule 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 candidate molecules included in the library, 1 × 10 ３Among the candidate molecules described above, examples of the range of the number of N-substituted amino acid residues contained in each candidate molecule include 1 to 10, 1 to 8, 1 to 7, and 2 to 6. Of the candidate molecules included in the library, 1 × 10 ３ In the above candidate molecules, the number of N-substituted amino acid residues contained in each candidate molecule is, for example, 1 to 13, preferably 3 to 12, more preferably 4 to 11, and most preferably 5 to 10.

[0120] If the candidate molecule is a peptide, then from the candidate molecules included in the library, 1 × 10 ３ In the above candidate molecules, the ClogP / amino acid residue count of each candidate molecule may be, for example, 1.0 or more. Of the candidate molecules included in the library, 1 × 10 ３ In the above candidate molecules, the ClogP / amino acid residue ratio of each candidate molecule may be, for example, 1.1 or more, or 1.2 or more. Of the candidate molecules included in the library, 1 × 10 ３ In the above candidate molecules, the upper limit of the ClogP / amino acid residue count for each candidate molecule may be, for example, 1.8 or less, 1.7 or less, 1.6 or less, or 1.5 or less. Of the candidate molecules included in the library, 1 × 10 ３ In the above candidate molecules, the range of ClogP / amino acid residue number for each candidate molecule is, for example, 1.0 to 1.8, preferably 1.0 to 1.7, more preferably 1.1 to 1.6, and most preferably 1.1 to 1.5.

[0121] If the candidate molecule is a cyclic peptide, then 1 × 10⁶ of the candidate molecules included in the library ３ In the above candidate molecules, the number of amino acid residues constituting the cyclic portion of each candidate molecule 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. ３In the above candidate molecules, the number of amino acid residues constituting the cyclic portion of each candidate molecule may be, for example, 5 to 15, preferably 9 to 15, more preferably 10 to 14, and most preferably 11 to 13.

[0122] If the candidate molecule is a cyclic peptide and the cyclic peptide has a linear portion, then from the candidate molecules included in the library, 1 × 10 ３ In the above candidate molecules, the number of amino acid residues constituting the cyclic portion of each candidate molecule may be, for example, 5 to 15, preferably 9 to 15, more preferably 10 to 14, and most preferably 11 to 13. Of the candidate molecules included in the library, 1 × 10 ３ In the above candidate molecules, the number of amino acid residues constituting the linear portion of each candidate molecule is, for example, 1 to 8, preferably 1 to 6, more preferably 1 to 4, and most preferably 1 to 3.

[0123] In this specification, among the candidate molecules included in the library, 1 × 10 ３ To determine whether the above candidate molecules fall within a specific range of values, we calculate the value that each candidate molecule in the library possesses, and the number of values ​​is 1 × 10⁻⁶. ３ This can be determined by checking whether the result is greater than or equal to the above. However, this is only applicable if the number of candidate molecules in the library is 1 × 10⁶. ３ If the above conditions are met, we assume all possible candidate molecules that could theoretically be included in the library, and of those, 1 × 10 ３ If more than 10 types of candidate molecules fall within a specific range, then from the candidate molecules included in the library, 1 × 10 ３ Assume that the above candidate molecules fall within a specific range of values.

[0124] (Method for carrying out the contact step) In step (2) (contact step), a candidate molecule or a candidate molecule-nucleic acid conjugate is brought into contact with the conjugate. In step (2) (contact step), it is preferable that the multiple types of target molecules are in close proximity to each other when in contact with the candidate molecule or the candidate molecule-nucleic acid conjugate. In step (2) (contact step), it is also preferable that the first target molecule and the second target molecule are in close proximity to each other when in contact with the candidate molecule or the candidate molecule-nucleic acid conjugate. When multiple types of target molecules are in close proximity to each other, or when the first target molecule and the second target molecule are in close proximity to each other, it is possible to efficiently obtain target molecules that can form complexes with multiple types of target molecules.

[0125] In one non-limiting aspect, "multiple types of target molecules being in close proximity to each other" means that the distance between target molecules is 60 nm or less. The distance between target molecules may be, for example, 55 nm or less, 50 nm or less, 45 nm or less, 40 nm or less, 35 nm or less, or 30 nm or less. Preferably, the distance between target molecules is 50 nm or less, more preferably 40 nm or less, and most preferably 30 nm or less. Also, the distance between target molecules may be, for example, 1 nm or more, preferably 5 nm or more, more preferably 10 nm or more, and most preferably 15 nm or more. Also, the distance between target molecules may be, for example, 1 nm to 60 nm, preferably 5 nm to 50 nm, more preferably 10 nm to 40 nm, and most preferably 15 nm to 30 nm. The efficiency of acquiring the target molecule is increased when the distance between target molecules is within the above range. The distance between target molecules is the distance of the shortest straight-line distance in the gap between target molecules. Note that the distance between target molecules may be 0 nm (i.e., the target molecules are in contact with each other). The distance between target molecules can be controlled depending on the means by which the target molecules are brought into close proximity. For example, if target molecules are linked together by a linker, the distance can be controlled by adjusting the length of the linker, or if multiple types of target molecules are arranged in close proximity on a solid support, the distance can be controlled by adjusting their density. For example, when multiple types of target molecules are linked via a linking portion, and the linking portion is a peptide with 15 α-amino acids bonded to it, its length is known to be approximately 5.7 nm (Advanced Drug Delivery Reviews, 2013, vol. 65, pp. 1357-1369). In this case, the maximum distance between the target molecules is 5.7 nm, which is the length if the peptide were to be stretched out in a straight line. Therefore, the multiple types of target molecules are in close proximity to each other.

[0126] Step (2) (contact step) may include a step of mixing the candidate molecule or the candidate molecule-nucleic acid conjugate with the conjugate.

[0127] In step (2) (contact step), the conjugate may have the first target molecule immobilized on a solid support. That is, in the case of embodiment (1) described above, the conjugate in which the first target molecule and the second target molecule are directly linked via linker Y before contact with the candidate molecule or candidate molecule-nucleic acid conjugate in step (2) (contact step) may be immobilized on a solid support. In the case of embodiment (2) described above, the first target molecule and the second target molecule are immobilized on a support that serves as part of the conjugate.

[0128] As a specific example of step (2) (contact step), for example, a method is to add a candidate molecule or a candidate molecule-nucleic acid conjugate to a liquid containing the conjugate and mix, and then incubate. The conditions for contacting the conjugate with the candidate molecule or candidate molecule-nucleic acid conjugate (incubation time, temperature during incubation, etc.) can be appropriately selected according to the type of conjugate, the type of candidate molecule or candidate molecule-nucleic acid conjugate, etc.

[0129] In one non-limiting embodiment, in step (2) (contact step), the candidate molecule or the candidate molecule-nucleic acid conjugate may be brought into contact with target molecules other than the first target molecule and the second target molecule contained in the conjugate, in addition to the conjugate. Furthermore, the target molecules other than the first target molecule and the second target molecule may be of the same type as one of the target molecules other than the first target molecule and the second target molecule contained in the conjugate. This can further reduce the recovery rate of the monospecific binder.

[0130] In one non-limiting embodiment, if the first target molecule and the second target molecule are peptides or proteins, the target molecules other than the first target molecule and the second target molecule may have an amino acid sequence having 80% or more sequence identity with one of the first target molecule and the second target molecule, and / or an amino acid sequence in which one to 20 amino acids are deleted, substituted, or added to the amino acid sequence of one of the first target molecule and the second target molecule. Sequence identity may be 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%. The number of deleted, substituted, or added amino acids may be one to 15, one to 10, one to 5, one to 4, one to 3, or one to 2.

[0131] In one non-limiting embodiment, the target molecules other than the first target molecule and the second target molecule may be individual first target molecules not immobilized on a solid phase support, or individual second target molecules not immobilized on a solid phase support. "Individual" means not forming the aforementioned linkage. The target molecules other than the first target molecule and the second target molecule are preferably individual first target molecules when recovery is performed via the first target molecule in step (4) (recovery step). When recovery is performed via the second target molecule in step (4) (recovery step), they are preferably individual second target molecules.

[0132] The target molecules other than the first target molecule and the second target molecule may be one type or two types, but preferably one type.

[0133] Target molecules other than the first and second target molecules do not need to be immobilized on the solid support. Furthermore, target molecules other than the first and second target molecules do not need to be bound to the solid support. For example, if the solid support has a biotin-binding protein on its surface, target molecules other than the first and second target molecules may be non-biotinylated molecules.

[0134] In step (2) (contact step), the ratio of the concentration of target molecules other than the first target molecule and the second target molecule to the concentration of the conjugate is, for example, 1 or more, preferably 3 or more, more preferably 5 or more, and most preferably 10 or more. The ratio of the concentration of target molecules other than the first target molecule and the second target molecule to the concentration of the conjugate is, for example, 1,000 or less, preferably 500 or less, more preferably 300 or less, and most preferably 100 or less. The ratio of the concentration of target molecules other than the first target molecule and the second target molecule to the concentration of the conjugate is, for example, 1 or more and 1,000 or less, preferably 3 or more and 500 or less, more preferably 5 or more and 300 or less, and most preferably 10 or more and 100 or less.

[0135] [Step (3): Cutting Step] In Step (3) (Cutting Step), the linkage is cut after the contact step. By performing the cutting step, the linkage between the first target molecule and the second target molecule via the linkage is lost. At this time, if the candidate molecule or candidate molecule-nucleic acid conjugate is a multispecific binder, the complex with the first target molecule, the second target molecule, and the candidate molecule or candidate molecule-nucleic acid conjugate is maintained, while if the candidate molecule or candidate molecule-nucleic acid conjugate is a monospecific binder, the complex is not maintained. Therefore, in Step (4) (Recovery Step), the recovery rate of the monospecific binder can be reduced by recovering the complex via either the first target molecule or the second target molecule. In particular, in Step (4) (Recovery Step), recovering the complex via the first target molecule can reduce the recovery rate of the monospecific binder with respect to the second target molecule, and recovering the complex via the second target molecule can reduce the recovery rate of the monospecific binder with respect to the first target molecule.

[0136] In step (3) (cleavage step), the linkage between the first target molecule and the second target molecule is cleaved. The cleavage in step (3) (cleavage step) may be performed without cleaving the bond between the first target molecule, which is immobilized on the solid support, and the solid support. The cleavage method can be appropriately selected depending on the configuration of the linkage. For example, if the linkage is a linker that can be cleaved by washing, a linker that can be cleaved using an enzyme, a linker that can be cleaved using light, a linker that can be cleaved by competitive elution, or a linker that can be cleaved by pH, these linkers can be cleaved by the method described in step (1) (linkage step). The cleavage in step (3) (cleavage step) may be performed by one or more methods selected from the group consisting of washing, competitive elution, enzyme use, and light use. Step (3) also includes a washing process. Here, the washing process includes, for example, washing the system containing the complex with a buffer solution, and applying a mild physical action to the complex, such as stirring or inverting the solution containing the complex. Furthermore, in the cleavage of step (3), the washing treatment may be carried out under conditions such that the recovery rate of candidate molecule-nucleic acid conjugates forming the complex is at least twice the recovery rate of candidate molecule-nucleic acid conjugates that bind only to the second target molecule. Such conditions can be achieved, for example, by appropriately setting the concentration of imidazole, histidine, polyhistidine, a solution with a pH of 6 or less, or a chelating agent added to the buffer used in the washing treatment.

