Evaluation Method for DNA-Encoded Libraries

Abstract

This invention provides a method for inducing a DNA-coding library (DEL) containing cleavable sites within the DNA strand into a crosslinker-modified double-stranded DEL and evaluating its effectiveness. [Solution] The present invention combines the advantages of both hairpin strand DEL and double-strand DEL by introducing a cleavable site, such as deoxyuridine, into the DNA strand. Furthermore, by easily inducing crosslinker-modified DEL, the present invention provides a compound screening technology that combines a "simple DEL synthesis method" and "expansion and improvement of DEL evaluation methods."

Description

Technical Field

[0001] The present invention relates to a method for evaluating a DNA-encoded library.

Background Art

[0002] A compound library is a group of compound derivatives systematically collected with compounds having a specific activity such as drug candidate compounds. This compound library is often synthesized based on the synthetic techniques and methodologies of combinatorial chemistry. Combinatorial chemistry is an experimental technique and a research field related thereto for efficiently synthesizing a variety of compounds in a systematic synthetic route from a series of compound libraries enumerated and designed based on combinatorial theory. As one type of compound library based on combinatorial chemistry, there is a DNA-encoded library. Hereinafter, the DNA-encoded library is appropriately abbreviated as DEL. In DEL, a DNA tag is added to each library-compounded compound. The DNA tag is designed in terms of sequence so that each structure of each compound can be identified and functions as a label for the compound (Patent Documents 1 to 3).

[0003] So far, a plurality of powerful compounds have been found in drug development by screening using DEL. Screening using DEL is carried out, for example, as follows (Non-Patent Documents 1 to 3). 1) Immobilize the target protein on an immobilization carrier. 2) Contact the target with DEL. 3) Wash and remove DEL having low affinity with the target. 4) Denature the target protein and elute DEL having high affinity. 5) Amplify the DNA sequence contained in the eluted DEL and identify the sequence. However, the screening methods described above have several drawbacks. First, because the target protein needs to be immobilized, the three-dimensional structure of some proteins may change after the immobilization process. In such cases, the compounds obtained through screening will not bind to the unimmobilized target protein, limiting the target applicability range of DEL (Patent Documents 4 and 5, Non-Patent Documents 4 and 5). Furthermore, while the above screening methods can recover binders with high affinity, binders with moderate affinity (for example, those with Kd values ​​on the order of μM) have low kinetic stability when complexed with the target protein and are difficult to recover after washing. As is well known to those skilled in the art, such binders with moderate affinity are also useful as hit compounds that can serve as the starting point for drug discovery, and are also valuable as structure-activity relationship information (Non-Patent Document 6).

[0004] In an effort to address the above-mentioned challenges, several screening methods using crosslinker-modified DELs have recently been reported. As will be shown later, several different methods have been reported, but they all share the commonality of reacting a target adjacent to an affinity library molecule with a crosslinker to form a covalent bond. This method is useful because it allows for DEL screening without (or before) immobilizing the target protein (Patent Documents 4 and 5, Non-Patent Documents 4 and 5, 7 to 9).

[0005] Let's briefly explain the DNA strand structure of DEL. The two most common known DNA strand structures of DEL are the hairpin strand and the double-stranded strand. The following outlines the basics, advantages, and disadvantages of double-stranded DEL and hairpin-stranded DEL. (1) Hairpin chain DEL DEL using hairpin strand DNA is a single-stranded structure in which two complementary DNA strands are linked together, and is synthesized using hairpin-type DNA having functional groups for introducing various building blocks as a starting material (headpiece) (Patent Document 3, Non-Patent Documents 1 and 2). (A) Strengths (a) Short DNA tags can be used. In this method, relatively short double-stranded DNA tags of about 9-13 mers with 2 mer sticky ends are often used, and these double-stranded DNA tags are introduced by a ligation reaction using DNA ligase. The use of such short DNA tags is possible because hairpin strand DNA forms a strong double helix within the molecule, and DNA regions other than the sticky ends do not interfere with the DNA tag. The use of short double-stranded DNA tags has several advantages in DEL synthesis. One advantage is that the cost of synthesizing the DNA tag is low. Another advantage is that using shorter DNA tags allows for a shorter overall DEL length when encoding the same number of reaction cycles. That is, even when encoding a larger number of cycles, the overall length of the DEL can be kept within a range that allows for efficient DNA sequence reading by next-generation sequencers. In fact, Non-Patent Literature 3 demonstrates the construction of a DEL using hairpin strand DNA that encodes as many as 6 reaction cycles. (b) High chemical stability Unlike double-stranded DNA, in hairpin strands, even if the double-stranded structure melts during a heating reaction, the double helix is ​​reformed within the original molecule under subsequent re-annealing conditions without strand exchange. Therefore, DEL using hairpin strand DNA has the advantage of being usable under a wider range of chemical conditions (Non-Patent Literature 2). In addition, generally, for the same chain length, hairpin strands form a stronger double helix than double-stranded DNA (higher Tm value). Therefore, under various chemical conditions when introducing building blocks, the chemical structures of hairpin strand DNA, especially the base region, should be more resistant to structural transformation than double-stranded DNA. (B) Disadvantages Hairpin strand DNA forms a double helix within the molecule, making it difficult to create a new double helix with another oligonucleotide strand. Therefore, it is difficult to convert it to crosslinker-modified double-stranded DEL by adding a newly crosslinker-modified oligonucleotide. (2) Double strand DEL DEL, which uses double-stranded DNA, is synthesized using single-stranded DNA (single-stranded DNA that is not a hairpin strand) or double-stranded DNA containing functional groups for introducing various building blocks as a starting material (headpiece). (A) Disadvantages In contrast to DELs that use hairpin strand DNA, relatively long single-stranded or double-stranded DNA tags of about 20-30 mers with 4-10 mers of sticky ends are often used (Patent Document 2, Non-Patent Document 10), and DELs that encode a reaction of about 3 cycles are common. (B) Strengths Double-stranded DNA can be converted to single-stranded DNA through denaturation or by undergoing a strand exchange reaction, which is advantageous because it can be converted into DNA structures suitable for various applications. Therefore, it is also possible to convert it to cross-linker modified double-stranded DEL by adding a newly cross-linker modified single-stranded oligonucleotide (Non-patent documents 7, 8, and 11).

[0006] Thus, while hairpin strand DNA and double-stranded DNA each have advantages in DEL synthesis and evaluation, no technology is known that combines these advantages.

[0007] The following are known examples of reports on screening using crosslinker-modified DEL.

[0008] Non-patent documents 7 and 8 describe the synthesis of single-stranded DEL with a library molecule at its 5' end, followed by the formation of a double helix with DNA having a photoreactive crosslinker at its 3' end, and subsequent screening to obtain a binder with moderate affinity. However, this method has the drawback that, because the DNA linked to the photoreactive crosslinker does not contain a coding sequence, exposing it to strong separation or elution conditions to remove non-specific binders may cause the double helix to dissociate, potentially preventing the acquisition of the sequence encoding the desired structure. Furthermore, because this method uses single-stranded DEL, it cannot leverage the advantages of hairpin-stranded DEL during synthesis.

[0009] Xiaoyu Li et al. have reported a screening method using crosslinker-modified DEL (Patent Document 5, Non-Patent Documents 4, 5, and 12). In Non-Patent Document 5, a single-stranded DEL with a library molecule at the 3' end is synthesized and double-stranded with a short DNA having a photoreactive crosslinker at the 5' end to create a photoreactive crosslinker-modified DEL. An advantage of this method is that, because the photoreactive crosslinker is located at the 5' end, the coding sequence is covalently linked to the target by the DNA polymerase extension reaction. Therefore, it is possible to include strong separation and elution conditions in the screening. However, no method has been reported for easily synthesizing single-stranded DEL with a library molecule at the 3' end. Similarly, this method does not take advantage of the benefits of hairpin strand DEL during synthesis.

[0010] Patent document 4 describes a hairpin chain DEL having a linking site with a crosslinker. Although this method can solve the problems described in non-patent document 5 above, it has another problem in that it is necessary to perform library synthesis without damaging the functional groups for crosslinker linking, which limits the usable reactions and / or library molecular structures.

[0011] Patent document 6 describes the synthesis of double-stranded DEL (which is thought to have properties equivalent to hairpin-strand DEL in terms of double-strand formation ability, etc.) crosslinked by reversible covalent bonds, and the conversion to crosslinker-modified DEL. As a reversible covalent bond, a covalent bond formed by [2 + 2] photocyclization between a special base such as cyanovinylcarbazole and a pyrimidine base is disclosed. However, photocyclized pyrimidine bases lose their aromaticity, and such pyrimidine bases that have lost their aromaticity are known to be chemically unstable and decompose under basic conditions (Non-patent document 13). Therefore, in this method, the reactions that can be used during DEL synthesis are limited, and the library molecular structures that can be constructed are also limited.

[0012] Thus, there is no known technology that has the advantages on the synthetic surface at the same level as the conventional hairpin - shaped DEL and is adaptable to screening with simply cross - linker - modified DEL.

Prior Art Documents

Patent Documents

[0013]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Patent Document 6

Non - Patent Documents

[0014]

Non - Patent Document 1

Non - Patent Document 2

Non - Patent Document 3

Non - Patent Document 4

[0015] This invention provides a method for inducing and evaluating DEL containing cleavable sites in a DNA strand into crosslinker-modified double-stranded DEL. [Means for solving the problem]

[0016] One aspect of nucleic acid chemistry, such as DNA cleavage techniques, involves the introduction of deoxyuridine into a DNA strand, which allows for selective cleavage by the USER® enzyme. As a result of diligent research, the inventors have discovered that by introducing a cleavable site, such as deoxyuridine, into the DNA strand, it is possible to achieve the advantages of both hairpin strand DNA and double-stranded DNA. Furthermore, by easily inducing crosslinker modification (DEL), the above-mentioned problems have been solved. Therefore, the present invention is as follows.

[0017] [1] A method for evaluating crosslinker-modified double-stranded DNA-coding libraries (DELs) derived from hairpin-type DNA-coding libraries (DELs) having "selectively cleavable sites," comprising the following steps: (1) The DEL is brought into contact with a biological target under conditions suitable for at least one library molecule of the DEL to bind to the biological target. (2) Crosslinking the library molecule bound to the biological target with the biological target, (3) Separating the complex of crosslinked library molecules and biological targets from the uncrosslinked library molecules. (4) Identify the sequences of oligonucleotides present in the library molecules within the recovered complex. (5) Using the sequences determined in (4), identify the structure of one or more compounds that bind to a biological target. A method consisting of the following. [2] The method according to [1], wherein the crosslinker of the crosslinker-modified double-stranded DEL is covalently linked to an oligonucleotide having a coding sequence. [3] The method according to [1] or [2], wherein the crosslinker of the crosslinker-modified double-stranded DEL is either directly attached to the 5' end of an oligonucleotide or attached via a bifunctional spacer. [4] The method by any of [1] to [3], wherein the induction to crosslinker-modified double-stranded DEL includes the following steps: (i) Cut at least one "selectively cleavable region" of a hairpin-type DEL having a "selectively cleavable region" to convert it into a double-stranded DEL. (ii) From the double-stranded DEL obtained in (i), remove the oligonucleotides that are not bound to the library molecule and convert them to single-stranded DEL. (iii)(ii) The single-stranded DEL obtained in (iii)(ii) is combined with cross-linker modified DNA to form a double strand, thereby inducing cross-linker modified double-stranded DEL. [5] The method by any of [1] to [3], wherein the induction to crosslinker-modified double-stranded DEL includes the following steps: (i) Cut at least one "selectively cleavable region" of a hairpin-type DEL having a "selectively cleavable region" to convert it into a double-stranded DEL. (ii) From the double-stranded DEL obtained in (i), remove the oligonucleotides that are not bound to the library molecule and convert them to single-stranded DEL. (iii)(ii) The single-stranded DEL obtained in (iii)(ii) is double-stranded with DNA having a reactive group for crosslinker modification. (iv) The reaction group for crosslinker modification is reacted with the crosslinker unit to induce the crosslinker-modified double-stranded DEL. [6] The method by any of [1] to [3], wherein the induction to crosslinker-modified double-stranded DEL is further comprising the following steps: (i) Cut at least one "selectively cleavable region" of a hairpin-type DEL having a "selectively cleavable region" to convert it into a double-stranded DEL. (ii) From the double-stranded DEL obtained in (i), remove the oligonucleotides that are not bound to the library molecule and convert them to single-stranded DEL. (iii)(ii) The single-stranded DEL obtained in (iii)(ii) is coated with a crosslinker-modified primer, and the coated primer is extended to induce crosslinker-modified double-stranded DEL. [7] The method by any of [1] to [3], comprising the following steps, for induction to crosslinker-modified double-stranded DEL. (i) Cut at least one "selectively cleavable region" of a hairpin-type DEL having a "selectively cleavable region" to convert it into a double-stranded DEL. (ii) From the double-stranded DEL obtained in (i), remove the oligonucleotides that are not bound to the library molecule and convert them to single-stranded DEL. (iii)(ii) The single-stranded DEL obtained in (iii)(ii) is given a modification primer having a reactive group for crosslinker modification, the given primer is extended, and it is converted into a double-stranded DEL having a reactive group for crosslinker modification. (iv) The reaction group for crosslinker modification is reacted with the crosslinker unit to induce the crosslinker-modified double-stranded DEL. [8] The method by any of [1] to [3], wherein the induction to crosslinker-modified double-stranded DEL includes the following steps: (i) Cut at least one "selectively cleavable region" of a hairpin-type DEL having a "selectively cleavable region" to convert it into a double-stranded DEL. (ii) The double-stranded DEL obtained in (i) is coated with a crosslinker-modified primer, and the coated primer is extended to induce crosslinker-modified double-stranded DEL. [9] The method by any of [1] to [3], comprising the following steps, for induction to crosslinker-modified double-stranded DEL. (i) Cut at least one "selectively cleavable region" of a hairpin-type DEL having a "selectively cleavable region" to convert it into a double-stranded DEL. (ii) The double-stranded DEL obtained in (i) is given a modification primer having a reactive group for crosslinker modification, the given primer is extended, and the reactive group for crosslinker modification reacts with the crosslinker unit to induce crosslinker-modified double-stranded DEL.

[10] The method described in [6] or [8], characterized by the following: (I) Use a hairpin-shaped DEL in which at least one "selectively cleavable site" is located 3' from the site to which the library molecule is bound. (II) Use a crosslinker-modified primer in which the crosslinker is either directly bound to the 5' end of the oligonucleotide or bound via a bifunctional spacer.

[11] The method described in [7] or [9], characterized by the following: (I) Use a hairpin-shaped DEL in which at least one "selectively cleavable site" is located 3' from the site to which the library molecule is bound. (II) Use a modification primer having a reactive group for crosslinker modification, in which the reactive group for crosslinker modification is directly bonded to the 5' end of the oligonucleotide or bonded via a bifunctional spacer.

[12] The method according to any one of [4] to [7], wherein in step (ii), the oligonucleotide to which the library molecule is not bound has a functional molecule and is removed by a treatment according to the function of the functional molecule.

[13] The method according to

[12] , wherein the functional molecule is biotin.

[14] The method according to any one of [4] to [7], wherein in step (ii), the removal of oligonucleotides not bound to library molecules is by degradation by exonuclease.

[15] The method according to

[14] , wherein the exonuclease is a lambda exonuclease.

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

[15] , wherein the crosslinker comprises at least one azide group, diazirine group, sulfonyl fluoride group, diazo group, cinnamoyl group, or acrylate group.

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

[15] , wherein the crosslinker comprises at least one azide group, a diazirine group, or a sulfonyl fluoride group.

[18] Crosslinker, equation (AA)~(AE): [ka] (In the formula, * represents the 5' end of the double-stranded DEL, or the binding site to the bifunctional spacer that is bound to the 5' end.) A method according to any of [1] to

[15] , comprising any of the structures.

[19] Crosslinker, equation (AA)~(AE): [ka] (In the formula, * represents the 5' end of the double-stranded DEL, or the binding site to the bifunctional spacer that is bound to the 5' end.) A method according to any of [1] to

[15] , which has one of the following structures.

[20] Crosslinker is formula (BA) or (BB): [ka] (In the formula, * represents the 5' end of the double-stranded DEL, or the binding site to the bifunctional spacer that is bound to the 5' end.) A method according to any of [1] to

[15] , comprising any of the structures.

[21] Crosslinker is formula (BA) or (BB): [ka] (In the formula, * represents the 5' end of the double-stranded DEL, or the binding site to the bifunctional spacer that is bound to the 5' end.) A method according to any of [1] to

[15] , which has one of the following structures.

[22] The method according to any one of [5], [7], [9] or

[11] , wherein the reactive group for crosslinker modification is a reactive group for a click reaction.

[23] The method according to [5], [7], [9] or

[11] , wherein the reactive group for crosslinker modification is an alkynyl group, an alkenyl group, an azide group or a tetradinyl group.

[24] The reactive group for crosslinker modification is given by formula (CA)~(CL): [ka] (In the formula, * indicates the 5' end of the double-stranded DEL, or the binding site to the bifunctional spacer that is bound to the 5' end.) The method described in [5], [7], [9], or

[11] , which has one of the following structures.

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

[19] , wherein the step of (2) "crosslinking the crosslinker of the library molecule bound to the biological target with the biological target" is replaced with the step of "crosslinking the crosslinker of the library molecule bound to the biological target with the biological target by light irradiation".

[26] The method according to

[25] , wherein the light irradiation conditions are light irradiation with a wavelength of 250 to 500 nm.

[27] The method according to

[25] , wherein the light irradiation condition is light irradiation with a wavelength of 365 nm.

[28] The method according to any of

[25] to

[27] , wherein the light irradiation conditions are light irradiation for 10 seconds to 180 minutes.

[29] The method according to any of

[25] to

[27] , wherein the light irradiation conditions are light irradiation for 30 seconds to 30 minutes.

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

[24] , wherein the step of (2) "crosslinking the crosslinker of the library molecule bound to the biological target with the biological target" is replaced with the step of "crosslinking the crosslinker of the library molecule bound to the biological target with the biological target by incubation".

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

[30] , wherein the step of (3) "separating the complex of crosslinked library molecules and biological targets from uncrosslinked library molecules" is replaced with the step of "separating the complex of crosslinked library molecules and biological targets from uncrosslinked library molecules by electrophoresis."

[32] The method according to

[31] , wherein the electrophoresis is gel electrophoresis.

[33] The method according to

[31] , wherein the electrophoresis is capillary electrophoresis.

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

[30] , wherein the step of (3) "separating the complex of crosslinked library molecules and biological targets from uncrosslinked library molecules" is "separating the complex of crosslinked library molecules and biological targets by immobilizing the biological targets onto an immobilization carrier and washing away the uncrosslinked library molecules."

[35] A hairpin-type DEL having a “part that can be selectively cut” is given by formula (I) [ka] (In the formula, X and Y are nucleotide chains, E and F are independent of each other. It is an oligomer composed of nucleotides or nucleic acid analogs, However, E and F contain complementary base sequences and form a double-stranded oligonucleotide. LP is the loop section, L is a linker, D is a divalent group derived from a reactive functional group, Sp is a bond or a bifunctional spacer, An is a substructure composed of at least one building block. It is a compound represented by the following: X and Y have sequences that can form a double helix in at least part of their structure. X binds to E at its 5' end. Y binds to F at its 3' end. (At least one of the parts E, F, or LP has at least one selectively cleavable portion.) DEL is represented as The method described in any of [1] to

[34] .

[36] A hairpin-type DEL having a “part that can be selectively cut” is given by formula (III) An-Sp-C-Bn (III) (In the formula, An and Sp have the same meaning as in

[35] , Bn represents a double-stranded oligonucleotide tag formed by oligonucleotide chain X and oligonucleotide chain Y. C is given by equation (I) [ka] (In the formula, E, LP, L, D, and F have the same meanings as in

[35] , except that D binds to An either directly or via a bifunctional spacer, and E and F bind to the corresponding terminals of the double-stranded oligonucleotide tag Bn.) DEL is represented as The method described in

[35] .

[37] An is the same as

[35] and is a substructure constructed of n building blocks α1 to αn (where n is an integer from 1 to 10), Bn is a double-stranded oligonucleotide tag formed by oligonucleotide chain X and oligonucleotide chain Y, and is a substructure containing an oligonucleotide with a base sequence that can identify the structure of An. The method described in

[35] or

[36] .

[38] LP, This is the loop region represented by (LP1)p-LS-(LP2)q, LS is a substructure selected from the group of compounds described in (A) to (C) below, (A) Nucleotides (B) Nucleic acid analogs (C) Trivalent C1-14 groups which may have substituents LP1 is a substructure selected individually or differently from the group of compounds described in (1) and (2) below, (1) Nucleotides (2) Nucleic acid analogs LP2 is each of q substructures selected individually or differently from the group of compounds described in (1) and (2) below. (1) Nucleotides (2) Nucleic acid analogs The total number of p and q is between 0 and 40. The method described in any of

[35] to

[37] .

[39] The method described in

[38] , wherein the total number of p and q is between 2 and 20.

[40] The method described in

[38] , wherein the total number of p and q is between 2 and 10.

[41] The method described in

[38] , wherein the total number of p and q is between 2 and 7.

[42] The method described in

[38] , wherein the total number of p and q is 0.

[43] LP1, LP2, and LS have the following structures, respectively: (A) Nucleotides or (B) Nucleic acid analogs that meet the following requirements (B11) to (B15) (B11) Having phosphoric acid (or equivalent part) and a hydroxyl group (or equivalent part), (B12) Composed of carbon, hydrogen, oxygen, nitrogen, phosphorus, or sulfur, (B13) Molecular weight is between 142 and 1500. (B14) The number of atoms between residues is 3 to 30. (B15) The bonding pattern between atoms in residues is either all single bonds, or one or two double bonds with the remainder being single bonds. A method according to any of

[38] to

[42] , wherein the structure is selected individually or differently from the others.

[44] LP1, LP2, and LS have the following structures, respectively: (A) Nucleotides or (B) Nucleic acid analogs that meet the following requirements (B21) to (B25) (B21) Having phosphoric acid and hydroxyl groups, (B22) Composed of carbon, hydrogen, oxygen, nitrogen, or phosphorus, (B23) Molecular weight is between 142 and 1000. (B24) The number of atoms between residues is 3 to 15. (B25) The bonding mode between atoms in each residue is all single bonds. A method according to any of

[38] to

[43] , wherein the structure is selected individually or differently from the others.

[45] LP1, LP2, and LS have the following structures, respectively: (A) Nucleotides or (B) Nucleic acid analogs that meet the following requirements (B31) to (B35) (B31) Having phosphoric acid and hydroxyl groups, (B32) Composed of carbon, hydrogen, oxygen, nitrogen, or phosphorus, (B33) Molecular weight is between 142 and 700. (B34) The number of atoms between residues is 4 to 7. (B35) The bonding mode between atoms in each residue is all single bonds. A method by which one or more of the structures are selected from

[38] to

[44] .

[46] LP1 and LP2 are as follows: (B41)d-Spacer, (B5) Polyalkylene glycol phosphate The method described in any of

[38] to

[45] , which is one of the following.

[47] The method according to any one of

[38] to

[46] , wherein LP1 and LP2 are each diethylene glycol phosphate ester or triethylene glycol phosphate ester.

[48] ​​The method according to any one of

[38] to

[47] , wherein LP1 and LP2 are each triethylene glycol phosphate esters.

[49] The method according to any of

[38] to

[46] , wherein LP1 and LP2 are each d-Spacer.

[50] The method according to any of

[38] to

[45] , wherein LP1 and LP2 are nucleotides, respectively.

[51] LS is given by equations (a) to (g): [ka] (In the formula, * represents the bond position with the linker, ** represents the bond position with LP1 or LP2, and R represents a hydrogen atom or a methyl group.) The method described in any of

[38] to

[50] , which is one of the following.

[52] LS is given by equation (h): [ka] (In the formula, * indicates the linker connection position, and ** indicates the linker connection position.) The method described in any of

[38] to

[50] .

[53] The method according to any one of

[38] to

[50] , wherein LS is a polyalkylene glycol phosphate ester.

[54] LS is given by equations (i) to (k): [ka] (In the formula, n1, m1, p1, and q1 are each independent integers between 1 and 20, * indicates the linker connection position, and ** indicates the linker connection position with LP1 or LP2.) The method described in any of

[38] to

[50] .

[55] LS is given by equation (l): [ka] (In the formula, * indicates the linker connection position, and ** indicates the linker connection position.) The method described in any of

[38] to

[50] .

[56] LS is (B42), (B43), or (B44): (B42) Amino C6 dT (B43)mdC(TEG-Amino) (B44) Uni-Link (Trademark Registered) Amino Modifier The method described in any of

[38] to

[50] , which is one of the following.

[57] The method according to any of

[38] to

[50] , wherein LS is a nucleotide.

[58] LS is a trivalent C1-14 group which may have a (C) substituent, and (C) has the following structure: (1) C1-10 aliphatic hydrocarbons which may have substituents and which may be replaced by 1-3 heteroatoms, (2) C6-14 aromatic hydrocarbons which may have substituents, (3) A C2-9 aromatic heterocycle which may have substituents, or (4) C2-9 non-aromatic heterocycles which may have substituents The method described in either

[38] -

[42] or

[46] -

[50] , which is one of the above.

[59] LS is a trivalent C1-14 group which may have a (C) substituent, and (C) has the following structure: (1) C1-6 aliphatic hydrocarbons which may have substituents, (2) C6-10 aromatic hydrocarbons which may have substituents, (3) C2-5 aromatic heterocycles which may have substituents The method described in any of

[38] -

[42] and

[46] -

[50] , which is one of the following.

[60] LS is a trivalent C1-14 group which may have a (C) substituent, and (C) has the following structure: (1) C1-6 aliphatic hydrocarbons, (2) Benzene, or (3)C2~5 nitrogen-containing aromatic heterocycle Here, (1) to (3) may be unsubstituted or substituted with one to three substituents selected individually or differently from substituent group ST1, wherein substituent group ST1 consists of C1-6 alkyl groups, C1-6 alkoxy groups, fluorine atoms, and chlorine atoms; however, if substituent group ST1 is substituted with an aliphatic hydrocarbon, alkyl groups are not selected from substituent group ST1. The method described in any of

[38] -

[42] and

[46] -

[50] , which is one of the following.

[61] LS is a trivalent C1-14 group which may have a (C) substituent, and (C) has the following structure: (1) C1-6 alkyl groups, or (2) Unsubstituted or benzenes substituted with one or two C1-3 alkyl or C1-3 alkoxy groups The method described in any of

[38] -

[42] and

[46] -

[50] , which is one of the following.

[62] LS is a trivalent C1-14 group which may have a (C) substituent, and (C) has the following structure: (1) C1-6 alkyl groups The method described in either

[38] -

[42] or

[46] -

[50] .

[63] E and F are oligomers composed independently of nucleotides or nucleic acid analogs, The chain lengths of E and F are 3 to 40, respectively. The method described in any of

[35] to

[62] .

[64] E and F are oligomers composed independently of nucleotides or nucleic acid analogs, The chain lengths of E and F are 4 to 30, respectively. The method described in any of

[35] to

[63] .

[65] E and F are oligomers composed independently of nucleotides or nucleic acid analogs, The chain lengths of E and F are 6 to 25, respectively. The method described in any of

[35] to

[64] .

[66] E and F are oligomers composed independently of nucleotides or nucleic acid analogs. E and F contain complementary base sequences, forming a double-stranded oligonucleotide. The E and F double-stranded oligonucleotides are the protruding ends. The method described in any of

[35] to

[65] .

[67] The method according to

[66] , wherein the projection of the projection end is two bases or longer.

[68] E and F are oligomers composed independently of nucleotides or nucleic acid analogs, E and F contain complementary base sequences, forming a double-stranded oligonucleotide. The double-chain oligonucleotides E and F have blunt ends. The method described in any of

[35] to

[65] .

[69] The method according to any of the

[35] -

[68] , wherein the chain lengths of the complementary base sequences contained in E and F are each 3 bases or more.

[70] The method according to any of the

[35] -

[69] , wherein the chain lengths of the complementary base sequences contained in E and F are each 4 bases or more.

[71] The method according to any of the

[35] -

[70] , wherein the chain lengths of the complementary base sequences contained in E and F are each 6 bases or more.

[72] The method according to any of

[35] to

[71] , wherein E and F are oligomers composed independently of nucleotides.