[0137] If the conjugate in which the first target molecule and the second target molecule are linked via a connecting portion is, for example, the conjugate in embodiment (1) in which the first target molecule and the second target molecule are directly linked via a linker Y, then in step (3) (cutting step), the linker Y is cut. Also, in embodiment (1), when the cutting step is performed, if the first target molecule is immobilized on the solid support via a linker X, it is preferable that linker X and linker Y are different types of linkers, and it is more preferable that linker X is a linker that is not cut under the conditions in which linker Y is cut. In the case of embodiment (1), the cutting in step (3) (cutting step) may be performed before the first target molecule is immobilized on the solid support. In this case, the first target molecule may be immobilized on the solid support with linker X after step (3) (cutting step) and before step (4) (recovery step).

[0138] In the case where the linked body formed by linking a first target molecule and a second target molecule via a linking portion is, for example, (2) a linked body in which the first target molecule and a solid support are linked via a linker X, and the solid support and the second target molecule are further linked via a linker Z, in step (3) (cutting step), only linker X may be cut, only linker Z may be cut, or both linker X and linker Z may be cut. From the viewpoint of facilitating the implementation of step (4) (recovery step), it is preferable to cut only linker X or only linker Z in step (3) (cutting step). In this case, it is preferable that linker X and linker Z are different types of linkers, and when only linker Z is cut, it is more preferable that linker X is a linker that is not cut under the conditions in which linker Z is cut, and when only linker X is cut, it is more preferable that linker Z is a linker that is not cut under the conditions in which linker X is cut.

[0139] In the aforementioned linkage, if the first target molecule and the second target molecule are linked by linker Y, linker Y may include a bond between one selected from the group consisting of His (histidine) consecutive His tags, HN tags consisting of His (histidine) and Asn (asparagine) consecutively, and HAT (Histidine Affinity Tag), and one selected from the group consisting of Ni-NTA, Ni-IDA, Co-NTA, and Co-IDA, particularly a bond between the His tag and Ni-NTA. In this case, the cleavage in step (3) (cleavage step) may be, for example, a washing treatment, a method using competitive elution, or a method of cleavage by pH, but the method using competitive elution or the method of cleavage by pH is preferred. In the method using competitive elution, for example, imidazole, histidine, or a chelating agent may be used as the competing substance, and the use of imidazole is preferred. The imidazole concentration during competitive elution may be, for example, greater than 0 mM and 200 mM or less, 1 mM to 100 mM, 5 mM to 50 mM, 10 mM to 30 mM, greater than 0 mM and 25 mM or less, 5 mM to 25 mM or less, 15 mM to 25 mM or less, or 20 mM. In the pH-based cleavage method, for example, it is preferable to use a solution with a pH of 4 or less.

[0140] [Step (4): Recovery Step] In step (4) (recovery step), the composite material is recovered after step (3) (cutting step).

[0141] The composite can be recovered by methods well known to those skilled in the art. For example, the composite can be recovered by affinity-based methods such as the pull-down method.

[0142] 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.

[0143] Step (4) (Recovery Step) may involve recovery via the first target molecule. Recovery via the first target molecule involves recovery using affinity with the first target molecule. In this case, molecules not bound to the first target molecule (for example, a complex of a candidate molecule and a second target molecule) will not be recovered.

[0144] In step (4) (recovery step), the first target molecule may be immobilized on the solid support, the first target molecule and the second target molecule may be immobilized on the solid support, or only the first target molecule may be immobilized on the solid support.

[0145] In one non-limiting embodiment, for example, a first target molecule can be biotin-modified and mixed with a solid support (e.g., beads) on which biotin-binding proteins are bound to the surface to initiate affinity binding between biotin and biotin-binding proteins. The beads can then be recovered by centrifugation or the like to recover a candidate molecule or a candidate molecule-nucleic acid conjugate that has formed a complex with the first target molecule.

[0146] Furthermore, if the first target molecule is already immobilized on the solid support at the end of step (3) (cleavage step), in step (4) (recovery step), the candidate molecule or candidate molecule-nucleic acid conjugate that has formed a complex with the first target molecule can be recovered by recovering the solid support by centrifugation or the like without newly mixing it with the solid support.

[0147] Examples of biotin-binding proteins include avidin, neutraavidin, and streptavidin.

[0148] [Step (5): Elution Step] In step (5) (elution step), the nucleic acid in the candidate molecule-nucleic acid conjugate recovered in step (4) (recovery step) is eluted.

[0149] Nucleic acids may be eluted in the form of the nucleic acid itself, a candidate molecule-nucleic acid conjugate, or a complex of the target molecule and the candidate molecule-nucleic acid conjugate.

[0150] In other words, in step (5) (elution step), the bond between the candidate molecule and the nucleic acid may be cleaved, the bond between the target molecule and the candidate molecule-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 candidate molecule 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 candidate molecule-nucleic acid conjugate, the nucleic acid can be eluted in the form of the candidate molecule-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 candidate molecule-nucleic acid conjugate.

[0151] If the bond between the solid support and the target molecule is cleaved, for example, the linker X between the solid support and the first target molecule may be cleaved.

[0152] The bond can be cleaved by one or more methods selected from the group consisting of, for example, enzyme-based methods, light-based methods, heat-based methods, and competitive elution methods. Preferably, the bond can be cleaved by a method not used in the cleavage step.

[0153] The cleavage of bonds by enzymes, light, heat, and competitive elution can be performed, for example, by using linkers that can be cleaved by enzymes, by light, by heat, and by competitive elution to cleave the bond between a candidate molecule and a nucleic acid, the bond between a target molecule and a candidate molecule-nucleic acid conjugate, or the bond between a solid support and a target molecule. These linkers and cleavage methods are as described in step (1) (coupling step). Bonds that can be cleaved by heat may include affinity-based bonds, and these bonds may be cleaved by thermal elution by applying heat (for example, 95°C for 10 minutes). If the first target molecule is immobilized on a solid phase by a biotin-avidin bond, a site that is cleaved during thermal elution may be included in the bond portion. The nucleic acid may also be thermally eluted by cleaving the bond between the candidate molecule and the target molecule with heat.

[0154] [Step (6): Amplification Step] In step (6) (Amplification Step), the nucleic acid in the candidate molecule-nucleic acid conjugate that has formed a complex is amplified. The amplification step may be performed after step (4) (Recovery Step), but is preferably performed after step (5) (Elution Step), and more preferably after step (5) (Elution Step) and before step (7) (Identification Step).

[0155] Nucleic acid amplification can be carried out, for example, by PCR using the recovered complex or eluted nucleic acid as a template and primers that bind to sequences used for amplification in the nucleic acid.

[0156] [Repeated Method] The screening method according to this embodiment may also involve generating a new candidate molecule-nucleic acid conjugate library from the nucleic acid amplified in step (6) (amplification step), and repeating steps (2) (contact step), (3) (cleavage step), (4) (recovery step), and (6) (amplification step) multiple times using the candidate molecule-nucleic acid conjugate library.

[0157] By repeating steps (2) (contact step), (3) (cutting step), (4) (recovery step), and (6) (amplification step), a library enriched with 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, specifically, for example, one, two, three, four, or five times. In one non-limiting embodiment, when the number of repetitions is four or more, the target molecule is usually highly enriched, so the target molecule can be obtained without performing step (7) (identification step).

[0158] In other embodiment screening methods, a new candidate molecule-nucleic acid conjugate library may be generated from the nucleic acid amplified in step (6) (amplification step), and steps (2) (contact step), (3) (cleavage step), (4) (recovery step), (5) (elution step), and (6) (amplification step) may be repeated multiple times using the candidate molecule-nucleic acid conjugate library.

[0159] By repeating steps (2) (contact step), (3) (cutting step), (4) (recovery step), (5) (elution step), and (6) (amplification step), a library enriched with the target molecule can be obtained. The number of repetitions may be two or more, three or more, four or more, or five or more, specifically, for example, two, three, four, or five times. In one non-limiting embodiment, if the number of repetitions is four or more, the target molecule is usually highly enriched, so the target molecule can be obtained without performing step (7) (identification step).

[0160] Furthermore, the first target molecule and the second target molecule may be swapped, and each of the second steps may be performed. For example, for a group of non-target molecules that bind only to the target molecule forming the complex and the second target molecule, step (4) in the repetition may be set to be recovery via the second target molecule instead of recovery via the first target molecule. This method may reduce the recovery rates of both the monospecific binder that binds only to the first target molecule and the monospecific binder that binds only to the second target molecule.

[0161] [Step (7): Identification Step] In step (7) (Identification Step), the target molecule is identified from the candidate molecule that has formed a complex or the candidate molecule-nucleic acid conjugate. Step (7) (Identification Step) may be performed after step (4) (Recovery Step). In the case of a candidate molecule-nucleic acid conjugate, it is preferably performed after step (5) (Elution Step) or after step (6) (Amplification Step), and more preferably after both step (5) (Elution Step) and step (6) (Amplification Step).

[0162] Step (7) (Identification step) may include determining the sequence of the nucleic acid eluted in step (5) (Elution step) or the nucleic acid amplified in step (6) (Amplification step). By determining the sequence of the nucleic acid, the type of candidate molecule that forms a conjugate with the nucleic acid can be identified.

[0163] Step (7) (identification step) may include comparing the recovery rate of the candidate molecule or candidate molecule-nucleic acid conjugate (recovery rate A) when the candidate molecule or candidate molecule-nucleic acid conjugate is brought into contact with the conjugate, with the recovery rate of the candidate molecule-nucleic acid conjugate (recovery rate B) when the candidate molecule or candidate molecule-nucleic acid conjugate is brought into contact with only one type of target molecule selected from multiple types of target molecules.

[0164] In this case, a candidate molecule can be identified as a target molecule when recovery rate A is 5 times or more, preferably 10 times or more, more preferably 20 times or more, and most preferably 30 times or more, compared to recovery rate B. When the ratio of recovery rate A to recovery rate B is within the above range, it can be said that the identified target molecule has a stronger property of forming complexes with multiple types of target molecules.

[0165] Here, "recovery rate" is the ratio (percentage: %) of the number of candidate molecules or candidate molecule-nucleic acid conjugates recovered by forming a complex with the target molecule (output molecules) to the number of candidate molecules or candidate molecule-nucleic acid conjugates (input molecules) that were brought into contact with the conjugate or one type of target molecule (amount of candidate molecules or candidate molecule-nucleic acid conjugates).

[0166] Furthermore, recovery rate B includes the recovery rate when the candidate molecule or candidate molecule-nucleic acid conjugate is contacted only with each of the multiple types of target molecules. For example, if there are two types of target molecules, recovery rate B includes recovery rate B1 when the candidate molecule or candidate molecule-nucleic acid conjugate library is contacted only with the first target molecule, and recovery rate B2 when the candidate molecule or candidate molecule-nucleic acid conjugate library is contacted only with the second target molecule. In this case, the candidate molecule can be identified as the target molecule when recovery rate A is 5 times or more, preferably 10 times or more, more preferably 20 times or more, and most preferably 30 times or more, compared to both recovery rates B1 and B2. When the ratio of recovery rate A compared to both recovery rates B1 and B2 is within the above range, it can be said that the identified target molecule has a stronger property of forming complexes with multiple types of target molecules.

[0167] Step (7) (identification step) may include comparing the recovery rate of the candidate molecule or candidate molecule-nucleic acid conjugate when the conjugate is brought into contact with the conjugate (recovery rate A) with the recovery rate of the candidate molecule or candidate molecule-nucleic acid conjugate in the absence of the target molecule (recovery rate C).

[0168] In this case, a candidate molecule can be identified as a target molecule when the recovery rate A is 5 times or more, preferably 10 times or more, more preferably 20 times or more, and most preferably 30 times or more, compared to the recovery rate C. When the ratio of recovery rate A to recovery rate C is within the above range, it can be said that the identified target molecule has a stronger property of forming complexes with multiple types of target molecules.

[0169] Step (7) (identification step) may include comparing (1) the recovery rate of the candidate molecule or candidate molecule-nucleic acid conjugate (recovery rate A) when the candidate molecule or candidate molecule-nucleic acid conjugate is brought into contact with the conjugate, and (2) the recovery rate of the candidate molecule or candidate molecule-nucleic acid conjugate (recovery rate D) when the candidate molecule or candidate molecule-nucleic acid conjugate is brought into contact with a first target molecule, which is selected from a plurality of target molecules and is immobilized on a solid support, and a second target molecule, which is different from the first target molecule and is not immobilized on a solid support.

[0170] In this case, a candidate molecule can be identified as a target molecule when the recovery rate A is 5 times or more, preferably 10 times or more, more preferably 20 times or more, and most preferably 30 times or more, compared to the recovery rate D. When the ratio of recovery rate A to recovery rate D is within the above range, it can be said that the identified target molecule has a stronger property of forming complexes with multiple types of target molecules.

[0171] Furthermore, the recovery rate D includes the recovery rate for each case in which one of the multiple types of target molecules is selected as the first target molecule. For example, if there are two types of target molecules, target molecule X and target molecule Y, the recovery rate D includes the recovery rate D1 when target molecule X is selected as the first target molecule and the recovery rate D2 when target molecule Y is selected as the first target molecule. In this case, a candidate molecule can be identified as the target molecule when the recovery rate A is 5 times or more, preferably 10 times or more, more preferably 20 times or more, and most preferably 30 times or more, compared to both recovery rates D1 and D2. When the ratio of recovery rate A compared to both recovery rates D1 and D2 is within the above range, it can be said that the identified target molecule has a stronger property of forming complexes with multiple types of target molecules.

[0172] Step (7) (Identification Step) may include all of the above-mentioned comparison of recovery rates A, B, C, and D.

[0173] Furthermore, step (7) (identification step) may include all of the above-mentioned comparison of recovery rate A with recovery rates B, C, and D. In this case, a candidate molecule can be identified as a target molecule when recovery rate A is 5 times or more, preferably 10 times or more, more preferably 20 times or more, and most preferably 30 times or more, compared to any of recovery rates B, C, and D. When the ratio of recovery rate A compared to any of recovery rates B, C, and D is within the above range, it can be said that the identified target molecule has a stronger property of forming complexes with multiple types of target molecules.

[0174] Furthermore, step (7) (identification step) may include comparing recovery rate D with recovery rate B as described above. In this case, a candidate molecule can be identified as a target molecule when recovery rate D is 5 times or more, preferably 10 times or more, more preferably 20 times or more, and most preferably 30 times or more, compared to recovery rate B. When the ratio of recovery rate D compared to recovery rate B is within the above range, it can be said that the identified target molecule has a stronger property of forming complexes with multiple types of target molecules.

[0175] The aforementioned screening method may be performed in vitro.

[0176] The aforementioned screening method may not use fluorescence induced by GFP or luminescence induced by luciferase. Furthermore, the aforementioned screening method may not use fluorescence induced by split-GFP or luminescence induced by split-luciferase.

[0177] The target molecule selected by the screening method according to the present invention is a molecule that can form a complex with multiple types of target molecules.

[0178] If the target molecule is a molecule that can form a complex with multiple types of target molecules, the value of the dissociation constant between the multiple types of target molecules in the presence of 10 μM of the target molecule will be smaller than the value of the dissociation constant between the multiple types of target molecules in the absence of the target molecule. In other words, in the present invention, the state in which the target molecule forms a complex with multiple types of target molecules means a state in which the value of the dissociation constant between the multiple types of target molecules in the presence of 10 μM of the target molecule is smaller than the value of the dissociation constant between the multiple types of target molecules in the absence of the target molecule.

[0179] The smaller the value of the dissociation constant between the multiple types of target molecules in the presence of 10 μM of the target molecule is compared to the value of the dissociation constant between the multiple types of target molecules in the absence of the target molecule, the stronger the property of the target molecule to form a complex with the multiple types of target molecules, and the more stable the complex formed by the target molecule and the multiple types of target molecules is.

[0180] Here, the value of the dissociation constant between the multiple types of target molecules in the presence of 10 μM of the target molecule may be, for example, less than 1 / 5, preferably less than 1 / 10, more preferably less than 1 / 20, and most preferably less than 1 / 30, compared to the value of the dissociation constant between the multiple types of target molecules in the absence of the target molecule. When the value of the dissociation constant between the multiple types of target molecules in the presence of 10 μM of the target molecule is within the above range compared to the value of the dissociation constant between the multiple types of target molecules in the absence of the target molecule, it can be said that the target molecule has a stronger property of forming a complex with the multiple types of target molecules, and that the complex formed by the target molecule with the multiple types of target molecules is in a more stable state.

[0181] Furthermore, the value of the dissociation constant between the multiple types of target molecules in the presence of 10 μM of the target molecule may be, for example, 30 μM or less, preferably 20 μM or less, more preferably 10 μM or less, and most preferably 5 μM or less. When the value of the dissociation constant between the multiple types of target molecules in the presence of 10 μM of the target molecule is within the above range, it can be said that the target molecule has a stronger tendency to form a complex with the multiple types of target molecules, and that the complex formed by the target molecule and the multiple types of target molecules is in a more stable state.

[0182] Furthermore, the value of the dissociation constant between the multiple types of target molecules in the presence of 10 μM of the target molecule may be, for example, 30 μM or less and less than one-fifth of the value of the dissociation constant between the multiple types of target molecules in the absence of the target molecule, preferably 20 μM or less and less than one-fifth of the value of the dissociation constant between the multiple types of target molecules in the absence of the target molecule, more preferably 10 μM or less and less than one-fifth of the value of the dissociation constant between the multiple types of target molecules in the absence of the target molecule, and most preferably 5 μM or less and less than one-fifth of the value of the dissociation constant between the multiple types of target molecules in the absence of the target molecule. When the value of the dissociation constant between the multiple types of target molecules in the presence of 10 μM of the target molecule is within the above range, it can be said that the target molecule has a stronger property of forming a complex with the multiple types of target molecules, and the complex formed by the target molecule with the multiple types of target molecules is in a more stable state.

[0183] Furthermore, the value of the dissociation constant between the multiple types of target molecules in the presence of 10 μM of the target molecule may be, for example, 30 μM or less and less than one-fifth of the value of the dissociation constant between the multiple types of target molecules in the absence of the target molecule, preferably 20 μM or less and less than one-tenth of the value of the dissociation constant between the multiple types of target molecules in the absence of the target molecule, more preferably 10 μM or less and less than one-twentieth of the value of the dissociation constant between the multiple types of target molecules in the absence of the target molecule, and most preferably 5 μM or less and less than one-thirtieth of the value of the dissociation constant between the multiple types of target molecules in the absence of the target molecule. When the value of the dissociation constant between the multiple types of target molecules in the presence of 10 μM of the target molecule is within the above range, it can be said that the target molecule has a stronger property of forming a complex with the multiple types of target molecules, and the complex formed by the target molecule with the multiple types of target molecules is in a more stable state.

[0184] The target molecule selected by the screening method according to the present invention may be one in which the dissociation constant between at least two target molecules selected from a plurality of target molecules in the presence of 10 μM of the target molecule is 10 μM or less, preferably 5 μM or less, more preferably 3 μM or less, and most preferably 1 μM or less. The smaller the dissociation constant, the stronger the property of the target molecule to form a complex with at least two target molecules selected from a plurality of target molecules.

[0185] Furthermore, the target molecules selected by the screening method according to the present invention may be those in which the dissociation constants of multiple types of target molecules in the presence of 10 μM of the target molecule are all 10 μM or less, preferably 5 μM or less, more preferably 3 μM or less, and most preferably 1 μM or less.

[0186] Furthermore, the target molecule selected by the screening method according to the present invention may have a dissociation constant value of 5 times or more compared to the dissociation constant value of 10 μM of the target molecule compared to the dissociation constant value of 10 μM of the target molecule compared to the dissociation constant value of 10 μM of the target molecule compared to the dissociation constant value of 10 μM or more compared to the dissociation constant value of 10 μM or more compared to the dissociation constant value of 10 μM or more compared to the dissociation constant value of 10 μM or more compared to the dissociation constant value of 10 μM or more compared to the dissociation constant value of 10 μM or more compared to the dissociation constant value of 10 μM or more compared to the dissociation constant value of 10 μM of the target molecule

[0187] In the present invention, the state in which the target molecule forms a complex with multiple types of target molecules means a state in which, in the presence of 10 μM of the target molecule, the dissociation constants of the multiple types of target molecules are 10 μM or less, and the value of the dissociation constants of the multiple types of target molecules in the absence of the target molecule is 5 times or more compared to the value of the dissociation constants of the multiple types of target molecules in the presence of 10 μM of the target molecule.

[0188] In another aspect of the present invention, the state in which the target molecule forms a complex with multiple types of target molecules means, for example, a state in which, in the presence of 10 μM of the target molecule, the dissociation constant between the multiple types of target molecules is 30 μM or less, and the value of the dissociation constant between the multiple types of target molecules in the absence of the target molecule is 5 times or more compared to the value of the dissociation constant between the multiple types of target molecules in the presence of 10 μM of the target molecule.

[0189] In another aspect of the present invention, the state in which the target molecule forms a complex with multiple types of target molecules preferably means a state in which, in the presence of 10 μM of the target molecule, the dissociation constant between the multiple types of target molecules is 20 μM or less, and the value of the dissociation constant between the multiple types of target molecules in the absence of the target molecule is 5 times or more compared to the value of the dissociation constant between the multiple types of target molecules in the presence of 10 μM of the target molecule.

[0190] In another aspect of the present invention, the state in which the target molecule forms a complex with multiple types of target molecules more preferably means a state in which, in the presence of 10 μM of the target molecule, the dissociation constant between the multiple types of target molecules is 15 μM or less, and the value of the dissociation constant between the multiple types of target molecules in the absence of the target molecule is 5 times or more compared to the value of the dissociation constant between the multiple types of target molecules in the presence of 10 μM of the target molecule.

[0191] In another aspect of the present invention, the state in which the target molecule forms a complex with multiple types of target molecules most preferably means a state in which, in the presence of 10 μM of the target molecule, the dissociation constants of the multiple types of target molecules are most preferably 10 μM or less, and the value of the dissociation constants of the multiple types of target molecules in the absence of the target molecule is 5 times or more compared to the value of the dissociation constants of the multiple types of target molecules in the presence of 10 μM of the target molecule.

[0192] Furthermore, the dissociation constants between the multiple types of target molecules can be measured by Surface Plasmon Resonance (SPR). For example, the dissociation constants between two types of target molecules in the presence of 10 μM of the target molecule can be determined by the following method. First, Biotin CAPture reagent is immobilized on all flow cells on the sensor chip, and then the first type of target molecule is immobilized on some of the flow cells. Next, a mixed solution of the second type of target molecule in a dilution series with a maximum concentration of 10 μM or 20 μM and 10 μM of the target molecule is prepared as an analyte, and added to all flow cells to obtain a binding sensorgram.