[73] The method according to any of the following

[35] -

[72] , wherein the nucleotide is a ribonucleotide or a deoxyribonucleotide.

[74] The method described in any of

[35] -

[73] , wherein the nucleotide is a deoxyribonucleotide.

[75] The method according to any one of

[35] -

[74] , wherein the nucleotide is deoxyadenosine, deoxyguanosine, thymidine, or deoxycytidine.

[76] The method according to any one of

[35] to

[71] , wherein E and F are oligomers composed independently of nucleic acid analogs.

[77] L, (1) C1-20 aliphatic hydrocarbons which may have substituents and which may be replaced by 1-3 heteroatoms, or (2) C6-14 aromatic hydrocarbons which may have substituents The method described in any of

[35] to

[76] .

[78] The method according to any one of

[35] to

[77] , wherein L is a C1-6 aliphatic hydrocarbon which may have substituents, a C1-6 aliphatic hydrocarbon which may be replaced by one or two oxygen atoms, or a C6-10 aromatic hydrocarbon which may have substituents.

[79] L is a C1-6 aliphatic hydrocarbon substituted with substituent group ST1, or a benzene substituted with substituent group ST1, where substituent group ST1 is a group consisting of C1-6 alkyl groups, C1-6 alkoxy groups, fluorine atoms and chlorine atoms (however, when substituent group ST1 is substituted with an aliphatic hydrocarbon, alkyl groups are not selected from substituent group ST1), or the method according to any one of

[35] to

[78] .

[80] The method according to any one of

[35] to

[79] , wherein L is a C1-6 alkyl group, or a benzene that is unsubstituted or substituted with one or two C1-3 alkyl groups or C1-3 alkoxy groups.

[81] The method according to any of

[35] to

[80] , wherein L is a C1-6 alkyl group.

[82] The reactive functional group of D is The method according to any one of

[35] to

[81] , wherein the functional group is a C-C, amino, ether, carbonyl, amide, ester, urea, sulfide, disulfide, sulfoxide, sulfonamide, or a reactive functional group capable of forming a sulfonyl bond.

[83] The method according to any one of

[35] to

[82] , wherein the reactive functional group of D is a C1 hydrocarbon having a leaving group, an amino group, a hydroxyl group, a precursor of a carbonyl group, a thiol group, or an aldehyde group.

[84] The method according to any one of

[35] to

[83] , wherein the reactive functional group of D is a C1 hydrocarbon having a halogen atom, a C1 hydrocarbon having a sulfonic acid leaving group, an amino group, a hydroxyl group, a carboxyl group, a halogenated carboxyl group, a thiol group, or an aldehyde group.

[85] The method according to any one of

[35] to

[84] , wherein the reactive functional group of D is -CH2Cl, -CH2Br, -CH2OSO2CH3, -CH2OSO2CF3, an amino group, a hydroxyl group, or a carboxyl group.

[86] The method according to any one of

[35] to

[85] , wherein the reactive functional group of D is a primary amino group.

[87] The selectively cleavable sites are deoxyribonucleosides other than deoxyadenosine, deoxyguanosine, thymidine, and deoxycytidine. The method described in any of

[35] to

[86] .

[88] The method according to any one of

[35] to

[87] , wherein the selectively cleavable sites are deoxyuridine, bromodeoxyuridine, deoxyinosine, 8-hydroxydeoxyguanosine, 3-methyl-2'-deoxyadenosine, N6-etheno-2'-deoxyadenosine, 7-methyl-2'-deoxyguanosine, 2'-deoxyxanthosine, or 5,6-dihydroxy-5,6-dihydrodeoxythymidine.

[89] The method according to any one of

[35] to

[88] , wherein the selectively cleavable site is deoxyuridine or deoxyinosine.

[90] The method according to any of

[35] to

[89] , wherein the selectively cleavable site is deoxyuridine.

[91] The method according to any of

[35] to

[89] , wherein the selectively cleavable site is deoxyinosine.

[92] The method according to any of

[35] to

[86] , wherein the selectively cleavable site is the second phosphodiester bond in the 3' direction from deoxyinosine.

[93] The method according to any of

[35] to

[86] , wherein the selectively cleavable site is a ribonucleoside.

[94] The method according to any one of

[35] to

[93] , wherein there is one selectively severable site.

[95] The method according to any one of

[35] to

[93] , wherein at least one cleavable portion is included in E or (LP1)p and at least one cleavable portion is included in F or (LP2)q.

[96] The cleavable portion included in E or (LP1)p and the cleavable portion included in F or (LP2)q are cleavable under different conditions, The method described in

[95] .

[97] An is a substructure constructed of n building blocks α1 to αn (where n is an integer from 1 to 10). The method described in any of

[35] to

[96] .

[98] The method according to any of

[35] to

[97] , wherein An is a low molecular weight organic compound.

[99] The method according to any of

[35] to

[98] , wherein the building block of An is a compound with a molecular weight of 500 or less.

[0100] The method according to any of

[35] to

[99] , wherein the building block of An is a compound with a molecular weight of 300 or less.

[0101] The method according to any of

[35] to

[0100] , wherein the building block of An is a compound with a molecular weight of 150 or less.

[0102] The method according to any one of

[35] to

[0101] , wherein An is an organic compound composed of elements selected individually or differently from the group of elements consisting of H, B, C, N, O, Si, P, S, F, Cl, Br, and I.

[0103] The method according to any one of

[35] to

[0102] , wherein An is a low molecular weight organic compound having substituents selected individually or differently from the group of substituents consisting of aryl groups, non-aromatic cyclyl groups, heteroaryl groups, and non-aromatic heterocyclyl groups.

[0104] The method according to any of

[35] to

[0103] , wherein An has a molecular weight of 5000 or less.

[0105] The method according to any one of

[35] to

[0104] , wherein An has a molecular weight of 800 or less.

[0106] The method according to any of

[35] to

[0105] , wherein An has a molecular weight of 500 or less.

[0107] The method described in any of

[35] to

[97] , wherein An is a polypeptide.

[0108] The method according to any one of

[35] to

[0107] , wherein Sp is a bifunctional spacer.

[0109] The two functional spacers are SpD-SpL-SpX, respectively. SpD is a divalent group derived from a reactive group that can constitute a C-C, amino, ether, carbonyl, amide, ester, urea, sulfide, disulfide, sulfoxide, sulfonamide, or sulfonyl bond. SpL may be polyalkylene glycol, polyethylene, C1-20 aliphatic hydrocarbons which may optionally be replaced by heteroatoms, peptides, oligonucleotides, or combinations thereof. The method according to any one of [1] to

[0107] , wherein SpX is a divalent group derived from a reactive group that forms an amino, carbonyl, amide, ester, urea, or sulfonamide bond.

[0110] The two functional spacers are SpD-SpL-SpX, respectively. SpD is a divalent group derived from a primary amino group, SpL is polyethylene glycol or polyethylene. SpX is a divalent group derived from a carboxyl group. The method described in any of [1] to

[0107] .

[0111] The method according to any one of

[35] to

[0110] , wherein oligonucleotide chain X and oligonucleotide chain Y are sequences capable of forming a double helix.

[0112] The method according to any one of

[35] to

[0111] , wherein oligonucleotide chain X and oligonucleotide chain Y contain complementary base sequences.

[0113] The method according to any of

[35] to

[0112] , wherein oligonucleotide chain X and oligonucleotide chain Y are each 1 to 200 bases in length.

[0114] The method according to any of

[35] to

[0113] , wherein oligonucleotide chain X and oligonucleotide chain Y are each 3 to 150 bases in length.

[0115] The method according to any of

[35] to

[0114] , wherein oligonucleotide chain X and oligonucleotide chain Y are each 30 to 150 bases in length.

[0116] The method according to any one of

[35] to

[0115] , wherein oligonucleotide chain X and oligonucleotide chain Y have blunt ends.

[0117] The method according to any one of

[35] to

[0115] , wherein oligonucleotide chain X and oligonucleotide chain Y have protruding ends.

[0118] The method according to

[0117] , wherein the protruding portion of the protruding end is 1 to 30 bases in length.

[0119] The method according to

[0117] or

[0118] , wherein the protruding portion of the protruding end is 2 to 5 bases in length.

[0120] The method according to any one of

[0117] to

[0119] , wherein oligonucleotide chain X and oligonucleotide chain Y have protruding ends, and a specific molecular identification sequence is further bound to the protruding ends.

[0121] The method according to any one of

[35] to

[0120] , wherein a functional molecule is attached to either X or Y.

[0122] The method according to any one of

[35] to

[0120] , wherein biotin is bound to either X or Y.

[0123] The method described in any of

[35] to

[0107] , wherein Sp is a bond. [Effects of the Invention]

[0018] This invention provides a method for inducing and evaluating double-stranded DNA molecules (DELs) containing cleavable sites in the DNA strand into crosslinker-modified DELs. In other words, it provides a compound screening technology that combines a simpler DEL synthesis method with an expanded and improved DEL evaluation method compared to conventional methods. Therefore, this invention expands the opportunities to obtain useful hit compounds in the development of pharmaceuticals, agrochemicals, and medical materials. [Brief explanation of the drawing]

[0019] [Figure 1]An exemplary DEL manufacturing method of Form 1 is shown. Starting with a headpiece containing a first oligonucleotide strand with a cleavable region in the DNA strand, a loop region, and a second oligonucleotide strand, the manufacturing of DEL is achieved by repeatedly binding building blocks and double-strand ligation of oligonucleotide tags corresponding to the building blocks (three times in Figure 1), and optionally double-strand ligation of oligonucleotide tags including primer regions. [Figure 2] An exemplary method for using DEL in Form 1 is shown. By using a cleavable region in the first oligonucleotide chain of the headpiece, and using a cleavage method such as an enzyme to cleave the cleavable region, the DEL can be converted into a double-stranded oligonucleotide that is not bound at the loop region, allowing for highly efficient PCR. [Figure 3] An exemplary method for using DEL in Form 2 is shown. For DEL containing cleavable sites in the second oligonucleotide chain of the headpiece, PCR can be performed with high efficiency by using a cleavage method such as an enzyme to cleave the cleavable sites and convert them into double-stranded oligonucleotides that are not bound at the loop site. [Figure 4] An exemplary method for using DEL in Form 3 is shown. For DEL containing cleavable sites in both the first and second oligonucleotide strands of the headpiece, PCR can be performed with high efficiency by using a cleavage method such as an enzyme to cleave both cleavable sites and convert them into double-stranded oligonucleotides that do not have a loop attached. [Figure 5] An exemplary use of the DEL of Form 4 is shown. For a DEL containing two different cleavable sites in the first and second oligonucleotide chains of the headpiece, the first or second oligonucleotide chain can be selectively cleaved by selecting the cleavage conditions. [Figure 6]An exemplary use of the DEL in Form 5 is shown. A cleavable site is provided near the end of the DNA tag, and a new overhang can be generated by cleaving this site as desired. This overhang can be used as an adhesive end to ligate a desired nucleic acid sequence, such as UMIs (Specific Molecular Identification Sequences), thereby conferring new functionality. [Figure 7] An exemplary use of DEL in Form 6 is shown. In this invention, a cleavable site can be used in combination with a modifying group or functional molecule, and for example, it is possible to prepare DEL in which hairpin strand DNA has been converted to single-stranded DNA. For example, a double-stranded oligonucleotide chain having a functional molecule (e.g., biotin) at its 3' end is ligated to the synthesized DEL compound (A), the cleavable site is cleaved (B), and a treatment according to the function of the functional molecule is applied (C). For example, if the functional molecule is biotin, streptavidin beads with biotin affinity are used to selectively remove the biotin-bound oligonucleotide chain from the system. This makes it possible to obtain DEL having single-stranded DNA. [Figure 8] An example of the use of DEL obtained in Form 6 is shown. DELs with single-stranded DNA obtained in Form 6 can be given new functions by forming a double helix with a modified oligonucleotide (e.g., crosslinker-modified DNA such as a photoreactive crosslinker) having a desired functional site. [Figure 9] An exemplary use of DEL in Form 7 is shown. In this invention, a crosslinker can be introduced by utilizing the cleavable site. A cleavable site can be cleaved from a synthesized DEL compound (A), a modifying primer can be attached (B), and a crosslinker-modified double-stranded DEL compound can be synthesized based on the attached primer (C). The crosslinker-modified double-stranded DEL compound can significantly improve the detection sensitivity in screening DEL libraries (see Non-Patent Documents 7, 11, etc.). [Figure 10]This graph shows the conversion rate of the cleavage reaction at each incubation time when the cleavage reaction of 10 hairpin-type DEL substructures containing deoxyuridine (U-DEL1-sh, U-DEL2-sh, U-DEL3-sh, U-DEL4-sh, U-DEL5-HP, U-DEL6-HP, U-DEL7-HP, U-DEL8-HP, U-DEL9-HP, and U-DEL10-HP) was verified using USER® enzyme in Example 1. [Figure 11] Examples 2, 3, 4, 5, and 7 show schematic diagrams illustrating the synthesis procedures for various hairpin DELs (U-DEL1, U-DEL2, U-DEL4, U-DEL7, U-DEL8, U-DEL9, U-DEL10, H-DEL, U-DEL5, U-DEL11, U-DEL12, U-DEL13, I-DEL1, I-DEL2, I-DEL3, R-DEL1, and BIO-DEL). Each hairpin DEL is synthesized using a corresponding headpiece as a raw material, through a two-step double-stranded ligation with double-stranded oligonucleotides Pr_TAG and CP. [Figure 12] This graph shows the Ct values ​​measured by real-time PCR for eight types of hairpin DELs (U-DEL1, U-DEL2, U-DEL4, U-DEL7, U-DEL8, U-DEL9, U-DEL10, and H-DEL) and double-stranded DELs (DS-DEL) in Example 2, broken down by sample volume. Samples treated with USER® enzyme are indicated as “USER(+)”, and untreated samples are indicated as “USER(-)”. The deoxyuridine-containing, cleavable hairpin DELs (U-DEL1, U-DEL2, U-DEL4, U-DEL7, U-DEL8, U-DEL9, and U-DEL10) show Ct values ​​equivalent to those of double-stranded DELs (DS-DEL) after USER® enzyme treatment. [Figure 13]The image shows a gel obtained by modified polyacrylamide gel electrophoresis, illustrating the progress of the cleavage reaction of six types of deoxyuridine-containing hairpin DELs (U-DEL5, U-DEL7, U-DEL9, U-DEL11, U-DEL12, and U-DEL13) by USER® enzyme in Example 3. The numbers in the figure indicate the lane numbers. [Figure 14] The image shows a gel obtained by denatured polyacrylamide gel electrophoresis, illustrating the progress of the cleavage reaction of hairpin DELs (I-DEL1, I-DEL2, I-DEL3, and I-DEL4) containing four types of deoxyinosine by endonuclease V in Example 4. The numbers in the figure indicate the lane numbers. [Figure 15] The image shows a gel obtained by denatured polyacrylamide gel electrophoresis, illustrating the progress of the cleavage reaction of hairpin DEL (R-DEL1) containing ribonucleoside by RNaseHII in Example 5. The numbers in the figure indicate the lane numbers. [Figure 16] This is a schematic diagram showing the synthesis procedure for a model library containing 3×3×3(27) compound species using U-DEL9-HP as a starting material. In Example 6, the model library is synthesized using U-DEL9-HP as a starting material through three split-and-pool steps (cycles A, B, and C). Each cycle includes a ligation reaction of a double-stranded oligonucleotide tag and a chemical reaction for introducing building blocks. [Figure 17] These are images of gels obtained by agarose gel electrophoresis, showing the progress of the ligation reaction in each cycle during the model library synthesis of Example 6. The numbers in the figures indicate the lane numbers. [Figure 18] Figure 18A shows the chromatograph obtained from the sample after cycle C was completed in the model library synthesis of Example 6. Figure 18B shows the deconvolution results of the MS spectrum obtained from the sample after cycle C was completed in the model library synthesis of Example 6. [Figure 19]This image shows the gel obtained by modified polyacrylamide gel electrophoresis, illustrating the progress of the cleavage reaction using the USER® enzyme from the model library in Example 6. The numbers in the figure indicate the lane numbers. [Figure 20] The image shows the gel obtained by modified polyacrylamide gel electrophoresis, illustrating the cleavage reaction of five DEL compounds having biotin at their 3' ends ("AAZ-BIO-DEL", "SABA-BIO-DEL", "ClSABA-BIO-DEL", "mSABA-BIO-DEL", and "Amino-BIO-DEL") by USER® enzyme in Example 7. The numbers in the figure indicate the lane numbers. [Figure 21] The image shows the results of a primer extension reaction performed in Example 7 using single-stranded DNA-containing DEL compounds ("SS-AAZ-DEL", "SS-SABA-DEL", "SS-ClSABA-DEL", "SS-mSABA-DEL", and "SS-Amino-DEL") and the photoreactive crosslinker-modified primer "PXL-Pr". The image is of a gel obtained by polyacrylamide gel electrophoresis. The numbers in the figure indicate the lane numbers. [Figure 22] In Example 8, to compare the binder recovery efficiency of various photoreactive crosslinker-modified double-stranded DELs with and without photocrosslinking, the graph shows the Ct value and ΔCt value (difference from the Ct value of the negative control) measured by real-time PCR for the recovered amount. Samples without UV irradiation are indicated as "UV(-)", and samples that underwent UV irradiation are indicated as "UV(+)". Also, "Solution S" is indicated as "S", and "Solution E" is indicated as "E". The notations in the graph correspond to each sample as follows: Notation in graph: Sample AAZ: "PXL-DS-AAZ-DEL", SABA: "PXL-DS-SABA-DEL", ClSABA: "PXL-DS-ClSABA-DEL", mSABA: "PXL-DS-mSABA-DEL", Amino: "PXL-DS-Amino-DEL" [Figure 23]The image shows the results of a primer extension reaction performed in Example 10 using single-stranded DNA-containing DEL compounds ("SS-AAZ-DEL", "SS-SABA-DEL", "SS-ClSABA-DEL", "SS-mSABA-DEL", and "SS-Amino-DEL") and the photoreactive crosslinker-modified primer "PXL-Pr2". The image is of a gel obtained by polyacrylamide gel electrophoresis. The numbers in the figure indicate the lane numbers. [Figure 24] The image shows the results of a primer extension reaction performed in Example 10 using single-stranded DNA DEL compounds ("SS-AAZ-DEL3", "SS-SABA-DEL3", "SS-ClSABA-DEL3", "SS-mSABA-DEL3", and "SS-Amino-DEL3") and the photoreactive crosslinker-modified primer "PXL-Pr3". The image shows the gel obtained by polyacrylamide gel electrophoresis. The numbers in the figure indicate the lane numbers. [Figure 25] This graph shows the ΔCt value (difference from the negative control) calculated using the Ct value measured by real-time PCR to compare the binder recovery efficiency of various photoreactive crosslinker-modified double-stranded DELs with different linker structures, with and without photocrosslinking. Samples without UV irradiation are indicated as "UV(-)", and samples that underwent UV irradiation are indicated as "UV(+)". The notations in the graph correspond to each sample as follows: Notation in graph: Sample mSABA-DEL2: "PXL-DS-mSABA-DEL2" mSABA-DEL3: "PXL-DS-mSABA-DEL3" [Figure 26]This graph shows the ΔCt value (difference from the negative control) calculated using the Ct value measured by real-time PCR to compare the binder recovery efficiency of various "photoreactive crosslinker-modified double-stranded DELs having a covalent bond between the crosslinker and the coding sequence" and various "photoreactive crosslinker-modified double-stranded DELs without a covalent bond between the crosslinker and the coding sequence" in Example 12. Each bar in the graph corresponds to a sample from left to right, as described below. Leftmost bar: "PXL-DS-SABA-DEL3" without UV irradiation Second bar from the left: "PXL-DS-ClSABA-DEL3" without UV irradiation Third bar from the left: "PXL-DS-mSABA-DEL3" without UV irradiation Fourth bar from the left: "PXL-DS-SABA-DEL3" with UV irradiation Fifth bar from the left: "PXL-DS-ClSABA-DEL3" with UV irradiation Sixth bar from the left: "PXL-DS-mSABA-DEL3" with UV irradiation Seventh bar from the left: "PXL-DS-SABA-DEL4" without UV irradiation Eighth bar from the left: "PXL-DS-ClSABA-DEL4" without UV irradiation Ninth bar from the left: "PXL-DS-mSABA-DEL4" without UV irradiation (10th bar from the left); "PXL-DS-SABA-DEL4" with UV irradiation (11th bar from the left); "PXL-DS-ClSABA-DEL4" with UV irradiation (far right bar); "PXL-DS-mSABA-DEL4" with UV irradiation. [Figure 27]This graph shows the ΔCt value (difference from the negative control) calculated using the Ct value measured by real-time PCR to compare the binder recovery efficiency of various "photoreactive crosslinker-modified double-stranded DELs having a covalent bond between the crosslinker and the coding sequence" and various "photoreactive crosslinker-modified double-stranded DELs without a covalent bond between the crosslinker and the coding sequence" in Example 13. Each bar in the graph corresponds to a sample from left to right, as described below. Leftmost bar: "PXL-DS-SABA-DEL3" without UV irradiation Second bar from the left: "PXL-DS-ClSABA-DEL3" without UV irradiation Third bar from the left: "PXL-DS-mSABA-DEL3" without UV irradiation Fourth bar from the left: "PXL-DS-SABA-DEL3" with UV irradiation Fifth bar from the left: "PXL-DS-ClSABA-DEL3" with UV irradiation Sixth bar from the left: "PXL-DS-mSABA-DEL3" with UV irradiation Seventh bar from the left: "PXL-DS-SABA-DEL4" without UV irradiation Eighth bar from the left: "PXL-DS-ClSABA-DEL4" without UV irradiation Ninth bar from the left: "PXL-DS-mSABA-DEL4" without UV irradiation Tenth bar from the left: "PXL-DS-SABA-DEL4" that underwent UV irradiation (11th bar from the left): "PXL-DS-ClSABA-DEL4" that underwent UV irradiation (far right bar): "PXL-DS-mSABA-DEL4" that underwent UV irradiation [Figure 28] This image shows the gel obtained by modified polyacrylamide gel electrophoresis, illustrating the progress of the cleavage reaction of the hairpin DEL compound ("mSABA-DEL5") by USER® enzyme in Example 14. The numbers in the figure indicate the lane numbers. [Figure 29] The image shows the results of a primer extension reaction performed in Example 14 using a single-stranded DNA DEL compound ("SS-mSABA-DEL5") and a photoreactive crosslinker-modified primer "PXL-Pr5," obtained by polyacrylamide gel electrophoresis. The numbers in the figure indicate the lane numbers. [Figure 30] The image shows the results of primer extension reactions performed in Example 15 using the single-stranded DNA-containing DEL compound "SS-mSABA-DEL" and the crosslinker-modified primer "TPD-Pr", as well as "SS-ClSABA-DEL" and "ACA-Pr". The gel images were obtained by polyacrylamide gel electrophoresis. The numbers in the figure indicate the lane numbers. [Figure 31] The image shows the results of a primer extension reaction performed in Example 16 using a single-stranded DNA DEL compound ("SS-mSABA-DEL") and a reaction group modification primer "BCN-Pr" for crosslinker modification. The gel image was obtained by polyacrylamide gel electrophoresis. The numbers in the figure indicate the lane numbers. [Figure 32] The image shows the results of a primer extension reaction performed in Example 17 using a single-stranded DEL-modified model library, the photoreactive crosslinker-modified primer "PXL-Pr", and the reaction group-modified primer "BCN-Pr" for crosslinker modification. The gel image was obtained by polyacrylamide gel electrophoresis. The numbers in the figure indicate the lane numbers. [Modes for carrying out the invention]

[0020] As stated above, and as a concept well known to those skilled in the art, in this invention, a compound library refers to a systematic collection of compound derivatives, such as drug candidate compounds, that may possess specific activity. This compound library is often synthesized based on combinatorial chemistry synthesis techniques and methodologies. Combinatorial chemistry is the field of experimental methods and related research for efficiently synthesizing a wide variety of compounds from a series of compound libraries enumerated and designed based on combinatorial theory through systematic synthetic routes.

[0021] As mentioned above, and as is well known to those skilled in the art, one type of compound library based on combinatorial chemistry is the DNA-coding library. The DNA-coding library is often abbreviated as DEL. Furthermore, DEL is essentially synonymous with DNA-coding compound library. In this invention, a DNA-coding library means a library in which each compound in the library is tagged with a DNA tag. The DNA tag is sequenced to identify each structure of each compound and functions as a label for the compound.

[0022] A nucleotide is generally understood as a substance in which a phosphate group is bonded to a nucleoside. While nucleotides and nucleosides are well-known terms to those skilled in the art, a nucleoside is generally understood as a substance in which a nucleic acid base, such as a purine base or pyrimidine base, is glycosidically bonded to the 1-position of a sugar, such as a pentose. Nucleosides and nucleotides are also the constituent units of nucleic acids such as DNA and RNA. Furthermore, nucleic acids are a well-known concept to those skilled in the art, and are generally understood as polymers of nucleotides. In one embodiment, the nucleic acid of the present invention is a polymer composed of nucleotides and nucleic acid analogs, as described later.

[0023] Furthermore, in this specification, nucleic acid polymers composed of nucleotides and nucleic acid analogs, as well as nucleic acid monomers such as nucleotides and nucleic acid analogs, may also be simply referred to as nucleic acids. The latter usage is also in accordance with common technical knowledge and can be understood by those skilled in the art in accordance with the appropriate context.

[0024] In a broad sense, nucleotides include not only naturally occurring nucleotides (original nucleotides) but also artificial nucleotides (various nucleic acid analogs). The broad definition of nucleotide in this invention includes the following embodiments. (A) Natural nucleoside nucleotides (Examples of such nucleosides include adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxyuridine, deoxyguanosine, deoxycytidine, inosine, or diaminopurine deoxyriboside.) (B) Nucleoside nucleotides having a nucleic acid base analog. (Examples of nucleosides having the nucleic acid base analog include 2-aminoadenosine, 2-thiothymidine, pyrrolopyrimidine deoxyriboside, 3-methyladenosine, C5-propynylcytidine, C5-propynyluridine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 6-O-methylguanosine, or 2-thiocytidine.) (C) Nucleotides having intercalated nucleic acid bases (D) Non-natural nucleotides having ribose or 2'-deoxyribose (E) Nucleotides having a modified sugar in the sugar portion (Examples of such modified sugars include modified ribose, modified 2'-deoxyribose, 2'-O-methylribose, 2'-fluororibose, D-threoninol, arabinose, hexose, anhydrohexitol, althritol, or mannitol.) (F) Nucleic acid analogs (Examples of such nucleic acid analogs include cyclohexanyl nucleic acid, cyclohexenyl nucleic acid, morpholino nucleic acid (PMO), locked nucleic acid (LNA), glycol nucleic acid (GNA), threose nucleic acid (TNA), serinol nucleic acid (SNA), acyclic threoninol nucleic acid (aTNA), or nucleic acids in which oxygen in ribose has been replaced.) The following provides a detailed explanation of each nucleic acid analog. (F1) PMO PMO is a nucleic acid analog that has a morpholine ring in the sugar region and an uncharged phosphorodiamidate structure in the phosphate diester region. (F2)LNA LNAs are nucleic acid analogs that have a cross-linking structure in the sugar portion. The most typical example is in which the 2'-hydroxyl of ribose is cross-linked to the 4'-carbon of the same ribose sugar by a C1-6 alkylene or C1-6 heteroalkylene. Examples of cross-linking structures include methylene, propylene, ether, or amino cross-linking structures. A typical example of a nucleotide nanoparticle (LNA) is 2',4'-BNA (2'-O,4'-C-methylated nucleic acid). (F3)GNA Glycol nucleic acids are also called GNAs. Examples include R-GNA and S-GNA. In these cases, ribose is replaced by glycol units bonded to a phosphodiester bond. (F4)TNA Treose nucleic acids are also called TNAs. In this case, ribose is replaced with α-L-treophranosyl-(3'→2'). (F5)SNA Serinol nucleic acids are also called SNAs. In this case, ribose is replaced by selinol units attached to a phosphodiester bond. (F6)aTNA Acyclic threoninol nucleic acid is also called aTNA. Examples include D-aTNA and L-aTNA. In this case, ribose is replaced by threoninol units bonded to a phosphodiester bond. (F7) Sugar in which oxygen in ribose has been replaced. Specific examples include substituted oxygen compounds with S, Se, or alkylenes (for example, methylene or ethylene). (G) Modified nucleotides (Examples of nucleotides with this modified skeleton include peptide nucleic acids (also known as PNAs; in this case, the 2-aminoethyl-glycine linkage replaces the ribose and phosphodiester skeletons).) (H) Nucleotides modified with a phosphate group (Examples of nucleotides modified with the phosphate group include phosphorothioates, 5'-N-phosphoamidites, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, phosphotryesters, cross-linked phosphoramidates, cross-linked phosphorothioates, or cross-linked methylene phosphonates.) In the following description, the oligonucleotide, oligonucleotide chain, double-stranded oligonucleotide, double-stranded oligonucleotide chain, and double-stranded DNA of the present invention are nucleotides as defined above.