[0193] Furthermore, HBS, 1 mM Dithiothreitol, 0.01% Tween 20, and 4% Dimethylsulfoxide (DMSO) can be used as the running buffer. 5 mM CaCl ２ You can also add additional ingredients.

[0194] Furthermore, the analysis of the obtained sensorgrams can be performed, for example, using T200 evaluation software (Cytiva). The sensorgram obtained in a flow cell without the first target molecule immobilized, and the sensorgram obtained when running buffer is added, are used as double references. After applying DMSO solvent correction, the resulting sensorgram is curve-fitted using a 1:1 binding model. The apparent target intermolecular interaction parameter when 10 μM of the target molecule is present in the analyte is determined by the binding rate constant (k). ａ ), dissociation rate constant (k ｄ ), and the dissociation constant K Ｄ It is possible to make a decision.

[0195] In one non-limiting embodiment, the target molecule selected by the screening method according to the present invention may be a candidate molecule in which the recovery rate A is five times or more compared to the recovery rate B.

[0196] In one non-limiting embodiment, the target molecule selected by the screening method according to the present invention may be a candidate molecule in which the recovery rate A is five times or more compared to the recovery rate C.

[0197] In one non-limiting embodiment, the target molecule selected by the screening method according to the present invention may be a candidate molecule in which the recovery rate A is five times or more compared to the recovery rate D.

[0198] In one non-limiting embodiment, the candidate molecules (target molecules) selected by the screening method according to the present invention may be (i) binding sites that are formed only when multiple types of target molecules are in close proximity to each other, (ii) having two or more different sites that bind to each of the multiple types of target molecules, or (iii) binding to one or more types of target molecules and promoting binding to other types of target molecules.

[0199] [Method for producing peptides] The method for producing peptides according to the present invention includes carrying out the screening method according to the present invention described above. This makes it possible to produce peptides that can form complexes with multiple types of target molecules.

[0200] The method for producing the peptide includes, for example, (i) a step of screening for a target molecule using the screening method according to the present invention described above, and (ii) a step of producing a peptide based on the amino acid sequence of the target molecule selected in step (i).

[0201] Step (ii) may include a step of decoding the base sequence encoding the target molecule to identify the amino acid sequence of the target molecule (decoding step), and a step of synthesizing the target peptide based on the identified amino acid sequence (synthesis step). The decoding step and the synthesis step can be carried out by methods well known to those skilled in the art.

[0202] [Method for Identifying or Selecting Candidate Molecules Having Desired Characteristics (Mini Library Assay)] The screening method according to the present invention can also be applied to the precise evaluation and selection of candidate molecules. In one embodiment, the method for identifying or selecting candidate molecules having desired characteristics according to the present invention is a step of carrying out the screening method according to the present invention, wherein a candidate molecule-nucleic acid conjugate minilibrary is used as the candidate molecule or the candidate molecule-nucleic acid conjugate, and at least one of the contact step, the cleavage step, and the recovery step is carried out under a plurality of conditions with mutually different parameters, and the amount of each candidate molecule-nucleic acid conjugate recovered under each of the plurality of conditions is quantified by analyzing the nucleic acid sequence bound thereto, and the recovery rates under each condition are compared to identify or select candidate molecules having desired characteristics. This method is also referred to herein as a Mini Library Assay (MLA). Mini-library assays are useful as a high-throughput evaluation method that enables comparative evaluation to select the best molecule by identifying subtle differences in properties such as binding affinity, specificity, or stability under specific conditions from a group of promising candidates enriched in initial screening (panning).

[0203] The "candidate molecule-nucleic acid conjugate minilibrary" (minilibrary) used in this embodiment may not be a large-scale one like those used in initial screening, but rather a deliberately constructed minilibrary with relatively small diversity. For example, the diversity of the minilibrary may be 2 or more, or 1 × 10⁻¹⁶. ８ The following is preferably 1 x 10 to 1 x 10 ８ degree, for example, 1 x 10 ３ The above 1 x 10 ７ The following are possible:

[0204] Such minilibraries can be intentionally constructed, for example, by one of the following methods: (i) A method of constructing a minilibrary by intentionally selecting two or more candidate molecules that have the desired properties from a group of candidate molecules enriched by performing one or more rounds of the screening method according to the present invention. In this method, if the candidate molecules are peptides or proteins and nucleic acids encode them, candidate molecule-nucleic acid conjugates may be reconstructed from the nucleic acids. This method enables comparative evaluation to identify slight differences in properties (e.g., binding strength, specificity) from a group of promising candidate molecules and to select the best molecule. (ii) A method of constructing a minilibrary as a candidate molecule by creating one or more child sequences by substituting one or more amino acid residues of a specific parent sequence with other amino acid residues using site-directed mutagenesis or the like, and selecting at least one from the group consisting of the parent sequence and child sequences. This method is useful for so-called affinity maturation and functional analysis of specific residues.

[0205] "Multiple conditions with mutually different parameters" means changing the parameters of any step in the screening method according to the present invention. As an example, conditions for changing the parameters in the cutting step can be cited. In particular, if the connecting portion contains a linker that can be cut by competitive elution, multiple conditions can be set in which the concentration of the competing substance used in the cutting step (or the associated washing step) is changed in stages.

[0206] Another example of "multiple different conditions" is the modification of parameters in the contact process. Specifically, multiple conditions can be set by changing the concentration of the first target molecule, the concentration of the second target molecule, pH, salt concentration, or temperature in the solution during the contact process. This makes it possible to select candidate molecules that function stably under specific environmental conditions (for example, under various concentration adjustments of the first and second target molecules, or under pH and salt concentrations that mimic those in living organisms or specific cell compartments) and to evaluate temperature sensitivity.

[0207] The step of "quantifying the amount of each candidate molecule-nucleic acid conjugate by analyzing the nucleic acid sequence bound to it" can be carried out by any method known to those skilled in the art, for example, by quantitative PCR (qPCR) using the recovered nucleic acid as a template, or by read counting using a next-generation sequencer (NGS).

[0208] Furthermore, by "comparing recovery rates," it becomes possible, for example, to select candidate molecules that show high recovery rates in the presence of specific competing substances as "molecules with high specificity," or to select "molecules with a desired binding profile" by comparing recovery rate profiles for multiple target molecules.

[0209] By the way, in WO2024 / 214825, different fragments obtained by cutting out a part of a single protein may be used as multiple types of target molecules. [Item 1] A method for screening target molecules, comprising (1) a step of preparing a conjugate by linking multiple types of target molecules together or by linking one or more types of target molecules with a candidate molecule-nucleic acid conjugate (linking step), and (2) a step of contacting a candidate molecule-nucleic acid conjugate with the conjugate formed by linking multiple types of target molecules together, or a step of contacting a conjugate formed by linking one or more types of target molecules with a candidate molecule-nucleic acid conjugate with a target molecule not used in the linking step (contact step). [Item 2] A method for screening target molecules, comprising (1) a step of preparing a conjugate by linking multiple types of target molecules together (linking step), and (2) a step of contacting a candidate molecule-nucleic acid conjugate with the conjugate prepared in the linking step (contact step). [Item 3] A method for screening target molecules, comprising the steps of (1) preparing a conjugate by linking one or more target molecules with candidate molecule-nucleic acid conjugates (linking step), and (2) contacting target molecules not used in the linking step with the conjugate prepared in the linking step (contact step). [Item 4] A method for screening target molecules, comprising the steps of contacting a candidate molecule-nucleic acid conjugate with a conjugate formed by linking multiple types of target molecules, or contacting a target molecule not included in a conjugate formed by linking one or more target molecules with candidate molecule-nucleic acid conjugates (contact step). [Item 5] A method for screening target molecules, comprising the step of contacting a candidate molecule-nucleic acid conjugate with a conjugate formed by linking multiple types of target molecules. [Item 6] A method for screening target molecules, comprising the step of contacting a target molecule not included in a conjugate formed by linking one or more target molecules with candidate molecule-nucleic acid conjugates. [Clause 7] The method according to Clause 1, 3, 4, or 6, wherein the one or more target molecules linked to the candidate molecule-nucleic acid conjugate are one type. [Clause 8] A method for screening target molecules, comprising the step of contacting a candidate molecule-nucleic acid conjugate library with a plurality of target molecules in close proximity to each other (contact step).[Clause 9] The method according to Clause 1, 2, 4, 5, or 8, wherein the multiple types of target molecules fuse with each other to form a target fusion molecule. [Clause 10] A method for screening a target molecule, comprising the step of contacting a candidate molecule-nucleic acid conjugate library with a target fusion molecule formed by the fusion of multiple types of target molecules (contact step). [Clause 11] The method according to Clause 8, wherein the multiple types of target molecules that are in close proximity to each other are immobilized on a solid support. [Clause 12] A method for screening a target molecule, comprising the step of contacting a candidate molecule-nucleic acid conjugate library with multiple types of target molecules immobilized on the same solid support (contact step). [Clause 13] The method according to Clause 8, wherein the contact step is the step of contacting a first target molecule, which is one selected from the multiple types of target molecules, with a conjugate library containing multiple types of conjugates, each of which is a candidate molecule-nucleic acid conjugate linked to a target molecule different from the first target molecule. [Clause 14] A method for screening a target molecule, comprising the step of contacting a conjugate library containing multiple types of conjugates, each of which a candidate molecule-nucleic acid conjugate is linked to a target molecule different from the first target molecule, with a first target molecule, which is one selected from a plurality of types of target molecules. [Clause 15] The method according to any one of Clauses 1 to 14, further comprising the step of recovering a complex of at least one target molecule selected from the plurality of types of target molecules and a candidate molecule-nucleic acid conjugate (recovery step). [Clause 16] The method according to Clause 15, further comprising the step of eluting the nucleic acid in the candidate molecule-nucleic acid conjugate recovered in the recovery step (elution step). [Clause 17] The method according to Clause 15 or 16, further comprising the step of amplifying the nucleic acid in the candidate molecule-nucleic acid conjugate that has formed the complex (amplification step). [Clause 18] The method according to Clause 17, comprising generating a new candidate molecule-nucleic acid conjugate library from the nucleic acid amplified in the amplification step, and repeating the contact step, recovery step, and amplification step multiple times using the candidate molecule-nucleic acid conjugate library. [Clause 19] The method according to any one of claims 15 to 18, further comprising the step of identifying a target molecule from the candidate molecule-nucleic acid conjugate that forms the complex (identification step). [Clause 20] The method according to any one of claims 1 to 19, wherein the candidate molecule is a peptide.[Clause 21] The method according to any one of Clauses 1 to 20, wherein the multiple types of target molecules are two to five types. [Clause 22] The method according to any one of Clauses 1 to 21, wherein the target molecule is a molecule that can form a complex with the multiple types of target molecules. [Clause 23] A method for producing a peptide, comprising the method according to any one of Clauses 1 to 22. Specifically, candidate molecules having the property of simultaneously binding to two or more different sites on a single protein molecule are obtained. For example, two different regions (fragments) derived from a single protein molecule can be used as the first and second target domains. The "first target domain" is designated as the first target molecule, and the "second target domain" as the second target molecule. The domain portions of these two target molecules are fused together to form a "target fusion molecule," or these two target molecules are linked via a "fusion portion" or "linker" to form a "target fusion molecule," thereby preparing a "target fusion molecule" in one form of a "conjugate" and using it for screening. Alternatively, a conjugate may be prepared by linking the first target molecule with a candidate molecule-nucleic acid conjugate. Here, "mutually distinct regions" is a broad concept that includes structurally and functionally distinct domains (e.g., kinase domains and bromodomains), different epitopes within the same domain, allosteric sites and active sites, regions with different post-translational modifications, or regions exhibiting different conformations.