[0025] In the present invention, when the term "nucleotide" is used without particular limitation, it means a natural nucleotide. Natural nucleotide is a term well known to those skilled in the art and is not particularly limited as long as it is a nucleotide that is essentially naturally occurring. In one embodiment, the natural nucleotide in the present invention is the nucleotide described in (A) above. (Nucleic acid analog) The term "nucleic acid analog" is well known to those skilled in the art, and the structure of the nucleic acid analog in this invention is not limited as long as it has the effects of the present invention. In one embodiment, a nucleic acid analog is a compound according to the embodiments of (B) to (H) described above. In one embodiment, the nucleic acid analog in the present invention is a compound having a phosphate-equivalent moiety and a hydroxyl group-equivalent moiety in a nucleic acid monomer. More preferably, the nucleic acid analog is a compound having a phosphate moiety and a hydroxyl group. In one embodiment, the nucleic acid analog in the present invention is a compound that can be used as a monomer in a nucleic acid synthesizer. As is well known to those skilled in the art, nucleic acid oligomers can be synthesized in a nucleic acid synthesizer by using a monomer in which the phosphate group (or equivalent site) of the nucleic acid analog is phosphoramiditeted and the hydroxyl group (or equivalent site) is protected with a protecting group. Furthermore, in nucleic acid analogs, substructures other than the phosphate group (or equivalent group) and the hydroxyl group (or equivalent group) can be called nucleic acid analog residues. The structure of nucleic acid analog residues is not limited as long as they have the effects of the present invention, but as a reference, if we examine the structural characteristics of natural nucleic acids (deoxyadenosine, thymidine, deoxycytidine, deoxyguanosine), we find that their molecular weights range from approximately 322 (thymidine monophosphate) to 347 (deoxyguanosine monophosphate), and the number of atoms between the hydroxyl group oxygen atom at the 3' position and the phosphorus atom at the 5' position that constitute the nucleic acid chain (including oxygen and phosphorus atoms; hereinafter also referred to as the number of atoms between residues) is 6. In addition, the following nucleic acid analogs are known to be usable in nucleic acid synthesizers. Amino C6 dT Molecular weight: 476, Number of residue atoms: 6 mdC(TEG-Amino) Molecular weight: 526, Number of residue atoms: 6 Uni-Link (trademark registered) Amino Modifier Molecular weight: 227, Number of atoms in residue: 6 (See Nucleic Acid Research, 1992, Vol. 20, pp. 6253-6259) d-Spacer Molecular weight: 198, Number of residue atoms: 6 Triethylene glycol phosphate (Spacer9) Molecular weight: 230, Number of atoms in residue: 11

[0026] For reference, the structures of each nucleic acid analog are listed below. [ka]

[0027] Therefore, in one embodiment, the nucleic acid analog is a compound (B1) characterized by the following: (B11) It has phosphoric acid (or an equivalent part) and a hydroxyl group (or an equivalent part). (B12) Composed of carbon, hydrogen, oxygen, nitrogen, phosphorus, or sulfur. (B13) The molecular weight is between 142 and 1500. (B14) The number of atoms between residues is 5 to 30. (B15) The bonding pattern between atoms in the residues is either all single bonds, or one or two double bonds with the remainder being single bonds.

[0028] In one embodiment, the nucleic acid analog is a compound (B2) characterized by the following: (B21) Contains phosphoric acid and hydroxyl groups. (B22) Composed of carbon, hydrogen, oxygen, nitrogen, or phosphorus. (B23) Molecular weight is between 142 and 1000. (B24) The number of atoms between residues is 5 to 20. (B25) The bonding mode between atoms in each residue is all single bonds.

[0029] In one embodiment, the nucleic acid analog is a compound (B3) characterized by the following: (B31) Contains phosphoric acid and hydroxyl groups. (B32) Composed of carbon, hydrogen, oxygen, nitrogen, or phosphorus. (B33) Molecular weight is between 142 and 700. (B34) The number of atoms between residues is 5 to 12. (B35) The bonding mode between atoms in each residue is all single bonds.

[0030] In one embodiment, the nucleic acid analog is one of the following compounds: (B41), (B42), (B43), (B44), (B5), (B51), or (B52). (B41)d-Spacer (B42) Amino C6 dT (B43)mdC(TEG-Amino) (B44) Uni-Link (Trademark Registered) Amino Modifier (B5) Polyalkylene glycol phosphate (B51) Diethylene glycol phosphate or triethylene glycol phosphate (B52) Triethylene glycol phosphate

[0031] In this invention, oligonucleotides and oligonucleotide chains mean polymers of nucleotides having one or more nucleotides at the 5' end, the 3' end, and the internal position between the 5' end and the 3' end.

[0032] Complementary base sequences refer to nucleotide sequences in nucleic acids that can form complementary base pairs, which are determined by hydrogen bonds between two oligonucleotides, such as adenine and thymine (or uracil), or guanine and cytosine. The formation of complementary base pairs is also called hybridization. Complementary base pairs are generally referred to as "Watson-Crick base pairs" or "natural base pairs." However, base pairs may be Watson-Crick type, Hoogsteen type base pairs, or base pairs formed by the formation of other hydrogen bonding motifs (e.g., diaminopurine and T, 5-methyl C and G, 2-thiothymine and A, 6-hydroxypurine and C, pseudoisocytosine and G). There are no restrictions on the sequences of "mutually complementary base sequences" as long as the two oligonucleotides can form a double helix and are usable for the purposes of the present invention, and there are no restrictions on the homology of the two sequences. Preferably, the homology is 99% or more, 98% or more, 95% or more, 90% or more, 85% or more, 80% or more, 70% or more, 60% or more, or 50% or more, in order of increasing preference.

[0033] To reiterate, in this invention, hybridization refers to the act of forming a double helix with oligonucleotides or oligonucleotide chains containing complementary base sequences, and the phenomenon of oligonucleotides or oligonucleotide chains containing complementary sequences forming a double helix.

[0034] In this invention, a double helix refers to a state in which two nucleic acid strands form complementary base pairs (hybridize). The two nucleic acid strands may originate from two separate nucleic acid strands, or from two nucleic acid sequences within a single nucleic acid strand molecule.

[0035] In this invention, a double-stranded oligonucleotide and a double-stranded oligonucleotide chain refer to a secondary structure formed by the hybridization of two or more different oligonucleotide chains. The two oligonucleotides may have different chain lengths and may have regions that are not hybridized. Furthermore, the region where two strands hybridize is a double helix.

[0036] In this invention, double-stranded DNA refers to a secondary structure formed by the hybridization of two different DNA strands. The lengths of the respective DNA strands may differ, and they may have regions that are not hybridized. The DNA strands are not limited to naturally occurring deoxyribonucleotides, but refer to all oligonucleotide strands that can be amplified by DNA polymerase.

[0037] In this invention, "forming a double helix" means that the nucleic acid forms a double helix under standard conditions for handling oligonucleotides, such as a temperature of 4 to 40°C, an aqueous solvent, and a pH of 4 to 10. For example, even if a double helix does not form under certain solvents or conditions, if the nucleic acid forms a double helix under standard conditions, then the nucleic acid is considered a double-helix-forming nucleic acid.

[0038] In this invention, the Tm value refers to the temperature at which half of the DNA molecules anneal with the complementary strand.

[0039] In this invention, a blunt end means that the ends of a double-stranded oligonucleotide are paired without either end protruding.

[0040] In this invention, a protruding end means that one of the ends of a double-stranded oligonucleotide has a protruding portion. The protruding portion of the protruding end can be of any length, but is preferably 1 to 50 bases, more preferably 1 to 30 bases, even more preferably 1 to 15 bases, and most preferably 2 to 6 bases. In certain embodiments, the protruding portion can be used as a hybridized region when performing ligation of sticky ends.

[0041] PCR stands for Polymerase Chain Reaction. PCR is a method for amplifying oligonucleotide chains and is a well-known technique to those skilled in the art. In general terms, the PCR process involves (1) dissociating the double-stranded oligonucleotide chain to be amplified into two single strands by heat treatment, and (2) adjusting the temperature to a level suitable for the enzymatic reaction, then synthesizing complementary strands to each single strand using an enzyme (such as DNA polymerase) present in the reaction system. In other words, one double-stranded oligonucleotide can be amplified into two. By repeating processes (1) and (2) through temperature control, oligonucleotide chains can be amplified with high efficiency in PCR.

[0042] In this invention, "primer" refers to an oligonucleotide that can be annealed to a template oligonucleotide chain and extended by polymerase in a template-dependent manner.

[0043] In this invention, the primer sequence for PCR refers to the sequence of the portion of the oligonucleotide chain that the primer anneals to, and is preferably a PCR-suitable sequence known in the art, and is preferably located at the end of the oligonucleotide chain.

[0044] In this invention, "nick" refers to a region in a double-stranded oligonucleotide chain where internucleotide bonds are lacking, resulting in a break in the oligonucleotide chain. The 5' end of this missing region may or may not have a phosphate group.

[0045] In this invention, a gap refers to a region in a double-stranded oligonucleotide chain where one or more consecutive nucleotides are deleted, causing the oligonucleotide chain to separate. The 5' end of the deleted region may or may not have a phosphate group.

[0046] In this invention, a hairpin strand is a single-stranded structure in which two complementary nucleic acid strands are linked together, and the characteristics of hairpin strands and hairpin strand DEL are as described above. The terms "hairpin region," "hairpin structure," and "hairpin type" used in this invention are understood to be terms derived from the hairpin, which is the same concept as the aforementioned "hairpin strand."

[0047] In this invention, nucleic acid ligation and nucleic acid linking reactions refer to reactions that link the ends of nucleic acids together.

[0048] Enzymatic nucleic acid ligation refers to a reaction in which the ends of nucleic acids are linked together using enzymes.

[0049] Enzymes that can be used in nucleic acid ligation reactions include, for example, DNA ligase, RNA ligase, DNA polymerase, RNA polymerase, or topoisomerase.

[0050] In one aspect, a DNA ligase is an enzyme that connects the ends of DNA strands with phosphate diester bonds. In another aspect, a DNA ligase is understood as a ligase belonging to EC number 6.5.1.1 or 6.5.1.2. DNA ligases are also called polydeoxyribonucleotide synthases or polynucleotide ligases. Examples of DNA ligases include DNA ligases I, II, III, IV, and T4 DNA ligase.

[0051] In one aspect, RNA ligases are enzymes that connect the ends of RNA chains with phosphate diester bonds. In another aspect, RNA ligases are understood as ligases belonging to EC number 6.5.1.3. Also, in another aspect, RNA ligases belong to the poly(ribonucleotide):poly(ribonucleotide) ligase family. RNA ligases are also called polyribonucleotide synthases or polyribonucleotide ligases.

[0052] In this invention, chemical ligation refers to a reaction that joins the ends of nucleic acids without the use of enzymes.

[0053] In chemical ligation, a linkage is formed when the ends of nucleic acids, which have functional groups that are paired for the chemical reaction, react with each other. The functional groups that pair for chemical reactions include, for example, pairs of an optionally substituted alkynyl group and an optionally substituted azide group; pairs of an optionally substituted diene having a 4π electron system (e.g., an optionally substituted 1,3-unsaturated compound, e.g., optionally substituted 1,3-butadiene, 1-methoxy-3-trimethylsilyloxy-1,3-butadiene, cyclopentadiene, cyclohexadiene, or furan) and an optionally substituted dienophile or optionally substituted heterodienophile having a 2π electron system (e.g., an optionally substituted alkenyl group or an optionally substituted alkynyl group); pairs of an optionally substituted amino group and a carboxylic acid group; pairs of a phosphorothioate group and an iodo group (e.g., a 3'-terminal phosphorothioate group and a 5'-terminal iodo group); or pairs of a phosphate group and a hydroxyl group (e.g., a 5'-terminal phosphate group and a 3'-terminal hydroxyl group, or a 5'-terminal hydroxyl group and a 3'-terminal phosphate group). Chemical ligation is a concept well known to those skilled in the art, and those skilled in the art can achieve chemical ligation appropriately based on common technical knowledge. See also Artificial DNA; PNA&XNA, 2014, Vol. 5, e27896, Current Opinion in Chemical Biology, 2015, Vol. 26, pp. 80-88.

[0054] In this invention, "selectively cleavable" means that, in a given compound, only a specific site can be selectively cleaved under predetermined conditions without altering the rest of the molecular structure of the compound.

[0055] In this invention, "selectively cleavable site" means a site in a compound that can be selectively cleaved under predetermined conditions.

[0056] In one embodiment, a preferred structure for the "selectively cleavable site" in the present invention is a "selectively cleavable nucleic acid." This site may be composed of multiple nucleic acids and be cleaved by a specific sequence, or it may be composed of a single nucleic acid. When the cleavable site is a nucleic acid, it is preferable from the viewpoint that (1) established manufacturing methods such as nucleic acid synthesizers can be used, resulting in good manufacturing efficiency, and (2) since the reaction conditions for constructing the building blocks of DEL require that the nucleic acid in the DNA tag portion not be degraded, if the cleavable site is a nucleic acid, it will also not be degraded.

[0057] A more preferred structure for the aforementioned "selectively cleavable nucleic acid" is a nucleic acid containing nucleotides not included in the DNA tag sequence of DEL. If the cleavable sites are nucleotides not included in the DNA tag sequence, it can be used without limiting the DNA tag sequence in order to avoid cleaving the DNA tag portion.

[0058] Deoxyadenosine, deoxyguanosine, thymidine, and deoxycytidine are preferred nucleic acids for use in DNA tag sequences. Therefore, the preferred structure for the selectively cleavable site is a nucleic acid that is neither deoxyadenosine, deoxyguanosine, thymidine, nor deoxycytidine.

[0059] An example of a "selectively cleavable site" is a "nucleotide containing a cleavable base." For example, in DEL, the N-glycosidic bond between the base and sugar portions of a "nucleotide containing a cleavable base" is cleaved by the action of DNA glycosylase, leaving a debasic site. The phosphodiester bond adjacent to the debasic site is cleaved by changes in chemical conditions (e.g., increased temperature, basic hydrolysis, etc.) or by enzymes with depurine / depyrimidine (AP) endonuclease activity or AP lyase activity (e.g., endonuclease III, endonuclease IV, endonuclease V, endonuclease VI, endonuclease VII, endonuclease VIII, APE1 (human-derived AP endonuclease), Fpg (formamidopyridine-DNA glycosylase), etc.), forming a one-base gap or nick.

[0060] Examples of "nucleotides with cleavable bases" include deoxyuridine, bromodeoxyuridine, deoxyinosine, 8-hydroxydeoxyguanosine, 3-methyl-2'-deoxyadenosine, N6-etheno-2'-deoxyadenosine, 7-methyl-2'-deoxyguanosine, 2'-deoxyxanthosine, and 5,6-dihydroxydeoxythymidine. Other nucleotides with cleavable bases are obvious to those skilled in the art. By incorporating these "nucleotides with cleavable bases" into DEL and using a DNA glycosylase that specifically recognizes their structure, the DEL can be selectively debased.

[0061] In this invention, DNA glycosylase refers to any enzyme having glycosylase activity that recognizes any nucleic acid base portion in an oligonucleotide, cleaves the N-glycosidic bond between the base portion and the sugar portion, and creates a debase site. Examples include uracil DNA glycosylase (recognizes deoxyuridine), alkyladenine DNA glycosylase (recognizes 3-methyl-2'-deoxyadenosine, 7-methyl-2'-deoxyguanosine, and deoxyinosine), Fpg (recognizes 8-hydroxydeoxyguanosine), endonuclease VIII (recognizes degraded pyrimidine bases such as 5,6-dihydroxydeoxythymidine and uracil glycol), and SUMG1 (abbreviation for single-strand selective uracil DNA glycosylase, which recognizes deoxyuridine).

[0062] In the present invention, more preferred examples of "selectively cleavable sites" include deoxyinosine and deoxyuridine.

[0063] In the present invention, a particularly preferred example of a "selectively cleavable site" is deoxyuridine.

[0064] In one embodiment, the "selectively cleavable site" in the present invention is preferably cleaved using an enzyme. Enzymes are generally preferred because they have high substrate specificity and do not recognize the DNA tag portion of DEL or the compound portion constructed from multiple building blocks as substrates, but only recognize and act on the "selectively cleavable site". Alternatively, cleavage using the enzyme may be achieved by first structurally altering the "selectively cleavable site" with an enzyme, and then changing the chemical conditions. Examples of such enzymes include glycosylases and nucleases.

[0065] In this invention, glycosylase is an enzyme that has the function of hydrolyzing glycosidic bonds (covalent bonds formed by dehydration condensation between a sugar molecule and another organic compound). Among these, DNA glycosylase, as mentioned above, is an enzyme that recognizes the nucleic acid base portion in oligonucleotides and hydrolyzes its glycosidic bonds.

[0066] In this invention, a nuclease is an enzyme that has the function of hydrolyzing the phosphodiester bond between the sugar and phosphate of nucleic acids. Nucleases include, for example, AP endonuclease, nickel endonuclease, and ribonuclease.

[0067] As described above, AP endonuclease cleaves phosphodiester bonds adjacent to debasement sites generated by the action of any DNA glycosylase. Therefore, in the present invention, it is preferable to use DNA glycosylase and AP endonuclease in combination.

[0068] Nicking endonucleases (e.g., Nb.BbvCI, Nb.BsmI, Nb.BsrDI, etc.) recognize specific DNA sequences and produce nicks in which the phosphodiester bond is cleaved on only one strand of the double helix. Endonuclease V can also produce nicks in which the second phosphodiester bond is cleaved in the 3' direction from deoxyinosine, and is useful in carrying out the present invention.

[0069] Ribonucleases are enzymes that degrade RNA. In this invention, ribonucleosides are used as "selectively cleavable sites," and can be utilized by acting with ribonucleases. RNaseHII, a type of ribonuclease, can create nicks by cleaving the phosphodiester bond at the 5' end of ribonucleotides incorporated into the DNA sequence, and is useful in carrying out this invention.

[0070] In this invention, USER (registered trademark) means "Uracil-Specific Excision Reagent" Enzyme. USER is an endonuclease cocktail that removes uracil, containing uracil DNA glycosylase (UDG) and endonuclease VIII. USER removes uracil from double-stranded DNA, creating a single-base gap and cleaving the DNA strand. In the USER process, UDG first removes the uracil base to create a debase site. Subsequently, the endonuclease breaks down the phosphodiester bond, releasing deoxyribose without a base and creating a single-base gap. In this specification, USER® enzyme and USER® Enzyme refer to USER® as defined above.

[0071] In this invention, an exonuclease is an enzyme that has the function of sequentially hydrolyzing phosphodiester bonds from the 5' or 3' end of a nucleic acid. Examples of exonucleases include lambda exonuclease, exonuclease III, and T7 exonuclease.

[0072] Lambda exonuclease is an enzyme that degrades double-stranded DNA in which the 5' end is phosphorylated. In this invention, it is useful in the process of converting double-stranded DEL to single-stranded DEL.

[0073] In this invention, a building block is a portion having a functional group that can constitute a part of a compound, and may be in the form of a compound.

[0074] In this invention, a nucleotide sequence that can identify each building block refers to a specific nucleotide sequence designed to correspond to the structure of each building block. Designing a sequence means assigning a nucleic acid nucleotide sequence to each structure, for example, nucleic acid nucleotide sequence AAA to building block structure A, nucleic acid nucleotide sequence TTT to structure B, and nucleic acid nucleotide sequence CGC to structure C. Sequences can be freely designed insofar as the objective of this invention is achieved. For example, any number of nucleotide sequences can be assigned to a single building block.

[0075] In this invention, an oligonucleotide tag is a substructure that includes an oligonucleotide containing a base sequence capable of identifying the structure of a substructure constructed by building blocks. In this invention, an oligonucleotide tag may be an oligonucleotide corresponding to each building block, or it may be a longer-chain oligonucleotide containing oligonucleotides corresponding to multiple building blocks. The nucleotides constituting the oligonucleotide tag of the present invention are not limited as long as they achieve the effects of the present invention, but it is desirable that they be nucleotides suitable for amplification by PCR and analysis by sequencer, in terms of ease of these operations. Examples of such preferred nucleotides include nucleotides having the aforementioned natural nucleic acid base as the base part and the aforementioned ribose or 2'-deoxyribose as the sugar part, and more preferred examples include deoxyadenosine, thymidine, deoxycytidine, or deoxyguanosine.

[0076] (Headpiece) In this invention, "headpiece" refers to a starting compound for the production of a compound library such as DEL. The structure of the headpiece of this invention is not limited insofar as it achieves the objectives of the invention, but in its most typical embodiment, it has at least one site to which building blocks can be attached, at least one site to which oligonucleotide tags can be attached, and further includes at least one selectively cleavable site in the structure. As described below, the DNA tag is preferably a double-stranded oligonucleotide chain, and there are preferably two sites where the oligonucleotide tag can be attached.

[0077] In one embodiment, the headpiece is a compound shown in the schematic diagram below. [ka]

[0078] In one aspect, it is desirable that the headpiece be chemically stable. Furthermore, in one embodiment, it is preferable that the headpiece has a structure that allows the DNA tag and building block to be placed in appropriate spaces. In one embodiment, it is preferable that the headpiece has a moderate degree of flexibility. Here, we will further explain appropriate spatial arrangement and flexibility (structural characteristics of the headpiece). Note that the structural characteristics of the headpiece described here may be achieved by the headpiece alone, or by combining the headpiece with a bifunctional spacer. In one embodiment, a preferred structural characteristic of the headpiece is one in which the headpiece and DNA tag do not inhibit the formation reaction of the building block, and conversely, the headpiece and building block do not inhibit the extension reaction of the DNA tag. In one embodiment, a preferred structural characteristic of the headpiece is one in which the headpiece or DNA tag portion does not affect the interaction between the building block compound (library compound) and the target (target protein, etc.). In one embodiment, a preferred structural characteristic of the headpiece is one in which the DNA tag and the building block region are oriented on opposite sides (for example, more than 90 degrees opposite). In one embodiment, a preferred structural characteristic of the headpiece is that the loop portion of the headpiece and the building block are separated by a few atoms to more than ten atoms in terms of the organic compound skeleton. In one embodiment, it is preferable that the headpiece has a moderate affinity for the DNA tag portion and the building block portion. Moderate affinity means, for example, chemical reactivity and stability that allow for the formation, maintenance, and cleavage of bonds under desired conditions in order to carry out the present invention. In this invention, a bifunctional spacer means a spacer portion having at least two reactive groups that enable bonding between the building block portion and the headpiece.

[0079] In this description of the present invention, the terms "headpiece," "headpiece compound," and "compound for headpiece" refer to compounds of the same concept. In the description of this invention, "compound used as a headpiece" can be understood essentially the same as "use of a compound as a headpiece" from the perspective of use, and essentially the same as "method of using a compound as a headpiece" from the perspective of method. The same applies to the compound library.

[0080] The following describes a preferred headpiece structure, but the headpiece structure is not limited as long as it achieves the effects of the present invention.

[0081] In one aspect, the headpiece is, (D) A reactive functional group having at least one site that can be directly connected to a building block or indirectly connected via a bifunctional spacer, (L) Linker extending from reactive functional group, (E) A first oligonucleotide chain having one binding site that can be linked to one of the oligonucleotide tags, (F) A second oligonucleotide chain having one binding site which can be linked to the other chain of the oligonucleotide tag, and (LP) Loop portion that binds to the linker and the two oligonucleotide chains, It is composed of, At least one of the parts E, F, or LP has at least one selectively cleavable portion.

[0082] In one embodiment, the headpiece is a compound represented by the following formula (I). [ka] (In the formula, E and F are independent of each other) It is an oligomer composed of nucleotides or nucleic acid analogs, However, E and F contain complementary base sequences and form a double-stranded oligonucleotide. LP is the loop section, L is a linker, D is a reactive functional group. It is a compound represented by the following: A compound having at least one selectively cleavable site at any one of the sites E, F, or LP.

[0083] In this invention, the substructure of the loop portion that connects to the linker may be referred to as the connecting portion or (LS). Furthermore, in this invention, E-LP-F may be collectively referred to as the hairpin portion.

[0084] (First and second oligonucleotide chains) Preferred embodiments of the first oligonucleotide chain (E) and the second oligonucleotide chain (F) are described below.

[0085] Preferably, the first oligonucleotide chain (E) and the second oligonucleotide chain (F) form a double helix intramolecularly via a loop region (LP), so that the headpiece forms a hairpin structure. The preferred chain length for intramolecular double helix formation is 3 bases or more, more preferably 4 bases or more, and even more preferably 6 bases or more. The chain lengths of E and F are, in one embodiment, 3 to 40, respectively. The chain lengths of E and F are each 4 to 40 in one embodiment. The chain lengths of E and F are each 6 to 25 in one embodiment.

[0086] The site to which the oligonucleotide tag is linked is preferably a structure suitable for enzymatic ligation or chemical ligation. In one embodiment, the ligation of the headpiece and the oligonucleotide tag is carried out by double-stranded ligation using an enzyme. In that case, the first and second oligonucleotide strands preferably form overhanging ends for ligation. The chain length of the overhanging end is preferably 2 bases or more, more preferably 2 to 10 bases, and even more preferably 2 to 5 bases. Therefore, one of the first and second oligonucleotide strands is preferably longer than the other strand by the chain length of the overhanging end. Also, for ligation by DNA ligase, the 5'-end of the strand having the 5'-end of the headpiece among the first and second oligonucleotide strands is preferably phosphorylated.

[0087] Also, the first and second oligonucleotide strands may contain part or all of a primer binding sequence for PCR. A suitable chain length as the primer binding sequence is 17 to 25 bases.

[0088] (Linker) Preferred embodiments of the linker (L) are described below. As described above, the linker is a site that extends from a reactive functional group and binds to a linking site. Typically, the linker is a divalent group (-L-) derived from the following embodiments.

[0089] In one embodiment, the linker is the following embodiment (L1). (L1) A C1-C20 aliphatic hydrocarbon which may have a substituent and may be replaced by 1 to 3 heteroatoms, or (2) a C6-C14 aromatic hydrocarbon which may have a substituent.

[0090] Other embodiments of L include the following embodiments (L2), (L3), (L4), or (L5). (L2) A C1-6 aliphatic hydrocarbon which may have substituents, a C1-6 aliphatic hydrocarbon which may be replaced by one or two oxygen atoms, or a C6-10 aromatic hydrocarbon which may have substituents. (L3) A C1-6 aliphatic hydrocarbon substituted with substituent group ST1, or a benzene substituted with substituent group ST1. Here, substituent group ST1 is composed of C1-6 alkyl groups, C1-6 alkoxy groups, fluorine atoms, and chlorine atoms. However, when substituent group ST1 is substituted with an aliphatic hydrocarbon, alkyl groups are not selected from substituent group ST1. (L4) Benzenes that are C1-6 alkyl or unsubstituted, or substituted with one or two C1-3 alkyl or C1-3 alkoxy groups. (L5) C1-6 alkyl groups.

[0091] (Reactive functional group) The following describes preferred embodiments of the reactive functional group (D). As described above, the reactive functional group has at least one site that can be directly linked to the building block or indirectly linked via a bifunctional spacer, and is a site that bonds to the linker group. Typically, the reactive functional group becomes a monovalent group (D-) in the headpiece and a "divalent group derived from the reactive functional group" (-D-) in the DEL. For example, if D is an amino group, the specific structure of (D-) is (R-HN-) (where R is a substituent as described below). For example, it reacts with an activated carboxyl group, a reactive sulfonyl group, or an isocyanate group to form an amide bond, a sulfonamide bond, or a urea bond, respectively. In this case, the specific structure of (-D-) becomes (-NR-). R is not limited insofar as the effects of the present invention are achieved, but in the following embodiments (D1) to (D5), R is preferably (1) a hydrogen atom, or (2) a C1-6 alkyl group that is unsubstituted or substituted with one to three substituents selected individually or differently from the group of substituents consisting of C1-6 alkoxy groups, fluorine atoms, and chlorine atoms. R is more preferably a hydrogen atom or a C1-3 alkyl group, and even more preferably a hydrogen atom. Furthermore, for example, if (D-) is a methylene group having a leaving group (X-), the specific structure of (D-) is (X-CH2-), and it reacts with nucleophiles such as an amino group, a hydroxyl group, or a thiol group to form a carbon-nitrogen bond, a carbon-oxygen bond, or a carbon-sulfur bond. In this case, the specific structure of (-D-) is (-CH2-). Also, for example, if (D-) is an aldehyde group, the specific structure of (D-) is (HOC-). The aldehyde group forms a carbon-nitrogen bond through a reductive amination reaction with an amino group, for example, in which case (-D-) becomes -CH2-; it forms a carbon-carbon double bond through a reaction with a phosphorus ylide group, for example, in which case (-D-) becomes -CH=; and it forms a carbon-carbon triple bond through a reaction with an α-diazophosphonate group, for example, in which case (-D-) becomes -C≡.

[0092] In one embodiment, part (D-) is as follows: (D1). (D1) C-C, amino, ether, carbonyl, amide, ester, urea, sulfide, disulfide, sulfoxide, sulfonamide, or functional group capable of forming a sulfonyl bond. (Literally, in this case, (-D-) represents a C-C bond, amino, ether, carbonyl, amide, ester, urea, sulfide, disulfide, sulfoxide, sulfonamide, or sulfonyl bond.)

[0093] Other embodiments include (D-) being the following embodiments (D2), (D3), (D4), or (D5). (D2) A C1 hydrocarbon having a leaving group, an amino group, a hydroxyl group, a precursor of a carbonyl group, a thiol group, or an aldehyde group. In this case, (-D-) could be -(C1 hydrocarbon)-, -NR-, -O-, -(C=O)-, -S-, -CH2-, -CH=, or -C≡, etc. (D3) C1 hydrocarbons having halogen atoms, C1 hydrocarbons having sulfonic acid leaving groups, amino groups, hydroxyl groups, carboxyl groups, halogenated carboxyl groups, thiol groups, or aldehyde groups. In this case, (-D-) could be -(C1 hydrocarbon)-, -NR-, -O-, -(C=O)-, -S-, -CH2-, -CH=, or -C≡, etc. (D4) -CH2Cl, -CH2Br, -CH2OSO2CH3, -CH2OSO2CF3, amino group, hydroxyl group, or carboxyl group. In this case, (-D-) will be -CH2-, -NR-, -O-, or -(C=O)-, respectively. (D5) Primary amino group. In this case, (-D-) becomes -NH-.

[0094] The preferred configuration of the loop portion (LP) is described below. The loop portion (LP) is preferably designed so that the first oligonucleotide chain (E) and the second oligonucleotide chain (F) form a double helix within the molecule, allowing the headpiece to form a hairpin structure. In other words, the loop portion (LP) is preferably designed to have a chain length and bond flexibility that makes the loop structure thermodynamically stable. Therefore, in one form, the loop section (LP) is as follows: LP, This is the loop region represented by (LP1)p-LS-(LP2)q, LS is a substructure selected from the group of compounds described in (A) to (C) below, (A) Nucleotides (B) Nucleic acid analogs (C) A trivalent group of C1 to C14 which may have a substituent LP1 is each partial structure selected independently or differently in p numbers from the group of compounds described in the following (1) and (2), (1) Nucleotide (2) Nucleic acid analog LP2 is each partial structure selected independently or differently in q numbers from the group of compounds described in the following (1) and (2), (1) Nucleotide (2) Nucleic acid analog The total number of p and q is 0 to 40.

[0095] A more preferred embodiment of the loop site is as described above. Hereinafter, the structure of the loop site will be further supplemented.

[0096] Here, the nucleotide is the natural nucleotide described above, and the nucleic acid analog is as described above.

[0097] Here, LP1 is each partial structure selected independently or differently in p numbers from the group of compounds described in the following (1) and (2), and LP2 is each partial structure selected independently or differently in q numbers from the group of compounds described in the following (1) and (2). (1) Nucleotide (2) Nucleic acid analog "Selected independently or differently in p numbers" means that, for example, when p is 4, LP1 can be selected independently or differently from the group of compounds described in (1) and (2), such as AATG, ATCG, TC(d-Spacer)G or A(d-Spacer)(d-Spacer)C. The same applies to LP2.

[0098] Also, the loop site may contain part or all of the primer binding sequence for PCR.

[0099] (Regarding LS) In one embodiment, LS is (A) a nucleotide or (B) a nucleic acid analog. When LS is (A) a nucleotide or (B) a nucleic acid analog, the loop region becomes a nucleic acid oligomer. The nucleic acid oligomer of the present invention is an oligomer formed by linking nucleotides or nucleic acid analogs as monomers. An oligomer can also be called a chain-like compound. Therefore, the nucleic acid oligomer of the present invention is any of the following: an oligonucleotide chain, a nucleic acid analog chain, or a mixed chain of nucleotides and nucleic acid analogs.

[0100] If the LS is (A) a nucleotide or (B) a nucleic acid analog, the loop region becomes a nucleic acid oligomer. In that case, the headpiece can be manufactured using a nucleic acid synthesizer, which is significantly preferable in practice.

[0101] When the LS is (A) a nucleotide or (B) a nucleic acid analog, in the manufacture of the headpiece, in one embodiment, a monomer for nucleic acid synthesis is prepared in which the linker site (L) and the reactive functional group site (D) are bound to the LS, and then the nucleic acid oligomer is synthesized. Examples of such nucleic acid synthesis monomers include the aforementioned Amino C6 dT, mdC (TEG-Amino), and Uni-Link (trademark registered) Amino Modifier. In this embodiment, for example, in the structure of the monomer mdC(TEG-Amino), the nucleotide portion corresponds to the linking site (LS), and the side chain portion extending from the base corresponds to the linker site (L) and the reactive functional group site (D). In preparation, the reactive functional group (D) may be protected with a protecting group.

[0102] In that case, one possible embodiment is the following compound (B6): (B6) A compound in which the (-LD) is bonded to the base portion of a nucleotide.

[0103] In one embodiment, the nucleic acid analog is one of the following compounds: (B61), (B62), (B63), (B64), or (B65). (B61)(-LD) is (-L1-D1) (B6) (B62)(-LD) is equal to (-L2-D2) (B6). (B63)(-LD) is (-L3-D3) (B6). (B64)(-LD) is (-L4-D4) (B6). A compound described in any of (B61) to (B64), wherein (B65)(-D) is (-D5).

[0104] When LS is (A) a nucleotide or (B) a nucleic acid analog, in the manufacture of the headpiece, one embodiment is to first synthesize the nucleic acid oligomer and then attach the linker site (L) and the reactive functional group site (D). In that case, it is preferable to include the "specific nucleic acid analog" to which the linker site binds as the linking site (LS) within the hairpin site (nucleic acid analog oligomer). Examples of such "specific nucleic acid analogs" include the aforementioned Amino C6 dT, mdC (TEG-Amino), and Uni-Link (trademark registered) Amino Modifier. In this embodiment, for example, mdC(TEG-Amino) itself corresponds to the linking site (LS), and the addition sites to which further bond from the base side chain correspond to the linker site (L) and the reactive functional group site (D).

[0105] (Regarding p and q) As described above, the chain length of the loop portion is preferably such that the first oligonucleotide chain (E) and the second oligonucleotide chain (F) form a double helix within the molecule, and the headpiece forms a hairpin structure. In one possible scenario, the total number of p and q ranges from 1 to 40. In one scenario, the total number of p and q is between 2 and 20. In one possible scenario, the total number of p and q is between 2 and 10. In one possible scenario, the total number of p and q is between 2 and 7.

[0106] In one embodiment, the loop portion of the present invention is (A) Nucleotides and consists of the following nucleic acid analogs (B41), (B42), (B43), (B44), or (B52). (B41)d-Spacer (B42) Amino C6 dT (B43)mdC(TEG-Amino) (B44) Uni-Link (Trademark Registered) Amino Modifier (B52) Triethylene glycol phosphate

[0107] In one embodiment, LS is preferably B42, B43, or B44. In another embodiment, LP1 and LP2 are preferably A, B41, or B52.

[0108] In one embodiment, the loop region is a nucleic acid oligomer with the sequences described in (X1) to (X9) below. (X1) A-B41-B42-B41-A (X2) A-B41-B43-B41-A (X3) A-B41-B44-B41-A (X4)B41-B41-B42-B41-B41 (X5)B41-B41-B43-B41-B41 (X6)B41-B41-B44-B41-B41 (X7)B52-B42-B52 (X8)B52-B43-B52 (X9)A52-A44-A52

[0109] In the aforementioned headpiece, the number of cuttable parts is preferably five or less, and more preferably one to two.

[0110] In the headpiece described above, if there are two or more cleavable sites, it is preferable that at least one cleavable site is located in the first oligonucleotide chain or between the first oligonucleotide chain and the linker binding site, and at least one cleavable site is located in the second oligonucleotide chain or between the second oligonucleotide chain and the linker binding site.

[0111] In one embodiment, in the headpiece described above, the location of the severable portion is preferably within 20 bases, more preferably within 10 bases, and even more preferably within 3 bases, starting from the binding portion between the loop portion and the first oligonucleotide chain or the second oligonucleotide chain.

[0112] In one aspect, the present invention provides appropriate conditions for a method of inducing and evaluating DEL containing cleavable sites in a DNA strand into crosslinker-modified double-stranded DEL. In one embodiment, in the headpiece, the location of the severable portion is in the 3' direction, starting from the binding portion between the loop portion and the first oligonucleotide chain or the second oligonucleotide chain, and is preferably within 20 bases, more preferably within 10 bases, even more preferably within 3 bases, and most preferably within 1 base.

[0113] Just to clarify, the preferred embodiment of the "selectively severable region" and preferred embodiments such as E, F, or LP are distinct concepts. That is, even if the location of the "selectively severable region" is included in E, the preferred embodiment of E does not necessarily apply to the "selectively severable region."

[0114] In one embodiment, the compound constituting DEL of the present invention is a compound represented by the following formula (II). [ka] (In the formula, X and Y are nucleotide chains, E and F are independent of each other. It is an oligomer composed of nucleotides or nucleic acid analogs, However, E and F contain complementary base sequences and form a double-stranded oligonucleotide. LP is the loop section, L is a linker, D is a divalent group derived from a reactive functional group, Sp is a bond or a bifunctional spacer, An is a substructure composed of at least one building block. It is a compound represented by the following: X and Y have sequences that can form a double helix in at least part of their structure. X binds to E at its 5' end. Y binds to F at its 3' end. A compound having at least one selectively cleavable site at any one of the sites E, F, or LP.

[0115] In one embodiment, preferred embodiments of E, F, LP, L, and D in the compound represented by formula (II) above are the same as preferred embodiments of E, F, LP, L, and D described with respect to formula (I) above. Preferred embodiments of X, Y, Sp, and An will be described separately.

[0116] (2 Functional Spacers) As described above, a bifunctional spacer is a spacer portion having at least two reactive groups that enable the bonding of a substructure An of the compound library to the headpiece. In one embodiment, the bifunctional spacer is SpD-SpL-SpX. SpX is a reactive group that forms a covalent bond with the reactive functional group of the headpiece. SpD is a reactive group that forms a covalent bond with the substructure An in the compound library. SpL is a chemically inert spacing portion. Furthermore, similar to the reactive functional group (D), the reactive group (SpX) is a monovalent group (-SpX) in the difunctional spacer alone (the reagent state before bonding with the headpiece), and in DEL (the state bonded with the headpiece), it becomes a "divalent group derived from the reactive group" (-SpX-) based on the aforementioned (-SpX). Similarly, the reactive group (SpD) is a monovalent group (SpD-) before bonding with An, and in DEL (the state bonded with An), it becomes a "divalent group derived from the reactive group" (-SpD-) based on the aforementioned (SpD-).

[0117] A preferred embodiment of SpX is a reactive group that forms an amino, carbonyl, amide, ester, urea, or sulfonamide bond. In one embodiment, SpX is a suitable reactive group when the reactive functional group of the headpiece is an amino group, and has the following structures: (SpX1), (SpX2), or (SpX3). (SpX1): Carboxylate group, halogenated carboxylate group, aldehyde group, or halogenated sulfonyl group (SpX2): Carboxylate group or halogenated sulfonyl group (SpX3): Carboxy group

[0118] The preferred embodiment of SpD is the same as that of D described above. In one embodiment, SpD is one of the aforementioned (D1), (D2), (D3), (D4), or (D5).

[0119] A preferred embodiment of SpL is as follows: In one embodiment, SpL is the aforementioned (L1), (L2), (L3), (L4), or (L5). In one form, SpL is (SpL1), (SpL2), or (SpL3). (SpL1) Polyalkylene glycol, polyethylene, C1-20 aliphatic hydrocarbons, peptides, oligonucleotides, or combinations thereof, which may optionally be replaced by heteroatoms. (SpL2) Polyalkylene glycol, polyethylene, C1-10 aliphatic hydrocarbons, or peptides (SpL3) Polyethylene glycol, or polyethylene

[0120] One embodiment of a bifunctional spacer is as follows: (Sp1):(D4)-(SpL1)-(SpX1) (Sp2):(D4)-(SpL2)-(SpX2) (Sp3):(D4)-(SpL3)-(SpX3) (Sp4):(D5)-(SpL1)-(SpX1) (Sp5):(D5)-(SpL2)-(SpX2) (Sp6):(D5)-(SpL3)-(SpX3)

[0121] In one embodiment, the (Sp-DL) portion of the compound constituting DEL is configured as follows: (SpDL1), (SpDL2), (SpDL3), (SpDL4), (SpDL5), (SpDL6), (SpDL7), (SpDL8), (SpDL9), or (SpDL10). (SpDL1):(D4)-(L1) (SpDL2):(D5)-(L1) (SpDL3):(D4)-(L2) (SpDL4):(D5)-(L2) (SpDL5):(Sp1)-(D5)-(L5) (SpDL6):(Sp2)-(D5)-(L5) (SpDL7):(Sp3)-(D5)-(L5) (SpDL8):(Sp4)-(D5)-(L5) (SpDL9):(Sp5)-(D5)-(L5) (SpDL10):(Sp6)-(D5)-(L5) In (SpDL1), (SpDL2), (SpDL3), and (SpDL4), Sp represents a connection.

[0122] In carrying out the present invention, it is advantageous that the headpiece can be synthesized in a nucleic acid synthesis apparatus. In such a carrying out, as described above, in one embodiment, a nucleic acid synthesis monomer in which the linker moiety (L) and the reactive functional group moiety (D) are bound to LS can be prepared, and then a nucleic acid oligomer can be synthesized. Examples of such nucleic acid synthesis monomers include the aforementioned Amino C6 dT, mdC (TEG-Amino), and Uni-Link (trademark registered) Amino Modifier. On the other hand, when using commercially available nucleic acid synthesis monomers or nucleic acid analogs usable with nucleic acid synthesizers as described above, the length of the linker region may be limited. In such cases, one embodiment is to introduce an appropriate bifunctional spacer, which makes it possible to adjust the distance between the headpiece and An, and is advantageous in carrying out the invention.

[0123] In the description of this invention, terms such as "C1-C6 alkyl group" and "C1-6 alkyl group" mean that the number of carbon atoms is 1 to 6. Similarly, when m and n are integers, the description "Cm-Cn" and "Cm-n" means that the number of carbon atoms is m to n. Therefore, "C1-C6 alkyl group" and "C1-6 alkyl group" mean an alkyl group having 1 to 6 carbon atoms, and "C1-C6 alkylene" and "C1-6 alkylene" mean an alkylene having 1 to 6 carbon atoms.

[0124] In this invention, "C1-6 alkyl" refers to a linear or branched alkyl group having 1 to 6 carbon atoms. Specific examples include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, and hexyl.

[0125] In this invention, "C1-3 alkyl" refers to a linear or branched alkyl group having 1 to 3 carbon atoms. Specific examples include methyl, ethyl, propyl, and isopropyl alkyl groups.

[0126] In this invention, "C1-6 alkoxy" refers to a linear or branched alkoxy having 1 to 6 carbon atoms. Specific examples include methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentyloxy, and hexyloxy.

[0127] In this invention, "C1-3 alkoxy" refers to a linear or branched alkoxy having one to three carbon atoms. Specific examples include methoxy, ethoxy, propoxy, and isopropoxy.

[0128] In this invention, "hydrocarbon" means a chain, branched chain, or cyclic saturated or unsaturated compound composed solely of carbon atoms and hydrogen atoms.

[0129] In this invention, "aliphatic hydrocarbon" means a hydrocarbon that is non-aromatic. "Aliphatic hydrocarbon" may be linear, branched, or cyclic, and may be saturated or unsaturated. Specific examples of structures include alkyl, alkenyl, alkynyl, cycloalkyl, or cycloalkenyl structures, or combinations thereof. In this invention, "C1-20 aliphatic hydrocarbons" means aliphatic hydrocarbons having 1 to 20 carbon atoms. In this invention, "C1-10 aliphatic hydrocarbons" means aliphatic hydrocarbons having 1 to 10 carbon atoms. In this invention, "C1-6 aliphatic hydrocarbons" means aliphatic hydrocarbons having 1 to 6 carbon atoms.

[0130] In this invention, "aromatic hydrocarbons" refers to hydrocarbons that are aromatic. In this invention, "C6-14 aromatic hydrocarbons" refers to aromatic hydrocarbons having 6 to 14 carbon atoms. Specific examples include benzene, naphthalene, and anthracene. In this invention, "C6-10 aromatic hydrocarbons" refers to aromatic hydrocarbons having 6 to 10 carbon atoms. Specific examples include benzene and naphthalene.

[0131] The aromatic heterocycle of the present invention is an aromatic heterocycle having, as heteroatoms within its ring structure, elements selected individually or differently from the group consisting of nitrogen, oxygen, and sulfur. In one embodiment, an aromatic heterocycle is a "C1-9 aromatic heterocycle" having 1 to 9 carbon atoms, and in another embodiment, a "C1-9 aromatic heterocycle" is an aromatic heterocycle with 5 to 10 members. In one aspect, an aromatic heterocycle is a "C1-5 aromatic heterocycle" having 1 to 5 carbon atoms, and in another aspect, a "C1-5 aromatic heterocycle" is an aromatic heterocycle with 5 to 6 members. In one embodiment, an aromatic heterocycle is a "C2-9 aromatic heterocycle" having 2 to 9 carbon atoms, and in another embodiment, a "C2-9 aromatic heterocycle" is an aromatic heterocycle with 5 to 10 members. In one aspect, an aromatic heterocycle is a "C2-5 aromatic heterocycle" having 2-5 carbon atoms, and in another aspect, a "C2-5 aromatic heterocycle" is an aromatic heterocycle with 5-6 members.

[0132] The nitrogen-containing aromatic heterocycle of the present invention is an aromatic heterocycle having nitrogen as a heteroatom within its ring structure. In one embodiment, a nitrogen-containing aromatic heterocycle is a "C1-5 nitrogen-containing aromatic heterocycle" having 1 to 5 carbon atoms, and in another embodiment, a "C1-5 nitrogen-containing aromatic heterocycle" is an aromatic heterocycle with 5 to 6 members. In one embodiment, a nitrogen-containing aromatic heterocycle is a "C2-5 nitrogen-containing aromatic heterocycle" having 2-5 carbon atoms, and in another embodiment, a "C2-5 nitrogen-containing aromatic heterocycle" is an aromatic heterocycle with 5-6 members.

[0133] The non-aromatic heterocycle of the present invention is a non-aromatic heterocycle having, as heteroatoms within its ring structure, elements selected individually or differently from the group consisting of nitrogen, oxygen, and sulfur. Non-aromatic heterocycles may contain partially unsaturated bonds. In one embodiment, a non-aromatic heterocycle is a "C2-9 non-aromatic heterocycle" having 2 to 9 carbon atoms, and in another embodiment, a "C2-9 non-aromatic heterocycle" is a "5-10 member non-aromatic heterocycle".

[0134] In this invention, "trivalent group of C1-14" means a trivalent group derived from a compound having 1 to 14 carbon atoms. The structure is not limited as long as the effects of the present invention are achieved.

[0135] In this invention, if it is stated that "it may be replaced by a heteroatom," a heteroatom means an atom other than carbon and hydrogen. The heteroatom is preferably an oxygen atom, a nitrogen atom, a silicon atom, a phosphorus atom, or a sulfur atom, and more preferably an oxygen atom, a nitrogen atom, or a sulfur atom. Therefore, taking propyl (-CH2-CH2-CH3) as an example of a hydrocarbon, the concept of "propyl that may be replaced by heteroatoms" refers to a structure that includes ethers ((-CH2-O-CH3) or (-O-CH2-CH3)) in which the methylene (-CH2-) in the alkyl group is replaced by oxygen, or amines ((-CH2-NH-CH3) or (-NH-CH2-CH3)) in which it is replaced by nitrogen.

[0136] If the present invention states that "substituents may be present," those substituents are not limited as long as they achieve the objectives of the present invention. The substituent is preferably a C1-6 alkyl group, a C1-6 alkoxy group, an amino group, a hydroxyl group, a nitro group, a cyano group, an oxo group, or a halogen atom. The substituent is more preferably a C1-6 alkyl group, a C1-6 alkoxy group, a fluorine atom, or a chlorine atom.

[0137] In this invention, polypeptides and peptides refer to compounds or substructures formed by the linking of amino acids. Amino acids are a general term for organic compounds that have both an amino group and a carboxyl group. The amino acids constituting the polypeptides and peptides of this invention are not particularly limited and include modified amino acids, etc. Following common usage in the field of life sciences, proline (classified as an imino acid) is also included as an amino acid in this invention. The amino acids constituting the polypeptides and peptides of this invention are preferably α-amino acids, and more preferably "amino acids that constitute proteins."

[0138] The halogen atoms of this invention include fluorine atoms, chlorine atoms, bromine atoms, and iodine atoms.

[0139] C-C, amino, ether, carbonyl, amide, ester, urea, sulfide, disulfide, sulfoxide, sulfonamide, and sulfonyl bonds are chemical bonds having chemical structures understood by their respective names. Those skilled in the art will understand, for example, that ether bonds are generally represented as "-O-", and carbonyl bonds are generally represented as "-C(=O)-". Amino, amide, and urea bonds have a hydrogen atom or other substituent on the nitrogen atom, but the structure on the nitrogen atom is not limited as long as it has the effect of the present invention. The substituent on the nitrogen atom is preferably a C1-6 alkyl group or a hydrogen atom, and more preferably a hydrogen atom. It goes without saying that CC bonds mean carbon-carbon bonds. CC bonds include single bonds, double bonds, and triple bonds. In one embodiment, in steps a and / or c of the manufacturing method of the present invention, a bond appropriately selected from the above 11 types is constructed. These 11 types of bonds are particularly basic bonding modes in organic chemistry, and the reactions for constructing them are well known to those skilled in the art. Therefore, in designing and constructing the partial structure An of the compound library of the present invention, those skilled in the art can use these 11 types of bonds in appropriate combinations.

[0140] An organic compound composed of elements selected individually or differently from the group of elements consisting of H, B, C, N, O, Si, P, S, F, Cl, Br, and I is an organic compound constructed by the bonding of the aforementioned 12 elements.

[0141] In one embodiment, the partial structure An of the compound library of the present invention is constructed from the 12 elements listed above. These 12 elements are particularly fundamental elements in organic compounds, and the reactions for constructing them are well known to those skilled in the art. Therefore, when designing and constructing the partial structure An of the compound library of the present invention, those skilled in the art can use these 12 elements in appropriate combinations.

[0142] Low molecular weight organic compounds having substituents selected individually or differently from the group consisting of aryl groups, non-aromatic cyclyl groups, heteroaryl groups, and non-aromatic heterocyclyl groups are low molecular weight organic compounds having a chemical structure understood by each name. Low molecular weight compounds are a concept well known to those skilled in the art, and examples of preferred molecular weights of low molecular weight compounds in the present invention will be mentioned separately.

[0143] The aryl group of the present invention is preferably a C6-10 aryl group, and more preferably a phenyl group.

[0144] The non-aromatic cyclyl group of the present invention is preferably a 5- to 8-membered non-aromatic cyclyl group, and more preferably a 5- or 6-membered non-aromatic cyclyl group. The non-aromatic cyclyl group may contain a partially unsaturated bond.

[0145] The heteroaryl group and non-aromatic heterocyclyl group of the present invention are groups having, as heteroatoms within the ring structure, elements selected individually or differently from the group consisting of nitrogen, oxygen, and sulfur. The heteroaryl group and non-aromatic heterocyclyl group of the present invention are preferably 5-membered to 8-membered groups, more preferably 5-membered or 6-membered groups, and the non-aromatic heterocyclyl group may contain a partially unsaturated bond.

[0146] In one embodiment, the partial structure An of the compound library of the present invention has the four groups described above. These four groups are particularly fundamental partial structures in organic compounds, and the reactions for constructing them in compounds are well known to those skilled in the art. Therefore, when designing and constructing the partial structure An of the compound library of the present invention, those skilled in the art can use these four groups in appropriate combinations.

[0147] The aforementioned preferred embodiments, namely compound libraries constructed with 11 types of bonds, 12 types of elements, and / or 4 types of groups, have particular core value. Those skilled in the art will understand that compound libraries constructed without these preferred embodiments generally have limited applications and, in many cases, limited commercial value.

[0148] The synthesis history of An refers to a record of all operations performed until An is synthesized, and in particular, the structure and sequence of building blocks used until An is synthesized. For example, when a reaction is carried out in two or more separate reaction vessels, each using different building blocks and / or different reaction conditions, the synthesis history is imprinted as sequence information of the oligonucleotide by ligating an oligonucleotide chain with a predetermined sequence to the product in each reaction vessel before or after the reaction. By repeating this operation until An is constructed, an oligonucleotide of Bn with the synthesis history of An is constructed.

[0149] Split-and-pool synthesis is a synthetic method developed by Geysen et al. during the early stages of combinatorial chemistry as a combinatorial chemical method for constructing peptide libraries using solid-phase synthesis. Split-and-pool synthesis is also known as the split-mix method.

[0150] Following the above process, let's explain using the synthesis of a peptide library using solid-phase synthesis as an example. In split-and-pool synthesis, at each peptide end-adding step, instead of cutting the sample from the solid-phase support to which the amino acids are bonded, N types of support are first mixed and homogenized, then divided equally to add the next N types of amino acids.

[0151] In other words, one type of peptide chain is generated for each carrier, and by applying all 20 natural amino acids at each stage, it becomes possible to construct a peptide library that allows for all possible combinations of peptides of a specific length.

[0152] If this peptide library is to be screened for antigen presentation or receptor binding, assays can be performed using peptides on a solid-phase support using methods such as ELISA. In other words, there is no need to cleave the sample peptide from the support; the support particles that react to the assay are picked up (for example, fluorescently labeled support particles of about 0.1 mm are picked up with an optical microscope). Then, the peptides in these particles can be analyzed using an instrumental analyzer (such as a peptide analyzer) to determine the target peptide sequence, or other combinatorial chemical identification methods (such as tagging) can be used to indirectly determine candidate peptide sequences for screening.

[0153] Furthermore, in the manufacturing method of the present invention, we will explain as an example the case in which v types of structures are synthesized when m is 2, and w types of structures are synthesized when m is 3, using split-and-pool synthesis. In this explanation, the process is repeated in the order of (c) and (d). (m=2) In the m=2 step, α2 is added to A1-Sp-C-B1 in step (c) and β2 in step (d), respectively, to produce A2-Sp-C-B2. Here, v types of α2 (α2(av)) structures and their corresponding v types of β2 (β2(av)) are prepared, and steps (c) and (d) are performed for each structure, respectively, to obtain v types of A2-Sp-C-B2 (A2(a)-Sp-C-B2(a), A2(b)-Sp-C-B2(b)...A2(v)-Sp-C-B2(v): i.e., A2(av)-Sp-C-B2(av)). In split-and-pool synthesis, the v types of A2-Sp-C-B2 are mixed and then divided into w parts. More specifically, division means dividing the mixture into w reaction vessels. (m=3) In step m=3, α3 is added to A2-Sp-C-B2 in step (c) and β3 in step (d), respectively, to produce A3-Sp-C-B3. Here, we prepare w types of α3 (α3(aw)) structures and w types of corresponding β3 (β2(aw)), and perform steps (c) and (d) on w (A2(av)-Sp-C-B2(av) mixtures). Then, through steps n=2 and n=3, (v×w) types of A3-Sp-C-B3 can be efficiently synthesized in (v+w) synthesis steps.