[0210] Another embodiment of the present invention is a specific and comprehensive screening method for identifying candidate molecules that bind to two different sites on a single protein molecule. This method essentially comprises the following steps (a) to (c): (a) preparing a conjugate by linking a first target molecule and a second target molecule from among the multiple types of target molecules, with two or more different regions derived from a single protein molecule as multiple types of target molecules (linking step); (b) contacting a candidate molecule or a candidate molecule-nucleic acid conjugate with the conjugate to form the complex (contact step); (c) selectively concentrating and recovering nucleic acids derived from the candidate molecules that formed the ternary complex by selectively concentrating and recovering nucleic acids derived from the candidate molecules that formed the ternary complex, through a cleavage step of the linkage of the present invention and a subsequent recovery step, and then appropriately repeating steps (5) (elution step) and (6) (amplification step). In step (a) above, "two distinct regions" includes structurally and functionally distinct domains, different epitopes within the same domain, allosteric sites and active sites, regions with different posttranslational modifications, or regions exhibiting different conformations.

[0211] The following describes preferred specific embodiments of the present invention as examples, but the present invention is not limited thereto. The following abbreviations were used in the examples: DIPEA: N,N-diisopropylethylamine DMF: N,N-dimethylformamide DMSO: dimethyl sulfoxide FA: formic acid HATU: 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate

[0212] [Preparation Example 1: Preparation of Proteins Used in the Study] <Preparation Example 1-1: Preparation of bio-Avi-TEV-FRB-FLAG-Sor-His> FRB expressed and purified in E. coli was used as the target protein for the study. Bio-Avi-TEV-FRB-FLAG-Sor-His was prepared by adding a TEV protease cleavage tag and a biotinylation enzyme recognition tag to the N-terminus of the FRB, and adding a purified tag containing a His tag to the C-terminus. ＊" represents biotinylated Lys.

[0213] Amino acid sequence of bio-Avi-TEV-FRB-FLAG-Sor-His (SEQ ID NO: 1) MGLNDIFEAQK ＊ IEWHESSGENLYFQGGGGSELIRVAILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFN QAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKQGGGSSDYKDDDDKGGSSLPMTGGGGSSHHHHHH

[0214] Specifically, an expression vector prepared by cloning the genes for bio-Avi-TEV-FRB-FLAG-Sor-His and biotin ligase (BirA) into pCDFDuet-1 was used to transform E. coli BL21 (DE3) and create an expression strain. OD ６００ The cultures were incubated in LB medium until the concentration was approximately 0.4 to 0.6. Induction was then carried out by adding 1 mM IPTG and 100 μM biotin to a final concentration, and the cultures were incubated at 18°C ​​for 16 hours. After that, the samples were centrifuged to collect the cultured cells.

[0215] The recovered cultured cells were suspended in a disruption buffer (50 mM Tris-HCl pH 7.0, 200 mM NaCl, 2 mM DTT, 0.1% CHAPS, 0.1 mg / mL lysozyme, 1 / 1000 (v / v) Benzone, Protein inhibitor cocktail), stirred at room temperature for 20 minutes, and then sonicated. This sample was centrifuged at 15000 × g for 30 minutes at 4°C, the supernatant was collected, and then filtered through a 0.22 μm filter to obtain the lysate sample.

[0216] Lysate samples were purified using 5 mL of Streptactin XT Superflow HC (IBA, #2-4030-010) resin and Tricorn 16 / 20 column. Equilibration buffer (50 mM Tris-HCl pH 7.5, 200 mM NaCl, 1 mM TCEP) and Elution buffer (50 mM Tris-HCl pH 7.5, 200 mM NaCl, 1 mM TCEP, 50 mM biotin) were used for purification. Fractions containing the target protein were recovered.

[0217] The collected samples were purified by gel filtration using HiLoad 26 / 600 Superdex 75 pg (Cytiva, #28-9893-34). A running buffer (50 mM Tris-HCl pH 7.5, 300 mM NaCl, 1 mM TCEP) was used for purification. The fraction containing the target protein was collected to obtain the target protein bio-Avi-TEV-FRB-FLAG-Sor-His. The samples were stored at -80°C.

[0218] <Preparation Example 1-2: Preparation of FKBP-FLAG-Sor-His> FKBP expressed and purified in Escherichia coli was used as the target protein for the study. FKBP-FLAG-Sor-His was prepared by adding a purified tag and a saltase recognition sequence to the C-terminus of the FKBP.

[0219] Amino acid sequence of FKBP-FLAG-Sor-His (SEQ ID NO: 2) MGVQVETISPGDGRTFPKRGQTCCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATTHGPGIIPPPHATLVFDVELLKLEGGGGSSDYKDDDDDKGGSSLPPMTGGGGGSSSHHHHHH

[0220] Specifically, an expression vector prepared by cloning the FKBP-FLAG-Sor-His gene sequence into pET11a was used to transform E. coli BL21 (DE3) and create an expression strain. ６００The cultures were incubated in LB medium until the concentration reached approximately 0.5, then induced by adding IPTG to a final concentration of 1 mM. After incubation at 18°C ​​for 18 hours, the sample was centrifuged to collect the cultured cells.

[0221] The recovered cultured cells were suspended in a disruption buffer (50 mM Tris-HCl pH 7.0, 200 mM NaCl, 2 mM DTT, 0.1% CHAPS, 0.1 mg / mL lysozyme, 1 / 1000 (v / v) Benzone, Protein inhibitor cocktail), stirred at room temperature for 20 minutes, and then sonicated. This sample was centrifuged at 15000 × g for 30 minutes at 4°C, and the supernatant was collected. The supernatant was further centrifuged at 15000 × g for 15 minutes at 4°C, and then passed through a 0.22 μm filter to obtain the lysate sample.

[0222] Lysate samples were purified using His Trap FF (Cytiva, 17531901). Binder buffer (100 mM Tris-HCl pH 8.0, 150 mM NaCl, 10% Glycerol) and Elution buffer (100 mM Tris-HCl pH 8.0, 150 mM NaCl, 10% Glycerol, 500 mM imidazole) were used for purification. Fractions containing the target protein were recovered and concentrated using Amicon Ultra-15 10K (MERCK, #UFC901024).

[0223] The concentrated sample was purified by gel filtration using HiLoad 26 / 600 Superdex 75 pg (Cytiva, #28-9893-34). A running buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM TCEP) was used for purification. The fraction containing the target protein was recovered, concentrated with Amicon Ultra-15 10K (MERCK, #UFC901024), and then filtered through a 0.22 μm filter to obtain the target protein FKBP-FLAG-Sor-His. The sample was stored at -80°C.

[0224] <Synthesis Example 1-1: Synthesis of DBCO-PEG(4)-NTA> The abbreviations and structures of the synthesized compounds are shown in Table 1 below.

[0225] The analytical conditions for LC-MS are as follows:

[0226] Synthesis of NTA (ST01) The compound was synthesized according to the method described in Non-Patent Literature (J. Am. Chem. Soc., 2006, Vol. 128, No. 7, pp. 2365-2372). LC-MS (ESI) m / z = 1049.9 (M + H) + Retention time: 0.26 min (Analytical conditions SQD FA05)

[0227] Synthesis of DBCO-PEG(4)-NTA(ST02) Under a nitrogen atmosphere, DBCO-PEG(4)-COOH (5.8 mg, 0.01 mmol) and HATU (34.9 mg, 0.03 mmol, 1.1 eq) were dissolved in DMF (50 μL), and then DIEPA (3.8 μL, 0.02 mmol, 2.0 eq) was added and the mixture was stirred at room temperature for 30 minutes. Then, a DMF solution (100 μL) containing NTA (35 mg, 3.0 eq) and DIEPA (22.8 μL, 0.12 mmol, 6.0 eq) was added and the mixture was stirred at room temperature for another 30 minutes. The reaction solution was purified directly by reverse-phase silica gel chromatography (0.1% formic acid aqueous solution / 0.1% formic acid acetonitrile) to obtain the title compound (DBCO-PEG(4)-NTA, 6.7 mg, 42%). LCMS (ESI) m / z=1612.3 (M+H)+ Retention time: 0.57 min (Analysis conditions SQD FA05)

[0228] <Preparation Example 1-3: Preparation of FKBP-TrisNTA> As the protein to be used for the study, FKBP12 (FKBP) having TrisNTA at the C-terminus was prepared. Specifically, a TrisNTA molecule having a saltase substrate site was prepared by a click reaction, and this was ligated with FKBP-FLAG-Sor-His prepared in Preparation Example 1-2 using saltase to prepare FKBP-TrisNTA. ＊ " is a modification of the C-terminus of Gly with TrisNTA as shown in (*1) or (*2) in Table 3.

[0229]

[0230] Amino acid sequence of FKBP-TrisNTA (SEQ ID NO: 3) MGVQVETISPGDGRTFPKRGQTCCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGAATGHHPGIIPPHATLVFDVELLKLEGGGGSSDYKDDDDKGGSSLPMTGGGG ＊

[0231] To prepare TrisNTA molecules containing a saltase substrate moiety, DBCO-PEG(4)-NTA synthesized in Synthesis Example 1-1 was linked to Picolyl Azide-Gly-Gly-Gly (Vector LABORATORIES, #CCT-1553) by a click reaction. Specifically, DBCO-PEG(4)-NTA and Picolyl Azide-Gly-Gly-Gly were mixed in DMSO to final concentrations of 40 mM and 20 mM, respectively, and reacted at 37°C for 24 hours. This reaction mixture was used as the crude product in the subsequent ligation reaction with saltase. The crude product was stored at -30°C until use in the ligation reaction.

[0232] For the ligation reaction with sortase, a reaction buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10 mM CaCl) is prepared to yield 40 μM FKBP-FLAG-Sor-His, 5 v / v% click reaction crude product DMSO solution, and 1 μM Sortase A (prepared by the method described in WO2022 / 138892). ２ It was added to ) and left to stand overnight at 4°C. Then, EDTA (Nacalai Tesque, #06894-14) was added to a final concentration of 20 mM to stop the reaction.

[0233] The reaction mixture was purified by gel filtration and affinity purification using Superdex 75 Increase 10 / 300 GL (Cytiva, #29148721) and 1 mL HisTrap (Cytiva, #290551021). A running buffer consisting of 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM DTT, and 10% Glycerol was used. The fraction containing the target protein was recovered and concentrated with Amicon Ultra-0.5 10K (MERCK, #UFC501096) to obtain the target protein FKBP-TrisNTA. The prepared samples were stored at -80°C.

[0234] <Preparation Example 1-4: Preparation of FKBP-FLAG-TEV-Avi-bio> FKBP expressed and purified in E. coli was used as the target protein for the study. FKBP-FLAG-TEV-Avi-bio was prepared by adding a purification tag, a TEV protease cleavage tag, and a biotinylation enzyme recognition tag to the C-terminus of the FKBP. ＊ " represents biotinylated Lys.

[0235] Amino acid sequence of FKBP-FLAG-TEV-Avi-bio (SEQ ID NO: 4) MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATTHGPGIIPPPHATLVFDVELLKLEGGGGSSDYKDDDDKSSGENLYFQSSGGGLNDIFEAQK ＊ IEWHE

[0236] Specifically, an expression vector prepared by cloning the FKBP-FLAG-TEV-Avi-bio and biotin ligase (BirA) genes into pCDFDuet-1 was used to transform E. coli BL21 (DE3) and create an expression strain. ６００ The cells were cultured in LB medium until the concentration was approximately 0.4 to 0.6. Induction was then carried out by adding 1 mM IPTG and 100 μM biotin to a final concentration, and the cells were cultured at 37°C for 3 hours. After that, the sample was centrifuged to collect the cultured cells.

[0237] The recovered cultured cells were suspended in a dispersion buffer (50 mM Tris-HCl pH 7.0, 200 mM NaCl, 2 mM DTT, 0.1% CHAPS, 0.1 mg / mL lysozyme, 1 / 1000 (v / v) Benzonase, Protein inhibitor cocktail), stirred at room temperature for 20 minutes, and then sonicated. This sample was centrifuged at 15000 × g for 30 minutes at 4°C, and the supernatant was collected. This supernatant was then centrifuged again at 15000 × g for 15 minutes at 4°C, and passed through a 0.22 μm filter to obtain the lysate sample.

[0238] Lysate samples were purified using 5 mL of Streptactin XT Superflow HC (IBA, #2-4030-010) resin and Econo-Pac Chromatography Columns (bio-rad, #7321010). Bending buffer (50 mM Tris-HCl pH 7.5, 200 mM NaCl, 1 mM TCEP) and Elution buffer (50 mM Tris-HCl pH 7.5, 200 mM NaCl, 1 mM TCEP, 50 mM biotin) were used for purification. The fraction containing the target protein was recovered and concentrated with Amicon Ultra-15 10K (MERCK, #UFC901024).

[0239] The concentrated sample was purified by gel filtration using HiLoad 26 / 600 Superdex 75 pg (Cytiva, #28-9893-34). A running buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM TCEP) was used for purification. The fraction containing the target protein was recovered, concentrated with Amicon Ultra-15 10K (MERCK, #UFC901024), and then filtered through a 0.22 μm filter to obtain the target protein FKBP-FLAG-TEV-Avi-bio. The sample was stored at -80°C.

[0240] <Preparation Example 1-5: Preparation of FRB-FLAG-TEV-Avi-bio> FRB expressed and purified in E. coli was used as the target protein for the study. FRB-FLAG-TEV-Avi-bio was prepared by adding a purification tag, a TEV protease cleavage tag, and a biotinylation enzyme recognition tag to the C-terminus of the FRB. ＊ " represents biotinylated Lys.

[0241] Amino acid sequence of FRB-FLAG-TEV-Avi-bio (SEQ ID NO: 5) MELIRVAILWHEMWHEGLEEASRLYFGERNNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKQGGGGSSDYKDDDDKSSGENLYFQSSGGGLNDIFEAQK ＊ IEWHE

[0242] Specifically, an expression vector prepared by cloning the genes for FRB-FLAG-TEV-Avi-bio and biotin ligase (BirA) into pCDFDuet-1 was used to transform E. coli BL21 (DE3) and create an expression strain. OD ６００ The cultures were incubated in LB medium until the concentration was approximately 0.4 to 0.6. Induction was then carried out by adding 1 mM IPTG and 100 μM biotin to a final concentration, and the cultures were incubated at 18°C ​​for 18 hours. After that, the samples were centrifuged to collect the cultured cells.

[0243] The recovered cultured cells were suspended in a dispersion buffer (50 mM Tris-HCl pH 7.0, 200 mM NaCl, 2 mM DTT, 0.1% CHAPS, 0.1 mg / mL lysozyme, 1 / 1000 (v / v) Benzonase, Protein inhibitor cocktail), stirred at room temperature for 20 minutes, and then sonicated. This sample was centrifuged at 15000 × g for 30 minutes at 4°C, and the supernatant was collected. The supernatant was then passed through a 0.22 μm filter to obtain the lysate sample.

[0244] Lysate samples were purified using 5 mL of Streptactin XT Superflow HC (IBA, #2-4030-010) resin and Econo-Pac Chromatography Columns (bio-rad, #7321010). Bending buffer (50 mM Tris-HCl pH 7.5, 200 mM NaCl, 1 mM TCEP) and Elution buffer (50 mM Tris-HCl pH 7.5, 200 mM NaCl, 1 mM TCEP, 50 mM biotin) were used for purification. The fraction containing the target protein was recovered and concentrated with Amicon Ultra-15 10K (MERCK, #UFC901024).

[0245] The concentrated sample was purified by gel filtration using HiLoad 26 / 600 Superdex 75 pg (Cytiva, #28-9893-34). A running buffer (50 mM Tris-HCl pH 7.5, 300 mM NaCl, 1 mM TCEP) was used for purification. The fraction containing the target protein was recovered, concentrated with Amicon Ultra-15 10K (MERCK, #UFC901024), and then filtered through a 0.22 μm filter to obtain the target protein FRB-FLAG-TEV-Avi-bio. The sample was stored at -80°C.

[0246] <Preparation Example 1-6: Preparation of bio-Avi-TEV-FLAG-FKBP-FRB-His> As the target protein to be used for the study, a fusion protein in which FKBP and FRB were linked with a linker was expressed and purified in E. coli and used as the target protein for panning. A purified tag, a TEV protease cleavage tag, and a biotinylated enzyme recognition tag were added to the N-terminus of this fusion protein, and a purified tag was added to the C-terminus to prepare bio-Avi-TEV-FLAG-FKBP-FRB-His. ＊ " represents biotinylated Lys.

[0247] The amino acid sequence of bio-Avi-TEV-FLAG-FKBP-FRB-His (SEQ ID NO: 6) is MGLNDIFEAQK ＊IEWHESSGENLYFQSGGGSSDYKDDDDKSGGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFD SSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL KLEGSGSSGLEVLFQGPSSGSSSGTELIRVAILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAM MERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKQGGGSSHHHHHHH

[0248] Specifically, an expression vector was prepared by cloning the gene sequences of bio-Avi-TEV-FLAG-FKBP-FRB-His and biotin ligase (BirA) into pCDFDuet-1, and an expression strain was created by transforming E. coli BL21 (DE3). OD ６００ The cultures were incubated in LB medium until the pH was approximately 0.4 to 0.6, then induction was induced by adding 1 mM IPTG and 100 μM biotin, followed by incubation at 18°C ​​for 18 hours. After incubation, the samples were centrifuged to collect the cultured cells.

[0249] The recovered cultured cells were suspended in a disruption buffer (50 mM Tris-HCl pH 7.0, 200 mM NaCl, 2 mM DTT, 0.1% CHAPS, 0.1 mg / mL lysozyme, 1 / 1000 (v / v) Benzonase, Protein inhibitor cocktail), stirred at room temperature for 20 minutes, and then sonicated. This sample was centrifuged at 15000 × g for 30 minutes at 4°C, and the supernatant was collected. The supernatant was further centrifuged at 15000 × g for 15 minutes at 4°C, and then passed through a 0.22 μm filter to obtain the lysate sample.

[0250] Lysate samples were purified using 5 mL of Streptactin XT Superflow HC (IBA, #2-4030-010) resin and Econo-Pac Chromatography Columns (bio-rad, #7321010). Bending buffer (50 mM Tris-HCl pH 7.5, 200 mM NaCl, 1 mM TCEP) and Elution buffer (50 mM Tris-HCl pH 7.5, 200 mM NaCl, 1 mM TCEP, 50 mM biotin) were used for purification. The fraction containing the target protein was recovered and concentrated with Amicon Ultra-15 10K (MERCK, #UFC901024).

[0251] The concentrated sample was purified by gel filtration using HiLoad 26 / 600 Superdex 75 pg (Cytiva, #28-9893-34). A running buffer (50 mM Tris-HCl pH 7.5, 300 mM NaCl, 1 mM TCEP) was used for purification. The fraction containing the target protein was recovered, concentrated on Amicon Ultra-15 10K (MERCK, #UFC901024), and then filtered through a 0.22 μm filter to obtain the target protein bio-Avi-TEV-FLAG-FKBP-FRB-His. The sample was stored at -80°C.

[0252] [Synthesis Example 2: Synthesis of Peptides Used in the Study] Four types of peptides, FF-01, FF-02, FK-01, and FK-02, were synthesized for use in the study.

[0253] The analytical conditions for LC-MS are as follows:

[0254] All starting materials and reagents were obtained from commercial suppliers or synthesized using known methods.

[0255] [Synthesis Example 2-1: Solid-Phase Synthesis of Peptides] The peptide was extended and the compound synthesized according to the Fmoc method described in WO2013 / 100132, WO2018 / 225864, and WO2022 / 234852. The process consists of five steps: 1) Peptide extension reaction from the N-terminus of the amino acid using the Fmoc method with the carboxylic acid of the Asp side chain supported on 2-chlorotrityllase resin; 2) Process of cleaving the peptide from the 2-chlorotrityllase resin; 3) Amide cyclization by condensation of the carboxylic acid of the Asp side chain detached from the 2-chlorotrityllase resin with the amino group at the N-terminus of the peptide chain; 4) Deprotection of protecting groups of side-chain functional groups contained in the peptide chain as needed; and 5) Purification of the compound by preparative HPLC. In this example, unless otherwise specified, the peptide was synthesized based on this synthesis method.

[0256] Cyclic peptides were synthesized according to the above synthesis conditions. Table 5 shows the amino acid sequences from the N-terminus in columns 1 to 11, and lists the exact mass (exact mass), LC-MS analysis conditions, observed mass spectrum (obs. mass; m / z), ionic form, and retention time (min) for each compound. All peptides are cyclic peptides in which an amide bond is formed between the N-terminal amino group and the C-terminal Asp side chain carboxyl group. The LC-MS analysis conditions are described in Table 4.

[0257]

[0258] Table 6 shows the structural formulas of the synthesized compounds FF-01, FF-02, FK-01, and FK-02.

[0259]

[0260] [Evaluation Example 1: Evaluation of Synthetic Peptides Using Surface Plasmon Resonance (SPR)] [Evaluation Example 1-1: Evaluation of FF-01 and FF-02] SPR was used to evaluate whether FF-01 and FF-02, synthesized in Synthesis Example 2-1, could form complexes with FKBP and FRB. Measurements were performed at 20°C using a BiacoreT200 (Cytiva).

[0261] Specifically, Biotin CAPture reagent was immobilized on all flow cells on the Via Core Sensor Chip Series S sensor chip CAP (Cytiva), and then FRB-FLAG-TEV-Avi-Bio was immobilized on some of the flow cells. As an analyte, a mixed solution of FKBP-FLAG-Sor-His in a dilution series with a maximum concentration of 10 μM and a 10 μM synthetic peptide was prepared and added to all flow cells to obtain binding sensorgrams. As a running buffer, a solution of HBS (Nacalai Tesque), 1 mM Dithiothreitol, 0.01% Tween 20, to which Dimethylsulfoxide (DMSO) was added to a final concentration of 4% was used.

[0262] The obtained sensorgrams were analyzed using T200 evaluation software (Cytiva). Sensorgrams obtained in a flow cell without immobilization of FRB-FLAG-TEV-Avi-Bio, and sensorgrams obtained with the addition of Running Buffer, were used as double references and subtracted, followed by DMSO solvent correction. The binding rate constant (ka), dissociation rate constant (kd), and dissociation constant KD were determined by curve fitting using a 1:1 binding model on the sensorgrams processed as described above. For some peptides, KD was determined by equilibrium value analysis. The KD obtained above was considered as the apparent dissociation constant of FRB and FKBP binding in the presence of 10 μM peptide, and was thought to be an indicator of the peptide inducing complex formation with the two proteins. No binding response was observed when a 10 μM FKBP-FLAG-Sor-His solution was added as an analyte.