[0154] (Biological assessment) When the w products obtained are mixed, a mixture of (v × w) types of A3-Sp-C-B3 compound libraries is obtained. For example, by performing a drug receptor binding test on this mixture, (v × w) types of compounds can be screened in a single step. By washing away the compounds that did not bind to the drug receptor, only the bound compounds can be isolated. In DEL as in the present invention, the DNA of the isolated A3-Sp-C-B3 compound is amplified to a sequence-decipherable amount, and the structure of A3 can be determined from the sequence information.

[0155] Furthermore, terms such as compound library, building block, and split-and-pool are well-known to those skilled in the art in fields such as combinatorial chemistry, and can be used as appropriate by referring to the following literature, etc. (1) Takashi Takahashi, Takayuki Doi, "Combinatorial Chemistry," Journal of the Society of Synthetic Organic Chemistry, 2002, Vol. 60, pp. 426-433. (2) Combinatorial Chemistry Research Group (ed.), "Combinatorial Chemistry", Kagaku Dojin

[0156] A DNA-coding library (or DEL) is a compound library consisting of a group of compounds labeled with DNA or oligonucleotides that have substantially equivalent function to DNA (DNA-coding compounds). Through the split-and-pool synthesis described above, the labeled DNA is imbued with the structure or synthesis history of each compound as sequence information. Due to these characteristics, a DNA-coding library is 10 2 ~10 20 By screening a mixture of various compounds and identifying the DNA sequences contained in the obtained compounds using methods known in the art (e.g., the use of next-generation sequencers and / or microarrays), it becomes possible to identify the structure of the compounds. One aspect of the screening method may involve contacting a target such as a protein with a DNA-coding library and selecting compounds that bind to the target.

[0157] While "biological target" is a term well known to those skilled in the art, in one aspect, in the present invention, "biological target" refers to a group of biological substances that can be targeted in the development of drugs such as pharmaceuticals and agrochemicals, and includes, for example, enzymes (e.g., kinases, phosphatases, methylases, demethylases, proteases, and DNA repair enzymes), proteins: proteins involved in protein-protein interactions (e.g., receptor ligands), receptor targets (e.g., GPCRs), ion channels, cells, bacteria, viruses, parasites, DNA, RNA, prions, or carbohydrates. "Biological activity evaluation" is a term well known to those skilled in the art, but in one aspect, in the present invention, "biological activity evaluation" means evaluating the presence or absence, or strength, of the biological activity (for example, the ability to bind to a biological target, the function of inhibiting enzyme activity, the function of promoting enzyme activity, etc.) of a compound. For specific examples of biological activity evaluation, please refer to the aforementioned Patent Documents 2 and 3, Non-Patent Documents 1 to 6, etc. "Functional evaluation" is a term well known to those skilled in the art, but in one aspect, in the present invention, "functional evaluation" means evaluating the presence or absence, or strength, of a specific function (e.g., binding ability, biological activity, luminescence properties, etc.) of a compound.

[0158] The present invention provides several methods with several advantages regarding DEL and methods for producing DEL by using DNA strands having cleavable sites. Forms 1 to 7 are described in detail below.

[0159] Form 1 The present invention provides a DEL using the aforementioned "hairpin-shaped headpiece having a severable portion".

[0160] As illustrated in Figure 1, in Form 1, DEL is produced by starting with a headpiece containing a first oligonucleotide strand with a cleavable region in the DNA strand, a loop region, and a second oligonucleotide strand, repeatedly performing the binding of building blocks and double-strand ligation of oligonucleotide tags corresponding to the building blocks (three times in Figure 1), and optionally performing double-strand ligation of oligonucleotide tags including a primer region.

[0161] As illustrated in Figure 2, in Form 1, PCR can be performed with high efficiency by using a cleavable site in the first oligonucleotide chain of the headpiece to cleave the cleavable site using a cleavage method such as an enzyme, thereby converting it into a double-stranded oligonucleotide that is not bound at the loop site.

[0162] (Regarding Form 2) As illustrated in Figure 3, in DEL using a "hairpin-shaped headpiece with a severable portion," the severable portion may be located on the second oligonucleotide chain. The features of Form 2 are the same as those of Form 1, except for the severable portion.

[0163] (Regarding Form 3) As illustrated in Figure 4, in DEL using a "hairpin-shaped headpiece with cleavable regions," the cleavable regions may be present on both the first and second oligonucleotide chains. In this embodiment, it is expected that PCR efficiency will be further improved by cleaving the loop region from both oligonucleotide chains.

[0164] (Regarding Form 4) As illustrated in Figure 5, in the present invention, cleavable sites may be present in both the first oligonucleotide chain (E) and the second oligonucleotide chain (F), and the structures of the cleavable sites may be different. In such cases, the cleavage sites can be controlled by utilizing the differences in the characteristics of the two (or more) cleavable sites. For example, deoxyuridine may be used as the cleavable site in the first oligonucleotide chain (E), and deoxyinosine may be used as the cleavable site in the second oligonucleotide chain (F). In this case, the USER enzyme can selectively cleave the deoxyuridine in the first oligonucleotide chain (E). [ka] On the other hand, using alkyladenine DNA glycosylase and endonuclease VIII, it is possible to selectively cleave the deoxyinosine-initiated cleavage site in the second oligonucleotide chain (F). [ka] In this way, by selecting the cutting site as desired, a wider range of DEL modifications becomes possible, and a wider range of evaluation methods can be applied thereafter. This can be expected.

[0165] (Regarding Form 5) As illustrated in Figure 6, the present invention also allows for the provision of a cleavable region in the DNA tag portion (e.g., oligonucleotide chain (Y)). By providing a cleavable region near the end of the DNA tag and cleaving the region as desired, a new protruding end can be generated. [ka] The protruding end can be used as an adhesive end to ligate a desired nucleic acid sequence, such as UMIs (Urban Misidentification Sequences). [ka] After biological evaluation, the selected DEL compounds are given UMIs regions as described above, and DNA sequencing is performed, enabling analysis with reduced amplification bias from PCR. Thus, the present invention provides unprecedented performance in the manufacturing and use of DEL compounds by having a site that can selectively cleave the nucleic acid sequence.

[0166] Here, UMIs (Specific Molecular Identifiers) are molecular identifiers that, when attached to DNA contained in a sample, assign a unique DNA sequence to each individual DNA molecule (see Nature Methods, 2012, Vol. 9, pp. 72-74). By attaching such molecular identifiers before PCR amplification, it becomes possible to identify PCR duplication (sequences originating from the same molecule) when quantifying the number of DNA molecules containing a specific sequence in a sample, thereby enabling quantification with reduced PCR amplification bias.

[0167] (Regarding Form 6) As illustrated in Figure 7, the present invention allows for the use of a combination of cleavable sites and modifying groups or functional molecules, making it possible, for example, to prepare DEL in which hairpin strand DNA has been converted to single-stranded DNA. As shown in Figure 7, a DEL compound using a headpiece with a severable portion in section E is given as an example. (Step A) A double-stranded oligonucleotide chain having a solid-supported, removable modifying group (e.g., biotin) at its 3' end is ligated to the synthesized DEL compound. (Step B) Cut off the parts that can be cut. (Step C) Apply a treatment according to the function of the modifying group. For example, in the case of biotin, use streptavidin beads with biotin affinity to selectively remove the biotin-bound oligonucleotide chain from the system. This makes it possible to obtain DEL containing single-stranded DNA.

[0168] Here, a functional molecule is a molecule that possesses a specific chemical or biological function (e.g., solubility, photoreactivity, substrate-specific reactivity, target protein degradation induction properties), and by conferring this function to DEL, it becomes possible to evaluate and purify DEL according to its function.

[0169] Here, "biotin" refers to all biotin compounds that bind to avidin, including not only vitamin B7 but also, for example, desthiobiotin.

[0170] In one aspect, the present invention provides appropriate conditions for a method of inducing and evaluating DEL containing cleavable sites in a DNA strand into crosslinker-modified double-stranded DEL. Another method for preparing DEL, which converts hairpin strand DNA to single-stranded DNA, involves using the following exonuclease. (Step A) A hairpin-shaped object having a "selectively cleavable site" is cleaved using an enzyme such as USER® Enzyme, which cleaves the object with the 5' end of the cleavage site phosphorylated after the cleavage reaction. (Step B) For example, one oligonucleotide strand with a phosphorylated 5' end is degraded and removed by treatment with lambda exonuclease. This yields single-stranded DEL (single-stranded DEL).

[0171] The single-stranded DEL obtained as described above is preferably a single-stranded DEL having a library molecule in the 3' direction of the oligonucleotide chain. This single-stranded DEL can undergo a primer extension reaction using a crosslinker-modified primer having a crosslinker at the 5' end. This method makes it possible to easily synthesize "crosslinker-modified double-stranded DEL" in which the crosslinker is covalently linked to an oligonucleotide having a coding sequence.

[0172] When obtaining a single-stranded DEL with library molecules in the 3' direction as described above, the "selectively cleavable sites" of the hairpin-shaped DEL used as raw material are located in the 3' direction from the site where the library molecules are bound.

[0173] As illustrated in Figure 8, single-stranded DNA-containing DELs can be given new functions by forming a double helix with modified oligonucleotides having a desired functional site (for example, crosslinker-modified DNA such as photoreactive crosslinkers, or crosslinker-modified primers such as photoreactive crosslinkers). Furthermore, if crosslinker-modified primers are used, the optionally attached primers may be extended to induce crosslinker-modified double-stranded DEL compounds. Such crosslinker-modified double-stranded DEL compounds are useful in the present invention because, after screening, a covalent bond is formed between the biological target and the coding sequence.

[0174] (Regarding Form 7) As illustrated in Figure 9, the present invention allows for the introduction of a crosslinker by utilizing a severable portion. As shown in Figure 9, a DEL compound using a headpiece with a severable portion in section E is given as an example. (Step A) Cut the cleavable parts of the synthesized DEL compound. (Step B) Apply a modified primer having the desired functional site (e.g., a crosslinker-modified primer such as a photoreactive crosslinker). (Step C) The applied primers are extended to synthesize a crosslinker-modified double-stranded DEL compound. In DEL evaluation, crosslinker-modified double-stranded DEL compounds can significantly improve detection sensitivity by allowing the crosslinker to further bind to the target protein when the building block compound (library small molecule compound) binds to the target protein (see Non-Patent Documents 7, 11, etc.). In the practical application of DEL technology, which evaluates a very large number of library compounds, enhancing the affinity of library compounds and improving detection sensitivity is extremely useful. This invention provides a novel and highly efficient method for producing crosslinker-modified double-stranded DEL compounds, and is extremely useful.

[0175] In one aspect, the present invention provides appropriate conditions for a method of inducing and evaluating DEL containing cleavable sites in a DNA strand into crosslinker-modified double-stranded DEL. In one embodiment, it is preferable that the crosslinker-modified double-stranded DEL compound has a crosslinker that is covalently linked to an oligonucleotide having a coding sequence. Such a "crosslinker-modified double-stranded DEL compound" is extremely useful because, after screening, a covalent bond is formed between the target and the coding sequence, and it is resistant to stronger separation and elution conditions than conventional compounds, such as for the removal of nonspecific binders.

[0176] (Crosslinker) In one embodiment, in the present invention, "crosslinker" means a reactive group that has the reactivity to form a covalent bond through reaction with a biological target such as a protein or nucleic acid molecule. For example, crosslinkers such as those listed in the Thermo Scientific Crosslinking Technical Handbook are known.

[0177] The crosslinker used in the present invention is preferably a reactive group comprising at least one azide group, a diazirine group, a sulfonyl fluoride group, a diazo group, a cinnamoyl group, or an acrylate group, and more preferably a reactive group comprising at least one azide group, a diazirine group, or a sulfonyl fluoride group.

[0178] In one embodiment, in the present invention, "having a crosslinker" and "crosslinker modification" mean having a substructure containing a crosslinker as a substituent.

[0179] In one preferred embodiment, the "crosslinker-modified double-stranded DEL," "crosslinker-modified DNA," and "crosslinker-modified primer" are such that the crosslinker is either directly bound to the 5' end of the "double-stranded DEL," "DNA," and "primer," respectively, or bound via a bifunctional spacer. In this case, the crosslinker is expressed by the following formulas (AA)~(AE) or (BA) or (BB) It is preferable that the structure be one of the following: [ka] [ka] (In the formula, * represents the 5' end of "double-stranded DEL", "DNA", or "primer", or the binding site to the bifunctional spacer that is bound to the 5' end.)

[0180] In one preferred embodiment, the crosslinker used in the present invention is a photoreactive crosslinker. In the present invention, a photoreactive crosslinker refers to a reactive group that changes into a highly reactive group (e.g., nitrene and carbene) upon light irradiation and forms a covalent bond with a nearby biological target. For example, azide groups and diazirine groups are known, and the structures of formulas (AA) to (AE) above are known.

[0181] In another preferred embodiment, the crosslinker used in the present invention is a reactive group containing at least one sulfonyl fluoride group. The sulfonyl fluoride group reacts with residues such as serine, threonine, tyrosine, lysine, cysteine, and histidine in biological target proteins to form a covalent bond. For example, the structures of formulas (BA) to (BB) described above are known.

[0182] In the present invention, the crosslinking reaction between the crosslinker and the biological target is preferably carried out in a temperature range in which the desired higher-order structure of the biological target does not change significantly. The preferred temperature range is, for example, 4 to 40°C.

[0183] (Crosslinker bifunctional spacer) As described above, the bifunctional spacer is a spacer portion having at least two reactive groups that enable the bonding of the compound library substructure An to the headpiece. Furthermore, in the present invention, the bifunctional spacer is a spacer portion having two reactive groups that enable the binding of the crosslinker to "double-stranded DEL," "DNA," or "primer." This embodiment of the bifunctional spacer is sometimes referred to as the "bifunctional spacer for the crosslinker." In contrast, the bifunctional spacer that binds to the aforementioned compound library is sometimes referred to as the "bifunctional spacer for the compound library." In one embodiment, the preferred form of the "bifunctional spacer for crosslinkers" is the same as the preferred form of the "bifunctional spacer for compound libraries" described above. Furthermore, in one embodiment, the "bifunctional spacer for the crosslinker" is preferably of a molecular chain length suitable for reacting the crosslinker with the biological target when the compound library binds to the biological target during screening, and preferably of a molecular chain length equivalent to that of the "bifunctional spacer for the compound library".

[0184] (Code array) In the present invention, "code sequence" refers to the sequence portion of the oligonucleotides contained in DEL that has a sequence capable of identifying the structure of the library molecule.

[0185] The term "reactive group for crosslinker modification" is not particularly limited as long as it is a reactive group that can react with the crosslinker unit described later. In one embodiment, the "reactive group for crosslinker modification" is a reactive group that exhibits reactivity selectivity with the crosslinker. By exhibiting reactivity selectivity with the crosslinker, it becomes possible to apply the present invention to reaction conditions in which the crosslinker cannot be used, such as when the crosslinker reacts first and undergoes structural transformation. That is, by introducing a unit having a reactive group for crosslinker modification into the process of the present invention, using reaction conditions in which the crosslinker cannot be used in the process of the present invention, and then reacting it with the crosslinker unit, the crosslinker required for the present invention can be introduced into the crosslinker-modified DEL of the present invention.

[0186] In one embodiment, the "reactive group for crosslinker modification" and the "reactive group paired with the reactive group for crosslinker modification" are a pair of reactive groups with high affinity in bonding reactions. When two compounds, each possessing such a pair, bond is formed, the pair reacts preferentially with high selectivity, even if various other functional groups are present in the compounds.

[0187] An example of the above pairings is the pairing of functional groups in a click reaction. The "click reaction" is a concept well known to those skilled in the art. (See HC Kolb, MG Finn & KB Sharpless: Angew. Chem. Int. Ed., 40, 2004(2001), etc.) "Click response" can be understood as one aspect of this phenomenon: A "click reaction" refers to a reaction that has at least the following characteristics: (1) the functional groups are orthogonal (i.e., the functional part reacts only with a reactive site complementary to that functional part, without reacting with other reactive sites), and (2) the resulting bond is irreversible (i.e., once the reactants react to form a product, it becomes difficult to decompose the product back into the reactants), or in some cases, the resulting bond may be reversible (i.e., under appropriate conditions, it can be returned to the reactants). Optionally, "click" chemistry may further have one or more of the following characteristics: (1) stereospecificity, (2) reaction conditions that do not require strict purification, atmospheric control, etc., (3) readily available starting materials and reagents, (4) availability of harmless solvents or no solvents at all, (5) isolation of the product by crystallization or distillation, (6) physiological stability, (7) high thermodynamic driving force (e.g., 10-20 kcal / mol), (8) a single reaction product, and (9) high chemical yield (e.g., over 50%).

[0188] In one embodiment, the "reactive group for crosslinker modification" and the "reactive group paired with the reactive group for crosslinker modification" are preferably reactive groups for click reactions, more preferably alkynyl groups, alkenyl groups, azide groups, or tetradinyl groups, and even more preferably any of formulas (CA) to (CL). [ka]

[0189] Here, the pairs of "reacting group for crosslinker modification" and "reacting group paired with the reacting group for crosslinker modification" are preferably an azide group paired with an alkynyl group and a tetradinyl group paired with an alkenyl group. These pairs have a so-called bolt-and-nut relationship and are interchangeable. For example, if an alkynyl group is used as the "reacting group for crosslinker modification," an azide group can be used as the "reacting group paired with the reacting group for crosslinker modification." The selection of these groups is well known to those skilled in the art. Preferred examples of "reactive groups for crosslinker modification" and "reactive groups paired with the reactive groups for crosslinker modification" include (CA) and (CH), (CB) and (CH), (CC) and (CH), (CE) and (CI), (CE) and (CJ), (CE) and (CK), (CE) and (CL), (CF) and (CI), (CF) and (CJ), (CF) and (CK), (CF) and (CL), (CG) and (CI), (CG) and (CJ), (CG) and (CK), (CG) and (CL).

[0190] The term "crosslinker unit" is not particularly limited as long as it has the aforementioned "reactive group paired with the reactive group for crosslinker modification" and a crosslinker.

[0191] In one embodiment, the "crosslinker unit" is composed of a "reactive group for crosslinker modification and a corresponding reactive group," a "bifunctional spacer," and a "crosslinker." The embodiment of the "bifunctional spacer" is as described above.

[0192] "DNA having a reactive group for crosslinker modification" is not particularly limited as long as it is a compound having the aforementioned "reactive group for crosslinker modification".

[0193] In one embodiment, "DNA having a reactive group for crosslinker modification" is composed of "a reactive group for crosslinker modification," a "bifunctional spacer," and "DNA." The embodiment of the "bifunctional spacer" is as described above.

[0194] The embodiment of the "modification primer having a reactive group for crosslinker modification" is the same as the "crosslinker modification primer" described above. However, "crosslinker" should be read as "reactive group for crosslinker modification" when referring to it.

[0195] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. The nucleic acids of various sequences in the examples can be prepared, for example, by an automated nucleic acid synthesizer according to standard procedures. An example of an automated nucleic acid synthesizer is the nS-8II (manufactured by GeneDesign). Furthermore, contract synthesis or contract laboratories can be used for nucleic acid preparation. Examples of well-known contract laboratories to those skilled in the art include GeneDesign and LGC Biosearch Technologies. Generally... These contract laboratories prepare nucleic acids with sequences specified by the client, under confidentiality agreements, and deliver them to the client.

[0196] Example 1 [Verification of the cleavage reaction of a hairpin-shaped DEL substructure containing deoxyuridine using USER® enzyme] Compounds with the sequences shown in Table 1 were prepared using the nucleic acid automated synthesizer nS-8II (manufactured by Gene Design). As will be apparent to those skilled in the art, in the sequence notation in Table 1, each sequence unit is connected by a phosphate diester bond, where "A" means deoxyadenosine, "T" means thymidine, "G" means deoxyguanosine, "C" means deoxycytidine, "(dU)" means deoxyuridine, "(p)" means phosphate, and "(amino-C6-dT)" is represented by the following formula (1) [ka] This refers to a modified nucleic acid represented by the following formula (2): "(amino-NC6-dT)" is given by the formula (2) below. [ka] This refers to modified nucleic acids represented by the following formula (3): [ka] It refers to the group represented by the following formula (4) [ka] This refers to the group represented by . In addition, amino-NC6-dT was synthesized according to the method described in (Journal of the American Chemical Society, 1993, Vol. 115, pp. 7128-7134) and is given by the following formula (5) [ka] The nucleic acid was introduced using nucleic acid synthesis reagents.

[0197] In Table 1, "No." in the left column represents the sequence number, and "Seq." in the right column represents the sequence. The left side of the sequence represents the 5' end, and the right side represents the 3' end. The names of the compounds corresponding to each sequence number (No.) are as follows. No.1: U-DEL1-sh No.2: U-DEL2-sh No.3: U-DEL3-sh No.4: U-DEL4-sh No. 5: U-DEL5-HP No. 6: U-DEL6-HP No.7: U-DEL7-HP No.8: U-DEL8-HP No.9:U-DEL9-HP No.10:U-DEL10-HP

[0198] [Table 1]

[0199] A 0.1 mM aqueous solution of each compound with the sequence shown in Table 1 was prepared, and the cleavage reaction with USER® enzyme was investigated using the following procedure.

[0200] A 1 μL 0.1 mM aqueous solution of the compound with the sequence shown in Table 1, 10 μL of CutSmart® Buffer (New England BioLabs, catalog number B7204S), and 79 μL of deionized water were added to a PCR tube. 10 μL of USER® enzyme (New England BioLabs, catalog number M5505S) was added to the solution, and the resulting solution was incubated at 37°C.

[0201] 20 μL samples were taken of each reaction solution 1 hour and 3 hours after the start of incubation. U-DEL1-sh, U-DEL5-HP, U-DEL6-HP, U-DEL7-HP, U-DEL8-HP, U-DEL9-HP, and U-DEL10-HP were also sampled at 20 hours. U-DEL8-HP and U-DEL9-HP were further incubated at 90°C for 1 hour before being sampled at 20 μL each.

[0202] Of the sampled solutions, U-DEL1-sh, U-DEL2-sh, U-DEL3-sh, and U-DEL4-sh were analyzed under the following analytical conditions 1, while U-DEL5-HP, U-DEL6-HP, U-DEL7-HP, U-DEL8-HP, U-DEL9-HP, and U-DEL10-HP were analyzed under the following analytical conditions 2.

[0203] Analysis condition 1: Equipment: maXis (manufactured by Bruker), UltiMate 3000 (manufactured by Dionex) Column: ACQUITY UPLC Oligonucleotide BEH C18 Column (130 Å, 1.7 μm, 2.1 × 50 mm) Column temperature: 50℃ solvent: Solution A: Water (0.75% v / v hexafluoroisopropanol; 0.038% v / v triethylamine; 5 μM ethylenediaminetetraacetic acid) Solution B: 90% v / v methanol aqueous solution (0.75% v / v hexafluoroisopropanol; 0.038% v / v triethylamine; 5 μM ethylenediaminetetraacetic acid) Gradient conditions: Measurement was started with a fixed flow rate of 0.36 mL / min and a mixing ratio of solution A and solution B of 95 / 5 (v / v). After 0.56 minutes, the mixing ratio of solution A and solution B was linearly changed to 40 / 60 (v / v) over 5.5 minutes. Detection wavelength: 260nm

[0204] Analysis conditions 2: Equipment: Waters ACQUITY UPLC / SQ Detector Column: ACQUITY UPLC Oligonucleotide BEH C18 Column (130 Å, 1.7 μm, 2.1 × 50 mm) Column temperature: 50℃ solvent: Solution A: Water (0.75% v / v hexafluoroisopropanol; 0.038% v / v triethylamine; 5 μM ethylenediaminetetraacetic acid) Solution B: 90% v / v methanol aqueous solution (0.75% v / v hexafluoroisopropanol; 0.038% v / v triethylamine; 5 μM ethylenediaminetetraacetic acid) Gradient conditions: Measurement was started with a fixed flow rate of 0.36 mL / min and a mixing ratio of solution A and solution B of 95 / 5 (v / v). After 0.56 minutes, the mixing ratio of solution A and solution B was linearly changed to 40 / 60 (v / v) over 5.5 minutes. Detection wavelength: 260nm

[0205] Tables 2 and 3 show the expected product sequences and theoretical molecular weights of the deoxyuridine moiety (debase-debased deoxyuridine moiety and cleaved fragments) in each reaction solution, as well as the molecular weights detected in each reaction solution. The notation for each column in Tables 2 and 3 is as follows:

[0206] “Entry” (far left): The experiment numbers are shown, and the substrates corresponding to each experiment number (Entry) are as follows: Entry.1:U-DEL1-sh Entry.2:U-DEL2-sh Entry.3:U-DEL3-sh Entry.4:U-DEL4-sh Entry.5:U-DEL5-HP Entry.6:U-DEL6-HP Entry.7:U-DEL7-HP Entry.8:U-DEL8-HP Entry.9:U-DEL9-HP Entry.10:U-DEL10-HP

[0207] “No.” (Second from the left): This represents the sequence number. Of the sequence numbers (No.), Nos. 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 are the substrates of the respective reaction solutions, Nos. 11, 14, 17, 20, 22, 25, 29, 31, 33, and 35 are the debase-debased forms of the deoxyuridine moiety of each substrate, and the remaining sequence numbers are fragments obtained by cleaving each substrate.

[0208] “Seq.” (Third from the left): This represents the arrangement, with the left side representing the 5' side and the right side representing the 3' side. Note that in the notation, "(B)" is the following equation (6) [ka] This represents the group (debase site) represented by , and other notations are the same as in Table 1.

[0209] “Expected MW.” (Fourth from the left): This represents the theoretical molecular weight (Da) of each sequence.

[0210] “Observed MW.” (Far right): This shows the numerical value of the molecular weight (Da) detected for each sequence. A "-" indicates that the sequence was not detected.

[0211] [Table 2]

[0212] [Table 3]

[0213] The conversion rates for the debase reaction and cleavage reaction were calculated from the area ratio of the peaks corresponding to each detected sequence. For all substrates, the debase reaction resulted in over 99% conversion at 37°C for 1 hour (the substrate peak was less than 1%, with the remaining peaks consisting only of the debased product and cleaved fragments). Figure 10 shows a graph illustrating the conversion rate of the cleavage reaction. As shown in the graph, for all substrates except U-DEL8-HP and U-DEL9-HP, the cleavage reaction proceeded to more than 95% within 20 hours at 37°C. For U-DEL8-HP and U-DEL9-HP, the cleavage reaction was completed to 100% by adding an incubation period of 1 hour at 90°C.

[0214] The results above indicate that the hairpin-type DEL substructures containing various deoxyuridines undergo a debase reaction by USER® enzyme at the deoxyuridine moiety, followed by a cleavage reaction.

[0215] Example 2 [Comparison of PCR efficiency between conventional hairpin DEL and severable hairpin DEL (hairpin-type DEL containing deoxyuridine)]

[0216] As shown in the schematic diagram in Figure 11, the compound (hairpin DEL) with the sequence shown in Table 4 was synthesized using the following procedure. Note that in the sequence notation in Table 4, "S" represents the following formula (7) [ka] This refers to the base represented by , and other notations are the same as in Table 1. The names of the compounds corresponding to each sequence number (No.) are as follows: No.37: U-DEL1 No.38: U-DEL2 No.39: U-DEL4 No.40: U-DEL7 No.41: U-DEL8 No.42: U-DEL9 No.43: U-DEL10 No.44: H-DEL [Table 4] The compound names of the raw material headpieces used to synthesize each hairpin DEL are as follows: Hairpin DEL: Raw material headpiece U-DEL1 :U-DEL1-HP U-DEL2: U-DEL2-HP U-DEL4: U-DEL4-HP U-DEL7: U-DEL7-HP U-DEL8: U-DEL8-HP U-DEL9: U-DEL9-HP U-DEL10: U-DEL10-HP H-DEL: H-DEL-HP Furthermore, the sequence numbers "No." and sequence "Seq" for U-DEL1-HP, U-DEL2-HP, U-DEL4-HP, and H-DEL-HP are as shown in Table 5 below. [Table 5]

[0217] The raw material headpieces shown in Table 5 were prepared using the nucleic acid automated synthesizer nS-8II (manufactured by Gene Design Co., Ltd.) in the same manner as in Example 1.