[0263] The obtained K Ｄ The following is shown.

[0264] The above results confirmed that FF-01 and FF-02 induce the formation of a complex between FKBP and FRB.

[0265] [Evaluation Example 1-2: Evaluation of FK-01 and FK-02] SPR was used to verify that FK-01 and FK-02 synthesized in Synthesis Example 2-1 can bind to FKBP, but cannot induce a complex of FKBP and FRB.

[0266] Verification was performed using SPR measurement with the Biocore T200. Specifically, after immobilizing Biotin CAPture reagent on all flow cells on the Biocore sensor chip Series S sensor chip CAP (Cytiva), approximately 560 RU of FKBP-FLAG-TEV-Avi-bio was immobilized on flow cell (Fc)-2, and 290 RU and 550 RU of FRB-FLAG-TEV-Avi-bio were immobilized on Fc3 and Fc4, respectively. To confirm the binding affinity of synthetic peptides FK-01 and FK-02 to FKBP and FRB, diluted series of synthetic peptides FK-01 and FK-02 were prepared as analytes with a maximum concentration of 10 μM. These were added to all flow cells, and sensograms of binding to FKBP and FRB were obtained. As a control, a diluted series of Rapamycin solutions with a maximum concentration of 1 μM was prepared, and binding was evaluated. On the other hand, to evaluate the ability to induce complex formation between FKBP and FRB, a mixed solution of diluted series of FKBP-FLAG-Sor-His with a maximum concentration of 10 μM and 10 μM of synthetic peptides FK-01 and FK-02 was prepared, added to all flow cells, and sensograms of binding were obtained. As a control, a mixed solution of FKBP-FLAG-Sor-His in a dilution series with a maximum concentration of 1 μM and 1 μM Rapamycin was prepared and added to all flow cells to obtain binding sensorgrams. As a running buffer, a buffer was used consisting of HBS (10 mM HEPES-NaOH, 150 mM NaCl, pH 7.4) (Nacalai Tesque), 1 mM Dithiothreitol, and 0.01% Tween 20, to which Dimethylsulfoxide (DMSO) was added to achieve a final concentration of 4%.

[0267] The obtained sensorgrams were analyzed using T200 evaluation software (Cytiva). To confirm the binding affinity of the synthetic peptide to FKBP and FRB, the sensorgrams obtained from Fc1 (where FKBP and FRB were not immobilized) and the sensorgram obtained with the addition of Running Buffer were used as double references to the sensorgrams of the synthetic peptide and Rapamycin binding obtained from Fc2, 3, and 4 (where FKBP-FLAG-TEV-Avi-bio and FRB-FLAG-TEV-Avi-bio were immobilized), and DMSO solvent correction was applied. If the sensorgrams after the above treatment showed a clear response, the binding rate constant (ka), dissociation rate constant (kd), and dissociation constant KD were determined by curve fitting using a 1:1 binding model. To evaluate the ability to induce complex formation between FKBP and FRB, sensorgrams obtained from Fc3 and Fc4, where FRB-FLAG-TEV-Avi-bio was immobilized, and sensorgrams obtained when running buffer was added were subtracted as double references from sensorgrams obtained from Fc1, where FKBP and FRB were not immobilized, and from sensorgrams obtained when running buffer was added. Finally, DMSO solvent correction was applied to obtain the final sensorgram.

[0268] Based on the evaluation of the binding affinity of synthetic peptides FK-01 and FK-02 to FKBP and FRB, the dissociation constants of the synthetic peptides' binding to FKBP were determined as follows. None of the analytes showed a binding response to FRB.

[0269] The following sensor gram was obtained by evaluating the ability of FKBP and FRB to induce complex formation.

[0270] While a mixture of FKBP and Rapaycin showed a binding response to FRB, mixtures of FK-01 and FK-02 with FKBP did not show a binding response.

[0271] Based on the above results, SPR confirmed that FK-01 and FK-02 can bind to FKBP, but do not induce complex formation between FKBP and FRB.

[0272] [Evaluation Example 2: Efficacy Verification by Clone Assay] In a system using target proteins brought into close proximity by the non-covalent interaction of Ni-NTA and His tag, we investigated whether mRNA display molecules of peptides that can form complexes with multiple targets can be recovered more efficiently than mRNA display molecules of peptides that can bind to only a single target, using FKBP and FRB as target proteins. In this study, FF-01 and FF-02 were used as peptides that can form complexes with FKBP and FRB, and FK-01 and FK-02 were used as peptides that can bind to FKB but cannot form a complex including FRB.

[0273] [Preparation Example 2-1: mRNA Preparation (FF-01, FF-02, FK-01, FK-02)] mRNA ((mR-1) to (mR-4)) was synthesized from template DNA ((D-1) to (D-4)) by in vitro transcription reaction using T7 RNA polymerase, and purified using RNAClean XP (BECKMAN COULTER).

[0274] Template DNA (D-1) base sequence (SEQ ID NO: 7) (DNA sequence: FF-01) GTAATACGACTCACTATAGGGTTTAATAAGGAGAATATAAATAGCATTCCGGGGTGAGTGCCATACTAGTTCCTTAAGCCCGACCGCGCACCGCGCACCGCAAAAAAAAA

[0275] Template DNA (D-2) base sequence (SEQ ID NO: 8) (DNA sequence: FF-02) GTAATACGACTCACTATAGGGTTTAAACTTTTAATAAGGAGAATATAAATATGTTTTTCGGGGAGTGCACTAACCACAGCCGTTGTTAGCCCGCACCGCGCACCGCGGCAAAAAAAAAA

[0276] Base sequence of template DNA (D-3) (Sequence ID 9) (DNA sequence: FK-01) GTAATACGACTCACTATAGGGTTTAAACTTTTAATAAGGAGAATATAAATGTTTGAGTTCGCTTTATTACTCCCGATGTGCTAGCCCGCACCGCGCACCGCGCAAAAAAAA

[0277] Template DNA (D-4) base sequence (SEQ ID NO: 10) (DNA sequence: FK-02) GTAATACGACTCACTATAGGGTTTAAACTTTTAATAAGGAGAATATAAATAGGGGTCATCGGGAACTTGCTGGAACCCCGAACTAGCCGACCGCGCACCGCGCACCGGCAAAAAAAAAA

[0278] mRNA (mR-1) base sequence (SEQ ID NO: 11) (RNA sequence: FF-01) GGGUUAACUUUAAUAAGGAGAAUAUAAUAUAUGCAAUUCGGGUGAGUGCCAAUACUAGUUCUAGCCGACCGGCACCGGCACCGGCAAAAAAAA

[0279] mRNA (mR-2) base sequence (SEQ ID NO: 12) (RNA sequence: FF-02) GGGUUAACUUUAAUAAGGAGAAUAUAAUAUAUUCGGGAGUGCCACUAACCAGCCGUUGUAGCCAGCCACCGGGCACCGGGCACCGGGCAAAAAAAAA

[0280] mRNA (mR-3) base sequence (SEQ ID NO: 13) (RNA sequence: FK-01) GGGUUAACUUUAAUAAGGAGAAUAUAAUAUAUUGUGAGUUCCUUAUAUACUCCGAUUGUCUAGCCAGACCGGCACCGGCACCGGCAAAAAAAA

[0281] mRNA (mR-4) base sequence (SEQ ID NO: 14) (RNA sequence: FK-02) GGGUUAACUUUAAUAAGGAGAAUAUAAUAUUGGUCAUCGGGAACUUGCUGGGAACCCCGAACUAGCCGCACCGGCACCGGCACCGGCAAAAAAAAA

[0282] [Preparation Example 2-2: Preparation of mRNA-peptide display molecules (FF-01, FF-02, FK-01, FK-02)] <Preparation Example 2-2-1: Preparation of acylated tRNA> Acylated tRNA for translation was prepared according to the methods described in WO2018 / 143145 and WO2018 / 225864. A mixture of elongator aminoacylated tRNAs was prepared using 19 amino acids consisting of Pro(4-pip-4-F2), Asp(SMe), Hph(3-Cl), cisPro(4-pip-4-F2), MeHnl(7-F2), Ser(3-F-5-Me-Pyr), MeSer(nPr), MeAla(3-Pyr), Phe(3-Cl), Pic(2), MeGly, Nle, MeSer(tBuOH), Ser(NtBu-Aca), Ser(iPen), nBuGly, MeHph, Ser(Ph-2-Cl), and D-MeSer. The final concentrations of each acylated tRNA in the translation solution ranged from 10 μM to 20 μM. The F-Pnaz-protected pCpA amino acids were processed after phenol extraction without deprotection. The initiator aminoacylated tRNA was the same compound as compound AAtR-3 (Acbz-MeCys(StBu)-tRNAfMetCAU) described in WO2017 / 150732, and was added to the translation solution to a final concentration of 25 μM.

[0283] <Preparation Example 2-2-2: Translation of mRNA-peptide display molecules (FF-01, FF-02, FK-01, FK-02)> The translation system used was the PURE system, a reconstituted cell-free protein synthesis system derived from prokaryotes.

[0284] Specific translation systems include 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, and 1 mg / mL E. tRNA derived from coli MRE600 (RNase-negative) (Roche Co., Ltd.) (Non-patent literature: Some tRNA was removed according to the method described in Yokogawa T et al. 2010), 3 μM in vitro transcription of E. coli tRNA Ala1B, 4 μg / mL creatine kinase, 6.72 U / mL myokinase, 2 units / mL inorganic pyrophosphatase, 1.1 μg / mL nucleoside diphosphate kinase, 0.4 Units / μL RNasin (registered trademark) Plus Ribonuclease Inhibitor (Promega), 0.26 μM EF-G, 2.7 μM IF1, 0.4 μM IF2, 1.5 μM IF3, 40 μM EF-Tu, 52 μM EF-Ts, 1 μM EF-P-Lys, 1.2 μM modified ribosome (WO2021 / 117848), 1.37 μM AlaRS, 1 μM GlyRS, 0.04 μM IleRS, 0.11 μM LysRS, 0.16 μM ProRS, 0.09 μM ThrRS, 0.5 μM mutant PheRS (WO2016 / 14804) 4) 1 μM mutant SerRS (WO2016 / 148044), 1 μM mutant ValRS (WO2016 / 148044), 250 μM glycine, 10 μM isoleucine, 250 μM lysine, 250 μM proline, 250 μM threonine, 2.5 mM N-methylalanine, 5 mM N-methylvaline, 5 mM N-methylserine, 5 mM N-methylphenylalanine, Elongator aminoacylated tRNA mixture prepared in Preparation Example 2-2-1, 25 μM prepared in Preparation Example 2-2-1 Initiator aminoacylated tRNA (WO2017 / 150732), 0.5 μM Penicillin G Amidase (PGA), and mRNA-puromycin conjugates prepared according to WO2013 / 100132 from mRNA prepared in Preparation Example 2-1 were added to the translation reaction mixture to a concentration of 1 μM, and translation was carried out by standing at 37°C for 1 hour.As the puromycin linker, axPM-27, as described in non-patent literature (J. Am. Chem. Soc., 2023, 145, pp. 24035-24051), was used. For FF-02 only, the translation system was modified to contain 7 mM magnesium acetate, 30 μM EF-Tu, and 40 μM EF-Ts before translation was performed.

[0285] The translated samples were subjected to cyclization, desulfurization, and reverse transcription according to the method described in WO2017 / 150732 to prepare mRNA-peptide display molecules.

[0286] [Evaluation Example 2-1: Pull-down assay of mRNA-peptide display molecules (FF-01, FF-02, FK-01, FK-02)] FKBP-Tris-NiNTA was prepared as the protein to be used for evaluation. Specifically, FKBP-TrisNTA prepared in Preparation Example 1-3 and NiCl ２ The aqueous solutions were mixed to final concentrations of 26 μM and 0.5 mM, respectively. The mixture was allowed to stand at 4°C for at least 30 minutes to obtain FKBP-Tris-NiNTA.

[0287] The FKBP-Tris-NiNTA prepared above and the bio-Avi-TEV-FRB-FLAG-Sor-His prepared in Preparation Example 1-1 were mixed in a buffer containing 1x TBS, 2 mg / mL BSA (Invitrogen), and 2 mM DTT to achieve final concentrations of 20 μM and 4 μM, respectively, and allowed to stand at 4°C for at least 30 minutes.

[0288] To a buffer containing 1×TBS, 2 mg / mL BSA (Invitrogen), and 2 mM DTT, 0.2 μM of mRNA-peptide display molecule prepared in Preparation Example 2-2 and the target protein were added to the concentrations listed in Table 10, and the mixture was allowed to stand at 4°C for 1 hour. Then, streptavidin-coated magnetic beads were mixed and inverted at 4°C for 10 minutes to immobilize the target protein. Biotin was then added to a total concentration of 0.08 mM and inverted at 4°C for another 10 minutes.

[0289]

[0290] Condition 1 used a fusion protein in which FKBP and FRB were linked by a linker. Under this condition, both FKBP and FRB were pulled down by beads. In conditions 2 to 8, biotinylated FRB was pulled down, and FKBP was also partially pulled down via the interaction of Ni-NTA and His tag.

[0291] Streptoavidin-coated magnetic beads immobilized with target proteins were collected using a magnet, and the supernatant was removed. These beads were then washed several times with the wash buffer shown in Table 11.

[0292] Under conditions 2 to 8, the concentration of imidazole in the wash buffer differs. It is expected that the addition of imidazole will attenuate the interaction between Ni-NTA and His tags in the wash.

[0293] After washing, the beads were treated with a TEV protease that recognizes and cleaves the TEV protease recognition sequence, and the target protein and the peptide-nucleic acid conjugate bound to it were eluted.

[0294] As a specific TEV eluate, 1× TEV buffer, 1 mM DTT, and 0.1 U / μL AcTEV protease (Thermo Fisher Scientific, #12575015) were added to washed streptavidin-coated magnetic beads and reacted. After the reaction, the supernatant was collected.

[0295] qPCR was performed using a portion of the TEV eluted products, and the recovery rate of each mRNA-peptide display molecule was evaluated. The primers used for qPCR were the primers described below (Fw-primer and Rv-primer). The qPCR reaction mixture consisted of 1× 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, #50513), and 0.025 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.

[0296] Fw-primer base sequence (SEQ ID NO: 15) GTAAATACGAACTCACTATAGGGGTTAACTTTAATAAGGAG

[0297] The nucleotide sequence of the RV-primer (SEQ ID NO: 16) is TTTTTTTTGCCGGTGCCGGTGCCGTCGGCTA

[0298] To construct a calibration curve for estimating library recovery rates, samples prepared by reverse transcription of mRNA-puromycin conjugates (prepared according to WO2022 / 138892) from a randomized double-stranded DNA library were diluted to 1E+8 / μL, 1E+6 / μL, and 1E+4 / μL, and subjected to qPCR under similar conditions.

[0299] The recovery rate was calculated by dividing the amount of nucleic acid of each display molecule recovered by binding to the target protein by the amount of nucleic acid of each display molecule present before mixing with the target protein (input value).

[0300] Recovery rate (%) = Amount of nucleic acid recovered (number of molecules) / Amount of nucleic acid (number of molecules) in input × 100

[0301] The recovery rates for each display molecule, FF-01, FF-02, FK-01, and FK-02, and the ratios obtained by dividing the recovery rate under condition 1 by the respective recovery rates under conditions 2 to 8, are summarized in Tables 12 to 15.

[0302]

[0303]

[0304]

[0305]

[0306] Comparing the results of Condition 1 and Condition 2, it was found that the recovery rates of FF-01 and FF-02, which can form complexes with FKBP and FRB, were about the same, while the recovery rates of FK-01 and FK-02, which can bind to FKB but cannot form complexes with FKB and FRB, were more than 10 times lower.

[0307] Furthermore, comparing conditions 1 and 5, in this experiment, adding 20 mM Imidazole to the Wash buffer resulted in a recovery rate of FF-01 and FF-02 decreasing by no more than 10 times compared to condition 1, while the recovery rate of FK-01 and FK-02 decreased by more than 100 times. This suggests that by adding substances that attenuate the interaction between Ni-NTA and His tags, such as Imidazole, to the Wash buffer at an appropriate concentration in the system, peptides that can form complexes with multiple targets can be recovered more selectively.

[0308] [Reference Example 1] To perform a pull-down assay of mRNA-peptide display molecules with higher throughput, as described herein, a library of candidate molecule-nucleic acid complexes with two or more diverse components may be used. In this case, the recovery rate of each sequence in the library is determined by analyzing the base sequences obtained after the amplification step. Normally, the contact step and amplification step are completed in one step, but in some cases, all or part of each step may be repeated multiple times.

[0309] By trying multiple pull-down conditions in parallel, the recovery rate of each sequence under each pull-down condition can be compared, as shown in evaluation example 2-1, and sequences exhibiting the desired properties can be selected as needed.

[0310] As one example, a Mini Library Assay (MLA) is performed. This assay is essentially a high-throughput evaluation method for efficiently identifying or selecting candidate molecules with desired properties, comprising the following steps (A) to (C): (A) preparing a deliberately constructed mini-library of candidate molecule-nucleic acid complexes (mini-library) with relatively small diversity; (B) subjecting the mini-library to a selection system containing target molecules under several different conditions; and (C) quantifying the amount of each candidate molecule recovered under each of the several conditions by analyzing the nucleic acid sequence bound to it, and identifying or selecting candidate molecules with desired properties by comparing the recovery rates under each condition. In step (A) above, "mini-library" refers to two or more, preferably 1 × 10 to 1 × 10 ８ degree, for example, 1 x 10 ３ ~1 x 10 ７This refers to a library with a relatively small degree of diversity. This minilibrary is characterized in that it is intentionally constructed by one of the following methods: (i) A method of intentionally selecting two or more candidate molecules that may have desired properties from a group of candidate molecules (peptides or proteins) enriched by performing one or more rounds of the screening method (panning) according to the present invention, and reconstructing them based on the nucleic acids that encode them. This method enables comparative evaluation to identify slight differences in properties (e.g., binding strength, specificity) from a group of promising candidate molecules and to select the best molecule. (ii) A method of creating one or more child sequences by substituting one or more amino acid residues of a specific parent sequence with other amino acid residues using site-directed mutagenesis or the like, and then selecting the parent sequence and / or child sequences to construct the library. This is useful for so-called affinity maturation and functional analysis of specific residues. The "multiple different conditions" in step (B) above means changing the parameters of any step in the screening method according to the present invention. For example, in the cleavage step described above, conditions such as adding competing substances at different concentrations, as shown in Evaluation Example 2-1, or in the contact step, conditions such as changing the concentration of the first target molecule, the concentration of the second target molecule, pH, salt concentration, temperature, etc., can be used. By "comparing the recovery rates" in step (C) above, it becomes possible to select, for example, a candidate molecule that shows a high recovery rate in the presence of a specific competing substance as a "molecule with high specificity," or to select a "molecule with a desired binding profile" by comparing the recovery rate profiles for multiple target molecules. Therefore, for example, (I) a library with limited diversity (especially multiple sequences selected from panning concentrates, or a site-directed mutagenesis library) can be used, (II) selection can be performed under multiple conditions, and (III) candidate molecules can be evaluated or selected by quantitatively comparing the recovery rates under each condition.

Claims

1. A method for screening target molecules that form complexes with multiple types of target molecules, comprising: (1) a step of preparing a conjugate by linking a first target molecule and a second target molecule from among the multiple types of target molecules via a connecting portion (linking step); (2) a step of contacting a candidate molecule or a candidate molecule-nucleic acid conjugate with the conjugate to form the complex (contact step); and (3) a step of cutting the connecting portion after step (2) (cutting step).

2. A method for reducing the recovery rate of non-target molecules that bind only to a second target molecule in order to screen for target molecules that form complexes with multiple types of target molecules, comprising: (1) a step of preparing a conjugate by linking a first target molecule and a second target molecule from among the multiple types of target molecules via a linking portion (linking step); (2) a step of contacting a candidate molecule or a candidate molecule-nucleic acid conjugate with the conjugate to form the complex (contact step); and (3) a step of cutting the linking portion after step (2) (cutting step).

3. (4) The method according to claim 1 or 2, further comprising a step of recovering the composite after step (3) (recovery step).

4. The method according to any one of claims 1 to 3, wherein in the linked body, the first target molecule and the second target molecule are directly linked via the linker Y which is the linking portion.

5. The method according to claim 4, wherein the conjugate is immobilized on a solid support before contact with the candidate molecule or candidate molecule-nucleic acid conjugate in step (2), after step (2) and before step (3), or after step (3) and before step (4).

6. The method according to any one of claims 1 to 3, wherein in the linked body, the first target molecule and the second target molecule are linked via the linking portion, which is linker X - solid support - linker Z, the linker X is a linker that links the first target molecule and the solid support, and the linker Z is a linker that links the second target molecule and the solid support.

7. The method according to claim 5 or 6, wherein the solid support and the first target molecule are linked by a linker X, and the linker Y is a different type of linker than the linker X.

8. The method according to claim 6 or 7, wherein the linker Z is a different type of linker than the linker X.

9. The method according to any one of claims 4 to 8, wherein the linker Y and / or the linker Z is a linker that can be cut by washing, a linker that can be cut by competitive elution, a linker that can be cut using an enzyme, a linker that can be cut using light, or a linker that can be cut by pH.

10. The method according to any one of claims 4 to 9, wherein the linker Y and / or the linker Z, as a connecting portion, includes a structure in which any of the metals nickel, cobalt, copper, and iron is immobilized using nitrilotriacetic acid (NTA) or iminodiacetic acid (IDA).

11. The method according to any one of claims 4 to 10, wherein the linker Y and / or the linker Z includes a His tag consisting of His (histidine) in sequence, an HN tag consisting of His (histidine) and Asn (asparagine) in sequence, or a HAT (Histidine Affinity Tag).

12. The method according to any one of claims 4 to 10, wherein the linker Y and / or the linker Z includes the combination of one selected from the group consisting of His (histidine) tags, HN tags consisting of His (histidine) and Asn (asparagine) tags, and HAT (Histidine Affinity Tag), and one selected from the group consisting of Ni-NTA, Ni-IDA, Co-NTA, and Co-IDA.

13. (5) The method according to any one of claims 1 to 12, further comprising a step of eluting the nucleic acid in the candidate molecule-nucleic acid conjugate recovered by the recovery step (elution step).

14. (6) The method according to any one of claims 1 to 13, further comprising the step of amplifying the nucleic acid in the candidate molecule-nucleic acid conjugate that forms the complex (amplification step).

15. (7) The method according to any one of claims 1 to 14, further comprising the step of identifying a target molecule from the candidate molecule-nucleic acid conjugate that forms the complex (identification step).

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