[0218] In a PCR tube, 2.0 μL of 1 mM aqueous solution of various raw material headpieces; 2.4 μL of 1 mM aqueous solution of Pr_TAG (prepared by annealing Pr_TAG_a and Pr_TAG_b synthesized in the same manner as in Example 1; sequences are shown in Table 6); 0.8 μL of 10X ligase buffer (500 mM Tris-HCl, pH 7.5; 500 mM sodium chloride; 100 mM magnesium chloride; 100 mM dithiothreitol; 20 mM adenosine triphosphate) and 2.0 μL of deionized water were added. 0.8 μL of a 10-fold diluted aqueous solution of T4 DNA ligase (Thermo Fisher, catalog number EL0013) was added to the solution, and the resulting solution was incubated at 16°C for 24 hours. The sequence notation in Table 6 is the same as in Table 1. The names of the compounds corresponding to each sequence number (No.) are as follows. No.49:Pr_TAG_a No.50:Pr_TAG_b [Table 6]

[0219] The reaction solution was treated with 0.8 μL of 5 M aqueous sodium chloride solution and 17.6 μL of cooled (-20°C) ethanol, and allowed to stand at -78°C for 2 hours. After centrifugation, the supernatant was removed, and the resulting pellets were air-dried. 2.0 μL of deionized water was added to each pellet to prepare the solution.

[0220] To each of the obtained solutions, 2.4 μL of a 1 mM aqueous solution of CP (prepared by annealing CP_a and CP_b synthesized in the same manner as in Example 1; sequences are shown in Table 7); 0.8 μL of 10X ligase buffer (500 mM Tris-HCl, pH 7.5; 500 mM sodium chloride; 100 mM magnesium chloride; 100 mM dithiothreitol; 20 mM adenosine triphosphate) and 2.0 μL of deionized water were added. 0.8 μL of a 10-fold diluted aqueous solution of T4 DNA ligase (Thermo Fisher, catalog number EL0013) was added to the solution, and the resulting solution was incubated at 16°C for 24 hours. The sequence notation in Table 7 is the same as in Table 1. The names of the compounds corresponding to each sequence number (No.) are as follows. No.51: CP_a No.52: CP_b

Table 7

[0221] The reaction solution was treated with 0.8 μL of 5 M aqueous sodium chloride solution and 17.6 μL of cooled (-20 °C) ethanol, and left standing at -78 °C for 2 hours. After centrifugation, the supernatant was removed, and the obtained pellet was air-dried. 10 μL of deionized water was added to the pellet to form a solution.

[0222] 1.0 μL of the obtained solution was sampled, diluted with deionized water, and then subjected to mass spectrometry by ESI-MS under the conditions of Analytical Condition 2 in Example 1 to identify the target substance (the theoretical molecular weights and the detected molecular weights of each sequence are shown in Table 4). After the remaining solution was lyophilized, deionized water was added to each to adjust the concentration to 20 μM.

[0223] Among the 8 types of hairpin-type DELs obtained above, H-DEL is a conventional hairpin DEL, and the remaining 7 types are cleavable hairpin DELs containing deoxyuridine. To compare the PCR efficiency before treatment with USER (registered trademark) enzyme and the PCR efficiency after treatment for various hairpin-type DELs, real-time PCR analysis was performed. Also, DS-DEL shown in Table 7 (prepared by annealing the compounds of Sequence Nos. 47 and 48) was used as the double-stranded DEL for comparison. In the sequence listing in Table 8, “(amino-C6-L)” means the group represented by the following formula (8)

Chemical formula

Table 8

[0224] <Treatment step by USER (registered trademark) enzyme> The treatment of 8 hairpin DELs and double-stranded DEL (DS-DEL) with USER (registered trademark) enzyme was performed according to the following procedure.

[0225] In a PCR tube, 1 μL of various DEL 20 μM aqueous solution; 1 μL of CutSmart (registered trademark) Buffer (manufactured by New England BioLabs, catalog number B7204S) and 7 μL of deionized water were added. 1 μL of USER (registered trademark) enzyme (manufactured by New England BioLabs, catalog number M5505S) was added to the solution, and the resulting solution was incubated at 37 °C for 1 hour.

[0226] <Preparation of DEL samples> Samples of various DELs before treatment with USER (registered trademark) enzyme and the reaction solutions after treatment were each diluted with deionized water to prepare 0.05 pM, 0.5 pM, and 5 pM DEL samples.

[0227] <Measurement of Ct values by real-time PCR> The Ct values of the various DEL samples obtained above were measured by real-time PCR, and the PCR efficiencies were compared. The conditions are as follows, and the results are shown in Figure 12. The Ct value is the number of cycles at which the fluorescence signal generated with the amplification of DNA reaches an arbitrary threshold value in real-time PCR. That is, when the initial number of DNA molecules is the same, the higher the PCR efficiency, the lower the Ct value.

[0228] Apparatus: 7500 Real-Time PCR System (manufactured by Applied Biosystems) Plate: MicroAmp 96-Well Plate (manufactured by Applied Biosystems, catalog number N8010560) PCR reaction solution: · TB Green Premix Ex taqII (manufactured by Takara Bio Inc., catalog number RR820): 10 μL · Forward primer (Table 9, SEQ ID NO: 55): 0.80 μL • Reverse primer (Table 9, SEQ ID NO: 56): 0.80 μL • ROX Reference Dye II (manufactured by Takara Bio, catalog number RR39LR): 0.40 μL • Aqueous solutions of various DEL samples (0.05 pM, 0.5 pM, 5 pM)*1: 2.0 μL Deionized water: 6.0 μL * 1: The mole amounts of the DEL sample are 0.1 amol, 1 amol, and 10 amol. Temperature conditions: After holding at 95°C for 2 minutes, the following cycle was repeated 35 times. 95℃ for 5 seconds 52℃ for 30 seconds 72℃ for 30 seconds [Table 9] Note that the arrangement notation in Table 9 is the same as in Table 1.

[0229] As shown in Figure 12, the Ct value of conventional hairpin DEL (H-DEL) did not change before and after USER® enzyme treatment, but the Ct value of cleavable hairpin DEL containing deoxyuridine (U-DEL1, U-DEL2, U-DEL4, U-DEL7, U-DEL8, U-DEL9, and U-DEL10) decreased to the same level as DS-DEL, which is a double-stranded DEL, after USER® enzyme treatment.

[0230] These results indicate that DEL cleaved with USER® enzyme exhibits improved PCR efficiency compared to before cleavage, and that cleavable hairpin DEL containing deoxyuridine is cleaved with high efficiency and selectivity by USER® enzyme.

[0231] Example 3 [Verification of the cleavage reaction of hairpin DEL containing deoxyuridine using USER(registered trademark) enzyme] <Combination of 4 types of hairpins DEL (U-DEL5, U-DEL11, U-DEL12, and U-DEL13)> The compound with the sequence shown in Table 10 (hairpin DEL) was synthesized using the following procedure. Note that in the sequence notation in Table 10, "[mdC(TEG-amino)]" is represented by the following formula (9). [ka] This refers to the base represented by , and other notations are the same as in Table 4. The names of the compounds corresponding to each sequence number (No.) are as follows: No. 57: U-DEL5 No. 58: U-DEL11 No. 59: U-DEL12 No. 60: U-DEL13 [Table 10] The compound names of the raw material headpieces used to synthesize each hairpin DEL are as follows: Hairpin DEL: Raw material headpiece U-DEL5: U-DEL5-HP U-DEL11: U-DEL11-HP U-DEL12 : U-DEL12-HP U-DEL13: U-DEL13-HP Furthermore, the sequence numbers "No." and sequence "Seq" for U-DEL11-HP, U-DEL12-HP, and U-DEL13-HP are as shown in Table 11 below. Note that the notation in Table 11 is the same as in Table 10.

[0232] [Table 11]

[0233] Of the raw material headpieces shown in Table 11, U-DEL12-HP and U-DEL13-HP were prepared using the nucleic acid automated synthesizer nS-8II (manufactured by Gene Design Co., Ltd.) in the same manner as in Example 1. U-DEL11-HP was also prepared in the same manner according to the standard method.

[0234] Similar to Example 2, two-step double-stranded ligation was performed using various raw material headpieces with double-stranded oligonucleotides Pr_TAG and CP.

[0235] A portion of the obtained solution was sampled, diluted with deionized water, and then mass spectrometry by ESI-MS was performed under the analytical conditions 3 shown below to identify the target product (the theoretical molecular weight of each sequence and the detected molecular weight are shown in Table 10). The remaining solution was freeze-dried, and then deionized water was added to each to prepare a 20 μM solution.

[0236] Analysis condition 3: Equipment: Waters ACQUITY UPLC / SQ Detector Column: ACQUITY UPLC Oligonucleotide BEH C18 Column (130 Å, 1.7 μm, 2.1 × 50 mm) Column temperature: 60℃ solvent: Solution A: Water (0.75% v / v hexafluoroisopropanol; 0.038% v / v triethylamine; 5 μM ethylenediaminetetraacetic acid) Solution B: 90% v / v methanol aqueous solution (0.75% v / v hexafluoroisopropanol; 0.038% v / v triethylamine; 5 μM ethylenediaminetetraacetic acid) Gradient conditions: Measurement was started with a fixed flow rate of 0.36 mL / min and a mixing ratio of solution A and solution B of 95 / 5 (v / v). After 0.56 minutes, the mixing ratio of solution A and solution B was linearly changed to 40 / 60 (v / v) over 5.5 minutes. Detection wavelength: 260nm Deconvolution: The ion signals were analyzed using ProMass for MassLynx Software (manufactured by Waters).

[0237] <USER (registered trademark) enzyme cleavage reaction> The cleavage reactions of hairpin DELs (U-DEL5, U-DEL7, U-DEL9, U-DEL11, U-DEL12, and U-DEL13) containing six types of deoxyuridine by USER (registered trademark) enzyme were examined according to the following procedure.

[0238] Into a PCR tube, 2 μL of various hairpin DEL 20 μM aqueous solution; 2 μL of CutSmart (registered trademark) Buffer (manufactured by New England BioLabs, catalog number B7204S) and 14 μL of deionized water were added. 2 μL of USER (registered trademark) enzyme (manufactured by New England BioLabs, catalog number M5505S) was added to the solution, and the resulting solution was incubated at 37 °C for 16 hours and then further incubated at 90 °C for 1 hour.

[0239] <Confirmation of the products after cleavage by LC-MS measurement> 5.0 μL of the obtained reaction solution was sampled, diluted with deionized water, and then mass spectrometry was performed by ESI-MS under analysis condition 3. Table 12 shows the sequences and theoretical molecular weights of the products after cleavage assumed in each reaction solution, and the molecular weights detected in each reaction solution. The substrates corresponding to each experimental number (Entry) are as follows, and other notations are the same as in Table 10. Entry.1: U-DEL5 Entry.2: U-DEL7 Entry.3: U-DEL9 Entry.4: U-DEL11 Entry.5: U-DEL12 Entry.6: U-DEL13

[0240]

Table 12

[0241] In all samples, no substrate MS was detected; instead, the MS of the cleavage product was observed as the main peak.

[0242] <Confirmation of cleavage reaction by gel electrophoresis> Furthermore, a portion of the obtained reaction solution was sampled and analyzed by modified polyacrylamide gel electrophoresis under the conditions shown below. The results shown in Figure 13 confirm that the cleavage reaction proceeded with high yield for all substrates. The samples in each lane of Figure 13 are as follows. Lane 1: 20 bp DNA Ladder (Lonza, catalog number 50330) Lane 2: U-DEL5 Lane 3: Sample after cleavage reaction of U-DEL5 Lane 4: U-DEL7 Lane 5: Sample after cleavage reaction of U-DEL7 Lane 6: U-DEL9 Lane 7: Sample after cleavage reaction of U-DEL9 Lane 8: U-DEL11 Lane 9: Sample after cleavage reaction of U-DEL11 Lane 10: U-DEL12 Lane 11: Sample after cleavage reaction of U-DEL12 Lane 12: U-DEL13 Lane 13: Sample after cleavage reaction of U-DEL13 Modified polyacrylamide gel electrophoresis: Gel: Novex (trademark) 10% TBE-Urea Gel (manufactured by Invitrogen by ThermoFisher SCIENTIFIC, catalog number EC68755BOX) Loading buffer: Novex (trademark) 10% TBE-Urea Sample Buffer (2x) (manufactured by Invitrogen by ThermoFisher SCIENTIFIC, catalog number LC6876) Temperature: 60℃ Voltage: 180V Electrophoresis time: 30 minutes Staining reagent: SYBER (trademark) GreenII Nucleic Acid Gel Stain (manufactured by Takara Bio, catalog number 5770A)

[0243] These results indicate that hairpin-type DELs containing various deoxyuridines undergo cleavage reactions by USER® enzyme at the deoxyuridine moiety.

[0244] Example 4 [Verification of the cleavage reaction of hairpin DEL containing deoxyinosine by endonuclease V] <Synthesis of hairpin DELs containing four types of deoxyinosine (I-DEL1, I-DEL2, I-DEL3, and I-DEL4)> The compound with the sequence shown in Table 13 (hairpin DEL) was synthesized using the following procedure. In the sequence notation in Table 13, "I" represents deoxyinosine, and the other notations are the same as in Table 2. The names of the compounds corresponding to each sequence number (No.) are as follows: No.73: I-DEL1 No.74: I-DEL2 No.75: I-DEL3 No.76: I-DEL4 [Table 13] The compound names of the raw material headpieces used to synthesize each hairpin DEL are as follows: Hairpin DEL: Raw material headpiece I-DEL1 : I-DEL1-HP I-DEL2: I-DEL2-HP I-DEL3 : I-DEL3-HP I-DEL4: I-DEL4-HP Furthermore, the sequence numbers "No." and sequence "Seq" for I-DEL1-HP, I-DEL2-HP, I-DEL3-HP, and I-DEL4-HP are as shown in Table 14 below. Note that the notation in Table 14 is the same as in Table 13.

[0245]

Table 14

[0246] The raw material headpiece shown in Table 14 was prepared according to a conventional method.

[0247] Similar to Example 2, two-step double-stranded ligation with double-stranded oligonucleotide Pr_TAG and CP was carried out using various raw material headpieces.

[0248] A part of the obtained solution was sampled, diluted with deionized water, and then subjected to mass spectrometry by ESI-MS under Analytical Condition 3 to identify the target substance (the theoretical molecular weights and the detected molecular weights of each sequence are shown in Table 13). The rest of the solution was lyophilized and then deionized water was added to each to prepare a solution with a concentration of 20 μM.

[0249] <Cleavage reaction by endonuclease V> The cleavage reaction of hairpin DEL (I-DEL1, I-DEL2, I-DEL3, I-DEL4) containing four kinds of deoxyinosine by endonuclease V was examined by the following procedure.

[0250] To a PCR tube, 1 μL of an aqueous solution of various hairpin DELs at 20 μM; 2 μL of NEBuffer (registered trademark) 4 (manufactured by New England BioLabs, catalog number B7004) and 15 μL of deionized water were added. 2 μL of Endonuclease V (manufactured by New England BioLabs, catalog number M0305S) was added to the solution, and the resulting solution was incubated at 37 °C for 24 hours.

[0251] <Confirmation of the product after cleavage by LC-MS measurement> 8.0 μL of the obtained reaction solution was sampled, diluted with deionized water, and then subjected to mass spectrometry by ESI-MS under analytical condition 3. Table 15 shows the expected sequence and theoretical molecular weight of the cleaved products for each reaction solution, as well as the molecular weight detected for each reaction solution. The substrates corresponding to each experimental number (Entry) are as follows, and other notations are the same as in Table 13. Entry 1: I-DEL1 Entry 2: I-DEL2 Entry 3: I-DEL3 Entry 4: I-DEL4

[0252] [Table 15]

[0253] In all samples, no substrate MS was detected; instead, the MS of the cleavage product was observed as the main peak.

[0254] <Confirmation of cleavage reaction by gel electrophoresis> Furthermore, a portion of the obtained reaction solution was sampled and analyzed by modified polyacrylamide gel electrophoresis under the same conditions as in Example 3. The results shown in Figure 14 confirm that the cleavage reaction proceeded with high yield for all substrates. The samples in each lane of Figure 14 are as follows: Lane 1: 20 bp DNA Ladder (Lonza, catalog number 50330) Lane 2: I-DEL1 Lane 3: Sample after I-DEL1 cleavage reaction Lane 4: I-DEL2 Lane 5: Sample after I-DEL2 cleavage reaction Lane 6: I-DEL3 Lane 7: Sample after I-DEL3 cleavage reaction Lane 8: I-DEL4 Lane 9: Sample after I-DEL4 cleavage reaction

[0255] These results indicate that, in hairpin-type DELs containing various deoxyinosines, the second phosphodiester bond from the deoxyinosine is cleaved in the 3' direction by endonuclease V.

[0256] Example 5 [Verification of the cleavage reaction of hairpin DEL containing ribonucleoside by RNaseHII] <Synthesis of hairpin DEL (R-DEL1) containing ribonucleoside> The compound with the sequence shown in Table 16 (hairpin DEL) was synthesized using the following procedure. In the sequence notation in Table 16, "u" represents uridine, and other notations are the same as in Table 2. The names of the compounds corresponding to the sequence number (No.) are as follows: No.87: R-DEL1 [Table 16] The compound names of the raw material headpieces used to synthesize each hairpin DEL are as follows: Hairpin DEL: Raw material headpiece R-DEL1: R-DEL1-HP Furthermore, the sequence number "No." and sequence "Seq" of R-DEL1-HP are as shown in Table 17 below. Note that the notation in Table 17 is the same as in Table 16.

[0257] [Table 17]

[0258] The raw material headpieces shown in Table 17 were prepared according to standard procedures.

[0259] Similar to Example 2, a two-step double-stranded ligation was performed using a raw material headpiece with double-stranded oligonucleotides Pr_TAG and CP.

[0260] A portion of the obtained solution was sampled, diluted with deionized water, and then subjected to mass spectrometry by ESI-MS under Analytical Condition 3 to identify the target substance (the theoretical molecular weights and the detected molecular weights of each sequence are shown in Table 16). After the remaining solution was lyophilized, deionized water was added to each to prepare a solution with a concentration of 200 μM.

[0261] <Cleavage Reaction by RNaseHII> The cleavage reaction of ribonucleoside-containing hairpin DEL (R-DEL1) by RNaseHII was examined according to the following procedure.

[0262] To a PCR tube, 0.5 μL of a 200 μM aqueous solution of hairpin DEL; 4.9 μL of ThermoPol® Reaction Buffer Pack (manufactured by New England BioLabs, catalog number B9004), and 43.6 μL of deionized water were added. 1 μL of RNase HII (manufactured by New England BioLabs, catalog number M0288S) was added to the solution, and the resulting solution was incubated at 37 °C for 8 hours.

[0263] <Confirmation of the Product after Cleavage by LC-MS Measurement> 10 μL of the obtained reaction solution was sampled and subjected to mass spectrometry by ESI-MS under Analytical Condition 3. The sequences, theoretical molecular weights, and detected molecular weights of the expected products after cleavage are shown in Table 18. The substrates corresponding to the experimental numbers (Entry) are as follows, and other notations are the same as those in Table 16. Entry.1: R-DEL1

[0264]

Table 18

[0265] For all samples, the MS of the substrate was not detected, and the MS of the product after cleavage was observed as the main peak.

[0266] <Confirmation of the Cleavage Reaction by Gel Electrophoresis> Furthermore, a portion of the obtained reaction solution was sampled and analyzed by modified polyacrylamide gel electrophoresis under the same conditions as in Example 3. The results shown in Figure 15 confirm that the cleavage reaction proceeded with high yield for all substrates. The samples in each lane of Figure 15 are as follows: Lane 1: 20 bp DNA Ladder (Lonza, catalog number 50330) Lane 2: R-DEL1 Lane 3: Sample after R-DEL1 cleavage reaction

[0267] These results indicate that hairpin-type DELs containing ribonucleosides undergo cleavage of the phosphodiester bond at the 5' end of the ribonucleotide by RNaseHII. Example 6 [Creating a model library using U-DEL9-HP as the raw material] As shown in the schematic diagram in Figure 16, a model library containing 3×3×3(27) compound species was synthesized using U-DEL9-HP as a starting material by split-and-pool synthesis, with the following reagents. ·U-DEL9-HP • Three types of building blocks (BB1, BB2, and BB3): [ka] • Ten types of double-stranded oligonucleotide tags (tag numbers in Table 19: Pr, A1, A2, A3, B1, B2, B3, C1, C2, and C3)

[0268] In Table 19, “Tag No.” (far left) represents the tag number, “No.” (second from the left) represents the sequence number, and “Seq.” (third from the left) represents the sequence. The sequence notation is the same as in Table 1.

[0269] Each double-stranded oligonucleotide tag was prepared by annealing two oligonucleotides with sequence numbers corresponding to each tag number, as shown in Table 19.

[0270] [Table 19]

[0271] <Synthesis of compound "AOP-U-DEL9-HP"> The compound "AOP-U-DEL9-HP" with the sequence shown in Table 20 was synthesized using the following procedure. Note that in the sequence notation in Table 20, "(AOP-AminoC7)" is represented by the following formula (10). [ka] This refers to the base represented by , and other notations are the same as in Table 2.

[0272] [Table 20]

[0273] Four bioremo centrifuge tubes were filled with a 2.5 mL, 1 mM solution of U-DEL9-HP in sodium borate buffer (150 mM, pH 9.4) cooled to 10°C. To each tube, 40 equivalents of N-Fmoc-15-amino-4,7,10,13-tetraoxaoctadecanoic acid (250 μL, 0.4 M N-dimethylacetamide solution) were added, followed by 40 equivalents of 4-(4,6-dimethoxy[1,3,5]triazine-2-yl)-4-methylmorpholinium chloride hydrate (DMTMM) (200 μL, 0.5 M aqueous solution). The resulting solutions were shaken at 10°C for 5 hours.

[0274] The above solutions were treated with 295 μL of 5 M aqueous sodium chloride solution and 9.7 mL of chilled (-20°C) ethanol, respectively, and allowed to stand overnight at -78°C. After centrifugation, the supernatant was removed, and the resulting pellets were air-dried. 2.75 mL of deionized water was added to each pellet to dissolve it, 306 μL of piperidine was added at 0°C, and the mixture was shaken at 10°C for 3 hours. After centrifugation of the mixture, the precipitate was removed by filtration, and the mixture was washed twice with 1.47 mL of deionized water. The resulting filtrates were treated with 600 μL of 5 M aqueous sodium chloride solution and 19.8 mL of chilled (-20°C) ethanol, respectively, and allowed to stand overnight at -78°C. After centrifugation, the supernatant was removed, and the resulting pellets were air-dried.

[0275] The obtained pellet was mixed with 10 mL of deionized water to form a solution. A portion of the solution was sampled, diluted with deionized water, and then mass spectrometry was performed by ESI-MS under the conditions of analytical condition 2 in Example 1 to identify the target compound (the theoretical molecular weight and detected molecular weight of the compound are shown in Table 20). The remainder of the solution was freeze-dried, and then deionized water was added to prepare a 5 mM solution.

[0276] <Introduction of the double-stranded oligonucleotide tag "Pr"> The compound "AOP-U-DEL9-HP-Pr" with the sequence shown in Table 21 was synthesized by ligating the compound "AOP-U-DEL9-HP" with the double-stranded oligonucleotide tag "Pr" using the following procedure. Note that the sequence notation in Table 21 is the same as in Table 20.

[0277] [Table 21]

[0278] In a Bioremo centrifuge tube, 40 μL of a 5 mM aqueous solution of the compound "AOP-U-DEL9-HP", 160 μL of a 100 mM sodium bicarbonate aqueous solution, 240 μL of a 1 mM aqueous solution of the double-stranded oligonucleotide tag "Pr", 80 μL of 10X ligase buffer (500 mM Tris-HCl, pH 7.5; 500 mM sodium chloride; 100 mM magnesium chloride; 100 mM dithiothreitol; 20 mM adenosine triphosphate) and 272 μL of deionized water were added. 8.0 μL of T4 DNA ligase (Thermo Fisher, catalog number EL0013) was added to the solution, and the resulting solution was incubated at 16°C for 24 hours.

[0279] The reaction solution was treated with 80 μL of 5 M aqueous sodium chloride solution and 2640 μL of cooled (-20°C) ethanol, and allowed to stand at -78°C for 2 hours. After centrifugation, the supernatant was removed, and 400 μL of deionized water was added to the resulting pellet. The resulting solution was concentrated using an Amicon® Ultra Centrifugal filter (30 kD cutoff). A portion of the resulting solution was sampled and mass spectrometry was performed by ESI-MS under the conditions of analysis condition 2 to identify the target compound (the theoretical molecular weight of the compound and the detected molecular weight are shown in Table 21). Through the above steps, 133 nmol of the compound "AOP-U-DEL9-HP-Pr" with a purity of 84.5% was obtained. The obtained compound "AOP-U-DEL9-HP-Pr" was prepared to a concentration of 1 mM by adding 100 mM aqueous sodium bicarbonate solution.

[0280] <Cycle A> In each of the three PCR tubes, 20 μL of a 1 mM solution of the compound "AOP-U-DEL9-HP-Pr" obtained above, 30 μL of a 1 mM aqueous solution of one of the double-stranded oligonucleotide tags A1-A3, 8.0 μL of 10X ligase buffer (500 mM Tris-HCl, pH 7.5; 500 mM sodium chloride; 100 mM magnesium chloride; 100 mM dithiothreitol; 20 mM adenosine triphosphate) and 21.6 μL of deionized water were added. 0.4 μL of T4 DNA ligase (Thermo Fisher, catalog number EL0013) was added to the solution, and the resulting solution was incubated at 16°C for 18 hours.

[0281] Each reaction solution was treated with 8.0 μL of 5 M aqueous sodium chloride solution and 264 μL of chilled (-20°C) ethanol, and allowed to stand at -78°C for 30 minutes. After centrifugation, the supernatant was removed, and the resulting pellets were dissolved in 20 μL of 150 mM sodium borate buffer (pH 9.4).

[0282] To each tube, 40 equivalents of one of the building blocks BB1-BB3 (4.0 μL, 200 mM N,N-dimethylacetamide solution), followed by 40 equivalents of 4-(4,6-dimethoxy[1.3.5]triazine-2-yl)-4-methylmorpholinium chloride hydrate (DMTMM) (4.0 μL, 200 mM aqueous solution), were added, and the mixture was shaken at 10°C for 2 hours. Furthermore, to each tube, 20 equivalents of building block (2.0 μL, 200 mM N,N-dimethylacetamide solution), followed by 20 equivalents of DMTMM (2.0 μL, 200 mM aqueous solution), were added, and the mixture was shaken at 10°C for 30 minutes.

[0283] Each reaction solution was treated with 3.2 μL of 5 M aqueous sodium chloride solution and 106 μL of chilled (-20°C) ethanol, and allowed to stand at -78°C for 30 minutes. After centrifugation, the supernatant was removed, and 18 μL of deionized water was added to each of the resulting pellets. The three solutions were then mixed in a single PCR tube.

[0284] To the mixed solution, 6.0 μL of piperidine was added at 0°C and shaken at room temperature for 1 hour. The reaction solution was treated with 6.0 μL of 5 M aqueous sodium chloride solution and 198 μL of chilled (-20°C) ethanol, and allowed to stand at -78°C for 18 hours. After centrifugation, the supernatant was removed, and 400 μL of deionized water was added to the resulting pellet. The resulting solution was concentrated using an Amicon® Ultra Centrifugal filter (30 kD cutoff), and 100 mM aqueous sodium bicarbonate solution was added to adjust the concentration to 1 mM, which was then used as the starting material for the next step.

[0285] <Cycle B> Each of the three PCR tubes contained 13.7 μL of 1 mM solution of the starting material obtained in cycle A; 20.6 μL of 1 mM aqueous solution of one of the double-stranded oligonucleotide tags B1-B3; 5.5 μL of 10X ligase buffer (500 mM Tris-HCl, pH 7.5; 500 mM sodium chloride; 100 mM magnesium chloride; 100 mM dithiothreitol; 20 mM adenosine triphosphate); and 14.8 μL of deionized water. 0.3 μL of T4 DNA ligase (Thermo Fisher, catalog number EL0013) was added to the solution, and the resulting solution was incubated at 16°C for 16 hours.

[0286] Each reaction solution was treated with 5.5 μL of 5 M aqueous sodium chloride solution and 181 μL of chilled (-20°C) ethanol, and allowed to stand at -78°C for 30 minutes. After centrifugation, the supernatant was removed, and the resulting pellets were dissolved in 13.7 μL of 150 mM sodium borate buffer (pH 9.4).

[0287] To each tube, one of the building blocks BB1-BB3 (5.5 μL, 200 mM N,N-dimethylacetamide solution) was added (80 equivalents), followed by 80 equivalents of DMTMM (5.5 μL, 200 mM aqueous solution), and the mixture was shaken at 10°C for 1 hour. Then, to each tube, 40 equivalents of building block (2.3 μL, 200 mM N,N-dimethylacetamide solution) was added (40 equivalents of DMTMM (2.3 μL, 200 mM aqueous solution)), and the mixture was shaken at 10°C for 2 hours.

[0288] Each reaction solution was treated with 2.5 μL of 5 M aqueous sodium chloride solution and 81.4 μL of chilled (-20°C) ethanol, and allowed to stand at -78°C for 30 minutes. After centrifugation, the supernatant was removed, and 12.3 μL of deionized water was added to each of the resulting pellets. The three solutions were then mixed in a single PCR tube.

[0289] To the mixed solution, 4.1 μL of piperidine was added at 0°C and shaken at room temperature for 3 hours. The reaction solution was treated with 4.1 μL of 5 M aqueous sodium chloride solution and 136 μL of chilled (-20°C) ethanol, and allowed to stand at -78°C for 3 hours. After centrifugation, the supernatant was removed, and 400 μL of deionized water was added to the resulting pellet. The resulting solution was concentrated using an Amicon® Ultra Centrifugal filter (30 kD cutoff), and 100 mM aqueous sodium bicarbonate solution was added to adjust the concentration to 0.48 mM, which was then used as the starting material for the next step.

[0290] <Cycle C> Each of the three PCR tubes contained 14.5 μL of a 0.48 mM solution of the starting material obtained in cycle B; 10.5 μL of a 1 mM aqueous solution of one of the double-stranded oligonucleotide tags C1-C3; and 2.8 μL of 10X ligase buffer (500 mM Tris-HCl, pH 7.5; 500 mM sodium chloride; 100 mM magnesium chloride; 100 mM dithiothreitol; 20 mM adenosine triphosphate). 0.14 μL of T4 DNA ligase (Thermo Fisher, catalog number EL0013) was added to the solution, and the resulting solution was incubated at 16°C for 16 hours.

[0291] Each reaction solution was treated with 2.8 μL of 5 M aqueous sodium chloride solution and 92 μL of chilled (-20°C) ethanol, and allowed to stand at -78°C for 30 minutes. After centrifugation, the supernatant was removed, and the resulting pellets were dissolved in 7.0 μL of 150 mM sodium borate buffer (pH 9.4).

[0292] To each tube, one of the building blocks BB1-BB3 (2.8 μL, 200 mM N,N-dimethylacetamide solution) was added (80 equivalents), followed by 80 equivalents of DMTMM (2.8 μL, 200 mM aqueous solution), and the mixture was shaken at 10°C for 1 hour. Furthermore, to each tube, 40 equivalents of building block (1.4 μL, 200 mM N,N-dimethylacetamide solution) was added (40 equivalents of DMTMM (1.4 μL, 200 mM aqueous solution)), and the mixture was shaken at 10°C for 2 hours.

[0293] Each reaction solution was treated with 1.3 μL of 5 M aqueous sodium chloride solution and 41.4 μL of chilled (-20°C) ethanol, and allowed to stand at -78°C for 30 minutes. After centrifugation, the supernatant was removed, and 6.3 μL of deionized water was added to each of the resulting pellets. The three solutions were then mixed in a single PCR tube.

[0294] To the mixed solution, 2.1 μL of piperidine was added at 0°C and shaken at room temperature for 2 hours. The reaction solution was treated with 2.1 μL of 5 M aqueous sodium chloride solution and 69 μL of chilled (-20°C) ethanol and allowed to stand at -78°C for 3 hours. After centrifugation, the supernatant was removed and 400 μL of deionized water was added to the resulting pellet. The resulting solution was concentrated using an Amicon® Ultra Centrifugal filter (30 kD cutoff), and 100 mM aqueous sodium bicarbonate solution was added to adjust the concentration to 0.41 mM, which was then used as the starting material for the next step.

[0295] <CPのライゲーション> In a PCR tube, 12.2 μL of a 0.41 mM solution of starting material obtained in cycle C was added; 6.0 μL of a 1 mM aqueous solution of CP (the same as used in Example 2); 2.1 μL of 10X ligase buffer (500 mM Tris-HCl, pH 7.5; 500 mM sodium chloride; 100 mM magnesium chloride; 100 mM dithiothreitol; 20 mM adenosine triphosphate) and 0.7 μL of deionized water were added. 0.1 μL of T4 DNA ligase (Thermo Fisher, catalog number EL0013) was added to the solution, and the resulting solution was incubated at 16°C for 16 hours.

[0296] The reaction solution was treated with 2.1 μL of 5 M aqueous sodium chloride solution and 69.6 μL of chilled (-20°C) ethanol, and allowed to stand at -78°C for 30 minutes. After centrifugation, the supernatant was removed, and 400 μL of deionized water was added to the resulting pellet. The resulting solution was concentrated using an Amicon® Ultra Centrifugal filter (30 kD cutoff), and deionized water was added to prepare a 20 μM solution.

[0297] <Result> Samples from each cycle after ligation of the double-stranded oligonucleotide tag were analyzed by electrophoresis using a 2.2% agarose gel (Lonza, FlashGel® cassette, catalog number 57031). The results shown in Figure 17 confirm that encoding by the double-stranded oligonucleotide tag was achieved with high efficiency in each cycle. The samples for each lane in Figure 17 are as follows: Lane 1: AOP-U-DEL9-HP-Pr Lane 2: Sample after A1 ligation of double-stranded oligonucleotides in cycle A. Lane 3: Sample after A2 ligation of double-stranded oligonucleotides in cycle A. Lane 4: Sample after A3 ligation of double-stranded oligonucleotides from cycle A. Lane 5: Sample after B1 ligation of double-stranded oligonucleotides in cycle B Lane 6: Sample after B2 ligation of double-stranded oligonucleotides in cycle B Lane 7: Sample after B3 ligation of double-stranded oligonucleotides in cycle B Lane 8: Sample after C1 ligation of double-stranded oligonucleotide tag in cycle C. Lane 9: Sample after C2 ligation of double-stranded oligonucleotide tag in cycle C. Lane 10: Sample after C3 ligation of double-stranded oligonucleotides in cycle C. Lane 11: Sample after CP ligation Lane 12: 20 bp DNA Ladder (Lonza, catalog number 50330)

[0298] The samples after the completion of cycle C were analyzed under analytical condition 3. Figure 18 shows the chromatograph and mass spectrum results. Deconvolution of the obtained mass spectrum revealed an average molecular weight of 35532.4. This result is consistent with the average molecular weight expected after the completion of cycle C (35514.2), indicating that the reactions for library synthesis (ligation of double-stranded oligonucleotide tags and introduction of building blocks) were achieved with high efficiency.

[0299] Based on the above synthesis procedure, a model library containing 3×3×3(27) compound species using U-DEL9-HP as a starting material was successfully synthesized.

[0300] <Disconnection of the obtained model library by USER(registered trademark)enzyme> The cleavage reaction using the USER(registered trademark)enzyme model library obtained above was performed using the following procedure.

[0301] 2.0 μL of a 20 μM aqueous solution of the model library, 2 μL of CutSmart® Buffer (New England BioLabs, catalog number B7204S), and 14 μL of deionized water were added to a PCR tube. 2 μL of USER® enzyme (New England BioLabs, catalog number M5505S) was added to the solution, and the resulting solution was incubated at 37°C for 16 hours, followed by incubation at 90°C for 1 hour.

[0302] A portion of the obtained reaction solution was sampled and analyzed by modified polyacrylamide gel electrophoresis under the same conditions as in Example 3. The results shown in Figure 19 confirm that the model library using U-DEL9-HP as a raw material undergoes a highly efficient cleavage reaction using USER® enzyme. The samples in each lane of Figure 19 are as follows: Lane 1: 20 bp DNA Ladder (Lonza, catalog number 50330) Lane 2: Model Library Lane 3: Sample after cleavage reaction by USER (registered trademark) enzyme in the model library

[0303] Example 7 [Conversion of hairpin DNA of DEL compound to single-stranded DNA and imparting of new functions] <Synthesis of raw material headpiece (AAZ-DEL-HP) of DEL compound> AAZ-DEL-HP of the sequence shown in Table 22 was synthesized by the following procedure. In the sequence listing in Table 22, "(AAZ-AOP-AminoC7)" represents the group represented by the following formula (11) [Chemical formula] and other notations are the same as those in Table 2. [Table 22]

[0304] Dimethyl sulfoxide (599 μL), 4-oxo-4-[(5-sulfamoyl-1,3,4-thiadiazol-2-yl)amino)butanoic acid (37.5 μL, 0.2 M dimethyl sulfoxide solution), sodium 1-hydroxy-2,5-dioxopyrrolidine-3-sulfonate (60 μL, 0.33 M dimethyl sulfoxide / deionized water (2:1, v / v) solution) were added to eight PCR tubes, respectively, and then 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (72 μL, 0.1 M dimethyl sulfoxide solution) was added, and the resulting solution was shaken at 30 °C for 30 minutes. Then, 150 μL of triethylamine hydrochloride buffer (500 mM, pH 10) and then an aqueous solution of AOP-U-DEL9-HP (synthesized in Example 6) (75 μL, 0.67 mM) were added to each solution, and the mixture was shaken at 37 °C for 6 hours.

[0305] The reaction solution was combined in a single Bioremo centrifuge tube and treated with 800 μL of 5 M sodium chloride aqueous solution and 26.3 mL of chilled (-20°C) ethanol, and allowed to stand at -78°C for 30 minutes. After centrifugation, the supernatant was removed, and the resulting pellet was air-dried. The pellet was dissolved in deionized water and purified by reverse-phase HPLC using a Phenomenex Gemini C18 column. The target product was eluted using a two-way mobile phase gradient profile with 50 mM triethylammonium acetate buffer (pH 7.5) and acetonitrile / water (100:1, v / v). The fractions containing the target product were collected, mixed, and concentrated. The resulting solutions were desalted using an Amicon® Ultra Centrifugal filter (3 kD cutoff), precipitated with ethanol, and then deionized water was added to the pellet to make a 1 mM aqueous solution.

[0306] A portion of the obtained solution was sampled, diluted with deionized water, and then subjected to mass spectrometry by ESI-MS under the conditions of analytical condition 2 in Example 2 to identify the target product, AAZ-DEL-HP (the theoretical molecular weight of the compound and the detected molecular weight are shown in Table 22).

[0307] <Synthesis of three DEL compound raw material headpieces (SABA-DEL-HP, ClSABA-DEL-HP, mSABA-DEL-HP)> The compounds with the sequences shown in Table 23 were synthesized using the following procedure. Note that in the sequence notation in Table 23, "(SABA-AOP-AminoC7)" is represented by the following formula (12). [ka] The group represented by this is "(ClSABA-AOP-AminoC7)" and is shown in the following equation (13). [ka] It refers to the group represented by "(mSABA-AOP-AminoC7)" as shown in equation (14) below. [ka] This refers to the base represented by , and other notations are the same as in Table 2. The names of the compounds corresponding to each sequence number (No.) are as follows: No. 114: SABA-DEL-HP No. 115: ClSABA-DEL-HP No.116: mSABA-DEL-HP [Table 23] The starting carboxylic acids required to synthesize each compound are as follows: Compound: Starting material carboxylic acid SABA-DEL-HP: 4-Sulfamoylbenzoic acid ClSABA-DEL-HP: 4-chloro-3-sulfamoylbenzoic acid mSABA-DEL-HP: 3-Sulfamoylbenzoic acid

[0308] The starting carboxylic acid (50 μL, 0.2MN,N-dimethylacetamide solution) was added to each PCR tube. 3-hydroxytriazolo[4,5-b]pyridine (16.7 μL, 0.6MN,N-dimethylacetamide solution), N,N'-diisopropylcarbodiimide (16.7 μL, 0.6MN,N-dimethylacetamide solution), and then N,N-diisopropylethylamine (16.7 μL, 0.6MN,N-dimethylacetamide solution) were added to each tube, and the resulting solutions were shaken at 25°C for 30 minutes. Then, a solution of AOP-U-DEL9-HP (synthesized in Example 6) in sodium borate buffer (250 mM, pH 9.4) (100 μL, 1 mM) was added to each solution, and the mixture was shaken at 25°C for 90 minutes.

[0309] The above solutions were treated with 20 μL of 5 M aqueous sodium chloride solution and 660 μL of cooled (-20°C) ethanol, respectively, and allowed to stand at -78°C for 30 minutes. After centrifugation, the supernatant was removed, and the resulting pellets were air-dried. Each pellet was dissolved in 50 mM triethylammonium acetate buffer (pH 7.5) and purified by reverse-phase HPLC using a Phenomenex Gemini C18 column. Using a two-way mobile phase gradient profile, the target product was eluted using 50 mM triethylammonium acetate buffer (pH 7.5) and acetonitrile / water (100:1, v / v). The fractions containing the target product were collected, mixed, and concentrated. Each of the resulting solutions was desalted using an Amicon® Ultra Centrifugal filter (3 kD cutoff), precipitated with ethanol, and then deionized water was added to the pellets to make a 1 mM aqueous solution.

[0310] A portion of the obtained solution was sampled, diluted with deionized water, and then subjected to mass spectrometry by ESI-MS under the conditions of analytical condition 2 in Example 2 to identify the target compounds (the theoretical molecular weight and detected molecular weight of each compound are shown in Table 23).

[0311] Synthesis of five DEL compounds with biotin at the 3' end ("AAZ-BIO-DEL", "SABA-BIO-DEL", "ClSABA-BIO-DEL", "mSABA-BIO-DEL", and "Amino-BIO-DEL") The DEL compounds with the sequences shown in Table 24 were synthesized using the following procedure. Note that in the sequence notation in Table 24, "(BIO)" represents the following formula (15). [ka] This refers to the base represented by , and other notations are the same as in Tables 2, 20, 22, and 23. The names of the compounds corresponding to each sequence number (No.) are as follows: No. 117: AAZ-BIO-DEL No. 118: SABA-BIO-DEL No.119:ClSABA-BIO-DEL No.120: mSABA-BIO-DEL No. 121: Amino-BIO-DEL [Table 24] The compound names of the raw material headpieces used to synthesize each DEL compound are as follows: DEL compound: Raw material headpiece AAZ-BIO-DEL :AAZ-DEL-HP SABA-BIO-DEL :SABA-DEL-HP ClSABA-BIO-DEL:ClSABA-DEL-HP mSABA-BIO-DEL :mSABA-DEL-HP Amino-BIO-DEL: AOP-U-DEL9-HP

[0312] In a PCR tube, 10 μL of 1 mM aqueous solution of various raw material headpieces was added; 12 μL of 1 mM aqueous solution of Pr_TAG2_CP-BIO (prepared by annealing Pr_TAG2_CP_a and Pr_TAG2_CP-BIO_b synthesized in the same manner as in Example 1; the sequences are shown in Table 25); 4 μL of 10X ligase buffer (500 mM Tris-HCl, pH 7.5; 500 mM sodium chloride; 10 mM magnesium chloride; 100 mM dithiothreitol; 20 mM adenosine triphosphate) and 10 μL of deionized water were added. 4 μL of a 10-fold diluted aqueous solution of T4 DNA ligase (Thermo Fisher, catalog number EL0013) was added to the solution, and the resulting solution was incubated overnight at 16°C. The sequence notation in Table 25 is the same as in Table 24. The names of the compounds corresponding to (No.) in each sequence number are as follows. No.122:Pr_TAG2_CP_a No.123:Pr_TAG2_CP-BIO_b [Table 25]

[0313] The reaction solution was treated with 4 μL of 5 M aqueous sodium chloride solution and 132 μL of cooled (-20°C) ethanol, and allowed to stand at -78°C for 30 minutes. After centrifugation, the supernatant was removed, the resulting pellet was air-dried, and the pellet was dissolved in deionized water. The resulting solution was desalted using an Amicon® Ultra Centrifugal filter (3 kD cutoff).

[0314] A portion of the obtained supernatant was sampled, diluted with deionized water, and then subjected to mass spectrometry by ESI-MS under the conditions of analytical condition 3 in Example 3 to identify the target compounds (the theoretical molecular weight and detected molecular weight of each compound are shown in Table 24).

[0315] <Cutting of five DEL compounds with biotin at their 3' ends (AAZ-BIO-DEL, SABA-BIO-DEL, ClSABA-BIO-DEL, mSABA-BIO-DEL, Amino-BIO-DEL) by USER® enzyme> The five DEL compounds obtained above, "AAZ-BIO-DEL," "SABA-BIO-DEL," "ClSABA-BIO-DEL," "mSABA-BIO-DEL," and "Amino-BIO-DEL," were cleaved using USER(registered trademark) enzyme according to the following procedure, converting them into DEL compounds "DS-AAZ-BIO-DEL," "DS-SABA-BIO-DEL," "DS-ClSABA-BIO-DEL," "DS-mSABA-BIO-DEL," and "DS-Amino-BIO-DEL," each possessing double-stranded nucleic acids with the sequences shown in Table 26. Note that the sequence notation in Table 26 is the same as in Table 24, meaning that the five compounds are formed by double-stranded oligonucleotide chains of SEQ ID NOs. 124 and 125, 126 and 127, 128 and 129, 130 and 131, and 132 and 133, respectively.

[0316] [Table 26]

[0317] 10 μL of 100 μM aqueous solutions of various DEL compounds, 100 μL of CutSmart® Buffer (New England BioLabs, catalog number 7240S), and 860 μL of deionized water were added to PCR tubes. 30 μL of USER® enzyme (New England BioLabs, catalog number 5505S) was added to each solution, and the resulting solutions were incubated at 37°C for 1 hour.

[0318] The resulting reaction solutions were desalted and concentrated using an Amicon® Ultra Centrifugal filter (3kD cutoff), followed by ethanol precipitation. Deionized water was then added to each of the resulting pellets to obtain aqueous solutions.

[0319] A portion of the obtained solution was sampled, diluted with deionized water, and then subjected to mass spectrometry by ESI-MS under analytical conditions 3 of Example 3. The target double-stranded nucleic acid-containing DEL compounds "DS-AAZ-BIO-DEL," "DS-SABA-BIO-DEL," "DS-ClSABA-BIO-DEL," "DS-mSABA-BIO-DEL," and "DS-Amino-BIO-DEL" were identified (theoretical molecular weights and detected molecular weights of the compounds are shown in Table 26).

[0320] Furthermore, a portion of the obtained reaction solution was sampled and analyzed by modified polyacrylamide gel electrophoresis under the same conditions as in Example 3. As shown in Figure 20, it was confirmed that "AAZ-BIO-DEL", "SABA-BIO-DEL", "ClSABA-BIO-DEL", "mSABA-BIO-DEL", and "Amino-BIO-DEL" were cleaved in high yield and converted to "DS-AAZ-BIO-DEL", "DS-SABA-BIO-DEL", "DS-ClSABA-BIO-DEL", "DS-mSABA-BIO-DEL", and "DS-Amino-BIO-DEL", respectively. The samples for each lane in Figure 20 are as follows. Also, concentration 1 and concentration 2 indicate that the samples were prepared so that the loading amount of the DEL compound was approximately 40 ng and approximately 80 ng, respectively. Lane 1: 20 bp DNA Ladder (Lonza, catalog number 50330) Lane 2: AAZ-BIO-DEL (Concentration 1) Lane 3: AAZ-BIO-DEL (Concentration 2) Lane 4: Sample after cleavage reaction with USER® enzyme from AAZ-BIO-DEL (concentration 1) Lane 5: Sample after cleavage reaction with USER® enzyme from AAZ-BIO-DEL (concentration 2) Lane 6: SABA-BIO-DEL (Concentration 1) Lane 7: SABA-BIO-DEL (Concentration 2) Lane 8: Sample after cleavage reaction with SABA-BIO-DEL's USER® enzyme (concentration 1) Lane 9: Sample after cleavage reaction with SABA-BIO-DEL's USER® enzyme (concentration 2) Lane 10: ClSABA-BIO-DEL (Concentration 1) Lane 11: ClSABA-BIO-DEL (Concentration 2) Lane 12: Sample after cleavage reaction of ClSABA-BIO-DEL with USER® enzyme (concentration 1) Lane 13: Sample after cleavage reaction of ClSABA-BIO-DEL with USER® enzyme (concentration 2) Lane 14: mSABA-BIO-DEL (Concentration 1) Lane 15: mSABA-BIO-DEL (Concentration 2) Lane 16: Sample after cleavage reaction with mSABA-BIO-DEL using USER® enzyme (concentration 1) Lane 17: Sample after cleavage reaction with USER® enzyme of mSABA-BIO-DEL (concentration 2) Lane 18: Amino-BIO-DEL (Concentration 1) Lane 19: Amino-BIO-DEL (Concentration 2) Lane 20: Sample after cleavage reaction with USER® enzyme from Amino-BIO-DEL (concentration 1) Lane 21: Sample after cleavage reaction with USER® enzyme from Amino-BIO-DEL (concentration 2) Lane 22: 20 bp DNA Ladder (Lonza, catalog number 50330)

[0321] <Preparation of single-stranded DNA-containing DEL using streptavidin beads> The double-stranded nucleic acid-containing DEL compounds "DS-AAZ-BIO-DEL", "DS-SABA-BIO-DEL", "DS-ClSABA-BIO-DEL", "DS-mSABA-BIO-DEL", and "DS-Amino-BIO-DEL" obtained above were treated with streptavidin beads, and single-stranded DNA-containing DEL compounds "SS-AAZ-DEL", "SS-SABA-DEL", "SS-ClSABA-DEL", "SS-mSABA-DEL", and "SS-Amino-DEL" were prepared according to the following procedure. The five compounds are oligonucleotide chains of SEQ ID NOs. 125, 127, 129, 131, and 133 in Table 26, respectively.

[0322] 450 μL of Magnosphere (trademark) MS160 / Streptavidin (JSR Life Sciences, catalog number J-MS-S160S) was added to each of the five PCR tubes. The supernatant was removed by magnetic separation, and then 900 μL of 1× binding buffer (10 mM Tris-HCl, pH 7.5; 0.5 mM ethylenediaminetetraacetic acid; 1 M sodium chloride; 0.05% v / v Tween20) was added. The supernatant was removed by magnetic separation. Each of the obtained particles was mixed with an aqueous solution of "DS-AAZ-BIO-DEL", "DS-SABA-BIO-DEL", "DS-ClSABA-BIO-DEL", "DS-mSABA-BIO-DEL", or "DS-Amino-BIO-DEL" (700 pmol, 450 μL each), and 450 μL of 2× binding buffer (20 mM Tris-HCl, pH 7.5; 1 mM ethylenediaminetetraacetic acid; 2 M sodium chloride; 0.1% v / v Tween20), and shaken at room temperature for 20 minutes.

[0323] The supernatant was removed from each mixture by magnetic separation, and the particles were washed with 900 μL of 1× binding buffer (10 mM Tris-HCl, pH 7.5; 0.5 mM ethylenediaminetetraacetic acid; 1 M sodium chloride; 0.05% v / v Tween20), followed by two rounds of supernatant removal by magnetic separation. Then, 900 μL of denaturation solution (0.1 M sodium hydroxide; 0.1 M sodium chloride) was added to each mixture, and the supernatant was collected by magnetic separation.

[0324] To each of the obtained supernatants, 900 μL of 3-(N-morpholino)propanesulfonic acid buffer (1.0 M, pH 7.0) was added, and desalting was performed using an Amicon® Ultra Centrifugal filter (3 kD cutoff). After ethanol precipitation of each obtained supernatant, deionized water was added to the pellet to form a solution.

[0325] A portion of the obtained solution was sampled, diluted with deionized water, and then subjected to mass spectrometry by ESI-MS under the conditions of analysis condition 3 of Example 3. Molecular weights of 24255.3, 24170.5, 24208.8, 24176.7, and 23984.8 were observed, and the target single-stranded DNA-containing DEL compounds "SS-AAZ-DEL," "SS-SABA-DEL," "SS-ClSABA-DEL," "SS-mSABA-DEL," and "SS-Amino-DEL" were identified.

[0326] <Synthesis of photoreactive crosslinker-modified primers> The photoreactive crosslinker-modified primer "PXL-Pr" with the sequence shown in Table 27 was synthesized using the following procedure. Note that in the sequence notation in Table 27, "X" represents the following formula (16). [ka] This refers to the base represented by , and other notations are the same as in Table 2. [Table 27]

[0327] A solution of L-Pr (synthesized in the same manner as in Example 1, sequence shown in Table 28) in sodium borate buffer (150 mM, pH 9.4) cooled to 10°C (200 μL, 1 mM) was added to a PCR tube. 40 equivalents of N-Fmoc-15-amino-4,7,10,13-tetraoxaoctadecanoic acid (20 μL, 0.4 M N-dimethylacetamide solution), followed by 40 equivalents of 4-(4,6-dimethoxy[1.3.5]triazine-2-yl)-4-methylmorpholinium chloride hydrate (DMTMM) (16 μL, 0.5 M aqueous solution) were added to the tube, and the resulting mixture was shaken at 10°C for 4 hours. The sequence notation in Table 28 is the same as in Table 8. [Table 28]

[0328] The reaction mixture was treated with 23.6 μL of 5 M aqueous sodium chloride solution and 778.8 μL of cooled (-20°C) ethanol, and allowed to stand overnight at -78°C. After centrifugation, the supernatant was removed, and the resulting pellet was air-dried. 180 μL of deionized water was added to the pellet to make a solution, then 20 μL of piperidine was added, and the mixture was shaken at 10°C for 3 hours.

[0329] The obtained solution was treated with 20 μL of 5 M aqueous sodium chloride solution and 660 μL of cooled (-20°C) ethanol, and allowed to stand at -78°C for 30 minutes. After centrifugation, the supernatant was removed, and 200 μL of deionized water was added to the resulting pellet to make a 1 mM solution.

[0330] To 100 μL of the solution obtained above, 75 μL of triethylamine hydrochloride buffer (500 mM, pH 10) was added, followed by 50 equivalents of 1-((3-(3-methyl-3H-diazilin-3-yl)propanoyl)oxy)-2,5-dioxopyrrolidine-3-sulfonate sodium (Sulfo-SDA) (25 μL, 200 mM aqueous solution), and the mixture was shaken at 37°C for 2 hours.

[0331] The obtained solution was treated with 20 μL of 5 M aqueous sodium chloride solution and 660 μL of cooled (-20°C) ethanol, and allowed to stand at -78°C for 30 minutes. After centrifugation, the supernatant was removed, and 100 μL of deionized water was added to the resulting pellet, followed by 75 μL of triethylamine hydrochloride buffer (500 mM, pH 10) and 50 equivalents of Sulfo-SDA (25 μL, 200 mM aqueous solution), and the mixture was shaken at 37°C for 1 hour and 20 minutes. Another 50 equivalents of Sulfo-SDA (25 μL, 200 mM aqueous solution) were added, and the mixture was shaken at 37°C for 40 minutes.

[0332] The obtained solution was treated with 22.5 μL of 5 M aqueous sodium chloride solution and 743 μL of chilled (-20°C) ethanol, and left to stand overnight at -78°C. After centrifugation, the supernatant was removed, and 100 μL of deionized water was added to the resulting pellet, followed by 75 μL of triethylamine hydrochloride buffer (500 mM, pH 10), and then 50 equivalents of Sulfo-SDA (25 μL, 200 mM aqueous solution), and the mixture was shaken at 37°C for 3 hours.

[0333] The obtained solution was treated with 20 μL of 5 M aqueous sodium chloride solution and 660 μL of chilled (-20°C) ethanol, and allowed to stand overnight at -78°C. After centrifugation, the supernatant was removed, and the resulting pellet was air-dried. The pellet was dissolved in 50 mM triethylammonium acetate buffer (pH 7.5) and purified by reverse-phase HPLC using a Phenomenex Gemini C18 column. The target substance was eluted using a two-way mobile phase gradient profile with 50 mM triethylammonium acetate buffer (pH 7.5) and acetonitrile / water (100:1, v / v). The fraction containing the target substance was collected, mixed, and concentrated. The resulting solution was desalted using an Amicon® Ultra Centrifugal filter (3 kD cutoff), precipitated with ethanol, and then 100 μL of deionized water was added to the pellet to make a solution.

[0334] A portion of the obtained solution was sampled, diluted with deionized water, and then subjected to mass spectrometry by ESI-MS under the conditions of analytical condition 3 in Example 3 to identify the target product, the photoreactive crosslinker-modified primer "PXL-Pr" (the theoretical molecular weight of the compound and the detected molecular weight are shown in Table 27).

[0335] <Synthesis of photoreactive crosslinker-modified double-stranded EL> The single-stranded DNA DEL compounds obtained above ("SS-AAZ-DEL", "SS-SABA-DEL", "SS-ClSABA-DEL", "SS-mSABA-DEL", and "SS-Amino-DEL") were used as template DNA, and primer extension reactions using "PXL-Pr" were performed according to the following procedure to synthesize the photoreactive crosslinker-modified double-stranded DEL compounds ("PXL-DS-AAZ-DEL", "PXL-DS-SABA-DEL", "PXL-DS-ClSABA-DEL", "PXL-DS-mSABA-DEL", and "PXL-DS-Amino-DEL") with the sequences shown in Table 29. Note that the sequence notation in Table 29 is the same as in Tables 26 and 27, meaning that the five compounds are formed by two oligonucleotide chains of SEQ ID NO: 136 and SEQ ID NO: 125, SEQ ID NO: 136 and SEQ ID NO: 127, SEQ ID NO: 136 and SEQ ID NO: 129, SEQ ID NO: 136 and SEQ ID NO: 131, and SEQ ID NO: 136 and SEQ ID NO: 133, respectively. [Table 29]

[0336] In a PCR tube, 30 μL of 10 μM aqueous solution of various single-stranded DNA DEL compounds, 0.505 μL of 594 μM aqueous solution of "PXL-Pr", 60 μL of 10×NEBuffer® 2 (New England BioLabs, catalog number B7002S), and 476 μL of deionized water were added. To the solution, 6 μL of DNA Polymerase I, Large (Klenow) Fragment (New England BioLabs, catalog number M0210) and 12 μL of Deoxynucleotide (dNTP) Solution Mix (New England BioLabs, catalog number N0447) were added, and the resulting solution was incubated at 25°C for 90 minutes.

[0337] The obtained solution was desalted using an Amicon® Ultra Centrifugal filter (3kD cutoff). Deionized water was added to the supernatant to make a 60 μL solution, which was then treated with 6 μL of 5 M sodium chloride aqueous solution and 198 μL of cooled (-20°C) ethanol, and allowed to stand at -78°C for 30 minutes. After centrifugation, the supernatant was removed, and the obtained pellet was air-dried. 30 μL of deionized water was added to the pellet to make a solution.

[0338] A portion of the obtained solution was sampled, diluted with deionized water, and then subjected to mass spectrometry by ESI-MS under the conditions of analytical condition 3 of Example 3. The target photoreactive crosslinker-modified double-stranded delta compounds "PXL-DS-AAZ-DEL", "PXL-DS-SABA-DEL", "PXL-DS-ClSABA-DEL", "PXL-DS-mSABA-DEL", and "PXL-DS-Amino-DEL" were identified (theoretical molecular weights of the compounds and detected molecular weights are shown in Table 29).

[0339] Furthermore, a portion of the obtained reaction solution was sampled and analyzed by polyacrylamide gel electrophoresis under the conditions shown below. As shown in Figure 21, it was confirmed that the primer extension reaction converted the solutions to "PXL-DS-AAZ-DEL," "PXL-DS-SABA-DEL," "PXL-DS-ClSABA-DEL," "PXL-DS-mSABA-DEL," and "PXL-DS-Amino-DEL" in high yield. The samples in each lane of Figure 21 are as follows. Also, concentration 1 and concentration 2 indicate that the samples were prepared so that the loading amount of the DEL compound was approximately 40 ng and approximately 80 ng, respectively. Lane 1: 20 bp DNA Ladder (Lonza, catalog number 50330) Lane 2: DS-AAZ-BIO-DEL (Concentration 1) Lane 3: SS-AAZ-DEL (Concentration 1) Lane 4: Sample after primer extension reaction of SS-AAZ-DEL (PXL-DS-AAZ-DEL) (Concentration 1) Lane 5: DS-AAZ-BIO-DEL (Concentration 2) Lane 6: SS-AAZ-DEL (Concentration 2) Lane 7: Sample after primer extension reaction of SS-AAZ-DEL (PXL-DS-AAZ-DEL) (concentration 2) Lane 8: DS-SABA-BIO-DEL (Concentration 1) Lane 9: SS-SABA-DEL (Concentration 1) Lane 10: Sample after primer extension reaction of SS-SABA-DEL (PXL-DS-SABA-DEL) (Concentration 1) Lane 11: DS-SABA-BIO-DEL (Concentration 2) Lane 12: SS-SABA-DEL (Concentration 2) Lane 13: Sample after primer extension reaction of SS-SABA-DEL (PXL-DS-SABA-DEL) (concentration 2) Lane 14: 20 bp DNA Ladder (Lonza, catalog number 50330) Lane 15: DS-ClSABA-BIO-DEL (Concentration 1) Lane 16: SS-ClSABA-DEL (Concentration 1) Lane 17: Sample after primer extension reaction of SS-ClSABA-DEL (PXL-DS-ClSABA-DEL) (Concentration 1) Lane 18: DS-ClSABA-BIO-DEL (Concentration 2) Lane 19: SS-ClSABA-DEL (Concentration 2) Lane 20: Sample after primer extension reaction of SS-ClSABA-DEL (PXL-DS-ClSABA-DEL) (Concentration 2) Lane 21: DS-mSABA-BIO-DEL (Concentration 1) Lane 22: SS-mSABA-DEL (Concentration 1) Lane 23: Sample after primer extension reaction of SS-mSABA-DEL (PXL-DS-mSABA-DEL) (Concentration 1) Lane 24: DS-mSABA-BIO-DEL (Concentration 2) Lane 25: SS-mSABA-DEL (Concentration 2) Lane 26: Sample after primer extension reaction of SS-mSABA-DEL (PXL-DS-mSABA-DEL) (Concentration 2) Lane 27: 20 bp DNA Ladder (manufactured by Lonza, Lonza 20 bp DNA Ladder, catalog number 50330) Lane 28: DS-Amino-BIO-DEL (Concentration 1) Lane 29: SS-Amino-DEL (Concentration 1) Lane 30: Sample after primer extension reaction of SS-Amino-DEL (PXL-DS-Amino-DEL) (Concentration 1) Lane 31: DS-Amino-BIO-DEL (Concentration 2) Lane 32: SS-Amino-DEL (Concentration 2) Lane 33: Sample after primer extension reaction of SS-Amino-DEL (PXL-DS-Amino-DEL) (Concentration 2)

[0340] Polyacrylamide gel electrophoresis: Gel: SuperSep (trademark) DNA 15% TBE Gel (manufactured by Fujifilm Wako Pure Chemical Corporation, catalog number 190-15481) Loading buffer: 6× Loading Buffer (manufactured by Takara Bio Inc., catalog number 9156) Temperature: Room temperature Voltage: 200 V Electrophoresis time: 50 minutes Staining reagent: SYBER (trademark) GreenII Nucleic Acid Gel Stain (manufactured by Takara Bio Inc., catalog number 5770A)

[0341] Example 8 [Comparison of binder recovery efficiency based on the presence or absence of photocrosslinking reaction of photocrosslinker-modified double-stranded DEL] <Preparation of DEL samples> The five types of photocrosslinker-modified double-stranded DEL obtained above were each diluted with deionized water to prepare 50 nM DEL samples.

[0342] <Photocrosslinking reaction> Equipment: CL-1000 Ultraviolet Crosslinker (manufactured by UVP, INC.) Reaction tube: 96-well bottom vial (manufactured by Technolaboss Co., Ltd., catalog number 96-V050FB) Photocrosslinking reaction solution: Salmon sperm DNA, sheared (Invitrogen, catalog number AM9680): 1.6 μL ·1M NaCl aqueous solution: 5.0μL D-PBS(-) (FUJIFILM, catalog number 045-29795): 32.4 μL ·Carbonic Anhydrase IX / CA9 (Sino Biologi Cal Corporation (catalog number 10107-H08H): 10.0 μL • 50 nM aqueous solution of various DEL samples: 1.0 μL Reaction conditions: A mixture of CA9 protein and DEL solution with the above composition was incubated on ice for 1 hour. A portion of the solution was then collected (Solution S). The remaining solution was kept on ice and irradiated with 365 nm UV light for 20 minutes.

[0343] <Recovery of DEL cross-linked with protein> • Dynabeads Histag Isolation & Pulldown (Invitrogen, catalog number 10104D): 10.0 μL • Tween20 (manufactured by Sigma, catalog number P7949 - 100ML) • 10% SDS (Manufactured by NIPPON GENE, catalog number 311-90271) • Wash buffer (D-PBS(-) diluted with Tween20 and 10% SDS to prepare a 0.2% solution)

[0344] The reaction solution after UV irradiation was mixed with Dynabeads his-tag pulldown and incubated at room temperature for 30 minutes. The mixture was then fixed to a magnetic stand, allowed to stand for 2 minutes, the supernatant was removed, and 200 μL of wash buffer was added to suspend the Dynabeads. This washing procedure was repeated five more times. 80 μL of D-PBS(-) was added to the washed Dynabeads, and the mixture was reacted at 95°C for 10 minutes. After the reaction, the Dynabeads were placed on the magnetic stand, and the supernatant was collected after 2 minutes (Solution E).

[0345] <Preparation of samples that do not undergo photocrosslinking> The above series of operations were carried out without UV irradiation, and solutions S and E were prepared as samples.

[0346] <Measurement of Ct value by real-time PCR> The Ct values ​​of the various DEL samples obtained above were measured by real-time PCR, and the PCR efficiency was compared. The conditions were as follows, and the results are shown in Figure 22.

[0347] Equipment: 7500 Real-Time PCR System (Applied Biosystems) Plate: MicroAmp 96-Well plate (Applied Biosystems, catalog number N8010560) PCR reaction solution: • TaqMan Gene Expression Master Mix (Applied Biosystems, catalog number 4369016): 10.0 μL • Forward primer 1 (Table 30, SEQ ID NO: 137): 1.0 μL • Reverse primer 1 (Table 30, SEQ ID NO: 138): 1.0 μL • Taqman MGB probe (Thermo Fisher, product number 4316034, TaqMan® probe with FAM® fluorescent dye labeled at the 5' end and NFQ and MGB labeled at the 3' end of the nucleotide sequence of SEQ ID NO: 139 in Table 30): 0.50 μL • Aqueous solutions of various DEL samples (S and E samples): 2.0 μL Deionized water: 5.5 μL Temperature conditions: The temperature was maintained at 50°C for 2 minutes, then at 95°C for 10 minutes, and this cycle was repeated 40 times. 95℃ for 15 seconds 59℃, 1 minute [Table 30]

[0348] The strength of affinity between the CA9 protein and each compound as a binder is assumed to be in the following order (Non-Patent Documents 6 and 7). "PXL-DS-AAZ-DEL" > "PXL-DS-SABA-DEL" > "PXL-DS-ClSABA-DEL" > "PXL-DS-mSABA-DEL" > "PXL-DS-Amino-DEL (Negative Control)"

[0349] As shown in the graph in Figure 22, in solution E without UV irradiation, "PXL-DS-AAZ-DEL" and "PXL-DS-SABA-DEL," which have high affinity binders, showed a significantly lower Ct value compared to the negative control. However, "PXL-DS-ClSABA-DEL" and "PXL-DS-mSABA-DEL," which have moderate affinity binders, did not show a significant change in Ct value compared to the negative control. These results suggest that, in DEL screening, if photocrosslinking is not performed, it is possible to obtain high affinity binders, but it is difficult to obtain moderate affinity binders.

[0350] On the other hand, in solution E, which underwent UV irradiation, all compounds showed a significantly lower Ct value compared to the negative control. In other words, these results suggest that photocrosslinking reactions can be used in DEL screening to obtain binders with moderate affinity.

[0351] These results suggest that photoreactive crosslinker-modified double-stranded delta elastomers (DELs) derived from hairpin-type delta elastomers (DELs) possessing "selectively cleavable sites" are useful in DEL screening using photocrosslinking reactions.

[0352] Example 9 [Preparation of single-stranded DNA-containing DEL using Lambda Exonuclease] The DEL compound "DS-Amino-BIO-DEL," which contains double-stranded nucleic acid, was converted to the DEL compound "SS-Amino-DEL," which contains single-stranded DNA, by Lambda Exonuclease treatment using the following procedure.

[0353] An aqueous solution of DS-Amino-BIO-DEL (500 pF), 5 μL of 10× Lambda Exonuclease Reaction Buffer (New England BioLabs, catalog number B0262), and 1 μL of Lambda Exonuclease (New England BioLabs, catalog number M0262) were added to a PCR tube. Deionized water was then added to prepare a solution of 50 μL in total volume. The resulting solution was incubated at 37°C for 30 minutes.

[0354] Ten μL of the resulting reaction solution was sampled and subjected to mass spectrometry by ESI-MS under the conditions of analysis condition 3 in Example 3. A reading of 24024.9 was observed, identifying the target single-stranded DNA-containing DEL compound, "SS-Amino-DEL".

[0355] MS of one of the oligonucleotide strands (SEQ ID NO: 132) in DS-Amino-BIO-DEL was not detected, and the MS of the product after single-stranding was observed as the main peak, confirming that the single-stranding reaction proceeded in high yield.

[0356] Example 10 [Synthesis of photo-reactive crosslinker-modified double-stranded DEL with a different linker structure from the one used in Example 8] <Preparation of DEL containing 5 types of single-stranded DNA>

[0357] Similar to Example 7, five DEL compounds containing single-stranded DNA ("SS-AAZ-DEL", "SS-SABA-DEL", "SS-ClSABA-DEL", "SS-mSABA-DEL", and "SS-Amino-DEL") were prepared. However, in the <synthesis of five DEL compounds with biotin at the 3' end> ​​step described in Example 7, Pr_TAG2_CP (prepared by annealing Pr_TAG2_CP_a and Pr_TAG2_CP_b synthesized in the same manner as in Example 1, the sequences of which are shown in Table 31) was used instead of Pr_TAG2_CP-BIO, and the <preparation of single-stranded DNA DEL using streptavidin beads> step was replaced with the same procedure as in Example 9 for the preparation of single-stranded DNA DEL. The sequence notation in Table 31 is the same as in Table 25. The names of the compounds corresponding to each sequence number (No.) are as follows. No.122:Pr_TAG2_CP_a No.140:Pr_TAG2_CP_b [Table 31]

[0358] <Synthesis of photoreactive crosslinker-modified primer "PXL-Pr2"> The photoreactive crosslinker-modified primer "PXL-Pr2" with the sequence shown in Table 32 was synthesized using the following procedure. Note that in the sequence notation in Table 32, "(X2)" represents the following formula (17). [ka] This refers to the base represented by , and other notations are the same as in Table 2. [Table 32]

[0359] 3-(3-methyl-3H-diazilin-3-yl)propanoic acid (5 μL, 0.2 mN, N-dimethylacetamide solution) was added to a PCR tube. 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (2.5 μL, 0.4 mN, N-dimethylacetamide solution) was added to the tube, followed by N,N-diisopropylethylamine (2.5 μL, 0.4 mN, N-dimethylacetamide solution). The resulting solution was shaken at 4°C for 10 minutes. Then, a solution of L-Pr (sequence shown in Table 28) in sodium borate buffer (250 mM, pH 9.5) (100 μL, 1 mM) was added to the resulting solution, and the mixture was shaken at 10°C for 30 minutes.

[0360] The above solution was treated with 11 μL of 5 M aqueous sodium chloride solution and 363 μL of cooled (-20°C) ethanol, and allowed to stand overnight at -78°C. After centrifugation, the supernatant was removed, and the resulting pellet was air-dried. The resulting pellet was dissolved in deionized water, and the solution was desalted using an Amicon® Ultra Centrifugal filter (3 kD cutoff).

[0361] A portion of the obtained supernatant was sampled, diluted with deionized water, and then subjected to mass spectrometry by ESI-MS under the conditions of analytical condition 3 in Example 3 to identify the target photoreactive crosslinker-modified primer "PXL-Pr2" (theoretical molecular weight of each compound and the detected molecular weight are shown in Table 32).

[0362] <Synthesis of photoreactive crosslinker-modified double-stranded EL> Using the single-stranded DNA DEL compounds obtained above ("SS-AAZ-DEL", "SS-SABA-DEL", "SS-ClSABA-DEL", "SS-mSABA-DEL", and "SS-Amino-DEL") as template DNA, a primer extension reaction using "PXL-Pr2" was performed in the same procedure as in Example 7 to synthesize photoreactive crosslinker-modified double-stranded DEL compounds with the sequences shown in Table 33 ("PXL-DS-AAZ-DEL2", "PXL-DS-SABA-DEL2", "PXL-DS-ClSABA-DEL2", "PXL-DS-mSABA-DEL2", and "PXL-DS-Amino-DEL2"). Note that the sequence notation in Table 33 is the same as in Tables 26 and 32, meaning that the five compounds are formed by two oligonucleotide chains of SEQ ID NO: 142 and SEQ ID NO: 125, SEQ ID NO: 142 and SEQ ID NO: 127, SEQ ID NO: 142 and SEQ ID NO: 129, SEQ ID NO: 142 and SEQ ID NO: 131, and SEQ ID NO: 142 and SEQ ID NO: 133, respectively. [Table 33]

[0363] A portion of the obtained solution was sampled, diluted with deionized water, and then subjected to mass spectrometry by ESI-MS under the conditions of analytical condition 3 of Example 3. The target photoreactive crosslinker-modified double-stranded DELs "PXL-DS-AAZ-DEL2", "PXL-DS-SABA-DEL2", "PXL-DS-ClSABA-DEL2", "PXL-DS-mSABA-DEL2", and "PXL-DS-Amino-DEL2" were identified (theoretical molecular weights of the compounds and detected molecular weights are shown in Table 33).

[0364] Furthermore, a portion of the obtained reaction solution was sampled and analyzed by polyacrylamide gel electrophoresis under the conditions shown in Example 7. As shown in Figure 23, it was confirmed that the primer extension reaction converted the compounds to "PXL-DS-AAZ-DEL2", "PXL-DS-SABA-DEL2", "PXL-DS-ClSABA-DEL2", "PXL-DS-mSABA-DEL2", and "PXL-DS-Amino-DEL2" in high yield. The samples for each lane in Figure 23 are as follows. The samples were prepared so that the loading amount of each DEL compound was approximately 40 ng. Lane 1: 20 bp DNA Ladder (Lonza, catalog number 50330) Lane 2: SS-AAZ-DEL Lane 3: Sample after primer extension reaction of SS-AAZ-DEL (PXL-DS-AAZ-DEL2) Lane 4: SS-SABA-DEL Lane 5: Sample after primer extension reaction of SS-SABA-DEL (PXL-DS-SABA-DEL2) Lane 6: SS-ClSABA-DEL Lane 7: Sample after primer extension reaction of SS-ClSABA-DEL (PXL-DS-ClSABA-DEL2) Lane 8: SS-mSABA-DEL Lane 9: Sample after primer extension reaction of SS-mSABA-DEL (PXL-DS-mSABA-DEL2) Lane 10: SS-Amino-DEL Lane 11: Sample after primer extension reaction of SS-Amino-DEL (PXL-DS-Amino-DEL2) Lane 12: 20 bp DNA Ladder (Lonza, catalog number 50330)

[0365] <Preparation of the single-stranded DNA-containing DEL compound "SS-Amino-DEL3"> The DEL compound "SS-Amino-DEL3" (the sequence is shown in Table 34) having a single-stranded DNA was prepared in the same manner as in Example 1 using a nucleic acid automatic synthesizer nS-8II (manufactured by Gene Design). Note that "SS-Amino-DEL3" has the same structure as the oligonucleotide derived by the same procedure as <Preparation of DEL having 5 kinds of single-stranded DNAs> using "U-DEL12-HP" (the sequence is shown in Table 11) as the raw material headpiece. Also, the sequence listings in Table 34 are the same as those in Table 10.

Table 34

[0366] <Synthesis of DEL Compound "SS-AAZ-DEL3" Having a Single-Stranded DNA> The DEL compound "SS-AAZ-DEL3" having a single-stranded DNA of the sequence shown in Table 35 was synthesized by the following procedure. In the sequence listings in Table 35, "[AAZ-mdC(TEG-amino)]" means the group represented by the following formula (18)

Chemical formula

Table 35

[0367] Similar to Example 7 <Synthesis of the Raw Material Headpiece (AAZ-DEL-HP) of the DEL Compound>, using "SS-Amino-DEL3" as the raw material, a condensation reaction with 4-oxo-4-[(5-sulfamoyl-1,3,4-thiadiazol-2-yl)amino)butanoic acid was carried out.

[0368] A part of the obtained solution was sampled, diluted with deionized water, and then subjected to mass spectrometry by ESI-MS under the conditions of Analysis Condition 3 in Example 3 to identify the DEL compound "SS-AAZ-DEL3" having the target single-stranded DNA (the theoretical molecular weight and the detected molecular weight of the compound are shown in Table 35).

[0369] Synthesis of DEL compounds containing three types of single-stranded DNA ("SS-SABA-DEL3", "SS-ClSABA-DEL3", and "SS-mSABA-DEL3") Three single-stranded DNA DEL compounds ("SS-SABA-DEL3", "SS-ClSABA-DEL3", and "SS-mSABA-DEL3") with the sequences shown in Table 36 were synthesized using the following procedure. Note that in the sequence notation in Table 36, "[SABA-mdC(TEG-amino)]" is represented by the following formula (19). [ka] It refers to the group represented by "[ClSABA-mdC(TEG-amino)]" as shown in the following formula (20). [ka] It refers to the group represented by "[mSABA-mdC(TEG-amino)]" as shown in the following formula (21). [ka] This refers to the group represented by , and other notations are the same as in Table 10. The names of the compounds corresponding to each sequence number (No.) are as follows: No. 145: SS-SABA-DEL3 No. 146: SS-ClSABA-DEL3 No. 147: SS-mSABA-DEL3 [Table 36] The starting carboxylic acids required to synthesize each compound are as follows: Compound: Starting material carboxylic acid SABA-DEL-HP: 4-Sulfamoylbenzoic acid ClSABA-DEL-HP: 4-chloro-3-sulfamoylbenzoic acid mSABA-DEL-HP: 3-Sulfamoylbenzoic acid

[0370] The starting carboxylic acid (4 μL, 0.2 mN, N-dimethylacetamide solution) was added to a PCR tube. 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (2 μL, 0.4 mN, N-dimethylacetamide solution) was added to the tube, followed by N,N-diisopropylethylamine (2 μL, 0.4 mN, N-dimethylacetamide solution), and the resulting solution was shaken at 10°C for 30 minutes. Then, a solution of SS-Amino-DEL3 in sodium borate buffer (250 mM, pH 9.5) (50 μL, 1 mM) was added to the resulting solution, and the mixture was shaken at 10°C for 2 hours.

[0371] The above solution was treated with 5.8 μL of 5 M aqueous sodium chloride solution and 192 μL of cooled (-20°C) ethanol, and allowed to stand at -78°C for 30 minutes. After centrifugation, the supernatant was removed, and the resulting pellet was air-dried.

[0372] The obtained pellet was dissolved in 50 mM triethylammonium acetate buffer (pH 7.5) and purified by reverse-phase HPLC using a Phenomenex Gemini C18 column. The target product was eluted using 50 mM triethylammonium acetate buffer (pH 7.5) and acetonitrile / 500 mM triethylammonium acetate buffer (9:1, v / v) using a two-way mobile phase gradient profile. The fraction containing the target product was collected, mixed, and concentrated. The resulting solution was desalted using an Amicon® Ultra Centrifugal filter (3kD cutoff), precipitated with ethanol, and then deionized water was added to the pellet to form a solution.

[0373] A portion of the obtained solution was sampled, diluted with deionized water, and then subjected to mass spectrometry by ESI-MS under the conditions of analytical condition 3 of Example 3 to identify the target compounds "SS-SABA-DEL3," "SS-ClSABA-DEL3," and "SS-mSABA-DEL3" (the theoretical molecular weight and detected molecular weight of each compound are shown in Table 36).

[0374] <Synthesis of photoreactive crosslinker-modified primer "PXL-Pr3"> The photoreactive crosslinker-modified primer "PXL-Pr3" with the sequence shown in Table 37 was synthesized using the same procedure as described in <Synthesis of Photoreactive Crosslinker-Modified Primer "PXL-Pr2">. However, instead of "L-Pr" as a starting material, "L-Pr3" (synthesized in the same way as in Example 1, with the sequence shown in Table 38) was used. The sequence notation in Table 37 is the same as in Table 32. Also, the sequence notation in Table 38 is the same as in Table 8. [Table 37]

[0375] [Table 38]

[0376] A portion of the obtained solution was sampled, diluted with deionized water, and then subjected to mass spectrometry by ESI-MS under the conditions of analytical condition 3 in Example 3 to identify the target photoreactive crosslinker-modified primer "PXL-Pr3" (theoretical molecular weight of each compound and the detected molecular weight are shown in Table 37).

[0377] <Synthesis of five types of photoreactive crosslinker-modified double-stranded DEL "PXL-DS-DEL3"> Using the single-stranded DNA DEL compounds obtained above ("SS-AAZ-DEL3", "SS-SABA-DEL3", "SS-ClSABA-DEL3", "SS-mSABA-DEL3", and "SS-Amino-DEL3") as template DNA, a primer extension reaction using "PXL-Pr3" was performed in the same procedure as in Example 7 to synthesize five photoreactive crosslinker-modified double-stranded DEL compounds with sequences shown in Table 39 ("PXL-DS-AAZ-DEL3", "PXL-DS-SABA-DEL3", "PXL-DS-ClSABA-DEL3", "PXL-DS-mSABA-DEL3", and "PXL-DS-Amino-DEL3"). Note that the sequence notation in Table 39 is the same as in Tables 34-37, meaning that the five compounds are formed by double-stranded oligonucleotide chains of SEQ ID NO: 150 and SEQ ID NO: 144, SEQ ID NO: 150 and SEQ ID NO: 145, SEQ ID NO: 150 and SEQ ID NO: 146, SEQ ID NO: 150 and SEQ ID NO: 147, and SEQ ID NO: 150 and SEQ ID NO: 143, respectively. [Table 39]

[0378] A portion of the obtained solution was sampled, diluted with deionized water, and then subjected to mass spectrometry by ESI-MS under the conditions of analytical condition 3 of Example 3. The target photoreactive crosslinker-modified double-chain DEL compounds "PXL-DS-AAZ-DEL3", "PXL-DS-SABA-DEL3", "PXL-DS-ClSABA-DEL3", "PXL-DS-mSABA-DEL3", and "PXL-DS-Amino-DEL3" were identified (theoretical molecular weights and detected molecular weights of the compounds are shown in Table 39).

[0379] Furthermore, a portion of the obtained reaction solution was sampled and analyzed by polyacrylamide gel electrophoresis under the conditions shown in Example 7. As shown in Figure 24, it was confirmed that the primers were converted to "PXL-DS-AAZ-DEL3", "PXL-DS-SABA-DEL3", "PXL-DS-ClSABA-DEL3", "PXL-DS-mSABA-DEL3", and "PXL-DS-Amino-DEL3" in high yield through the primer extension reaction. The samples for each lane in Figure 24 are as follows. The samples were prepared so that the loading amount of each DEL compound was approximately 40 ng. Lane 1: 20 bp DNA Ladder (Lonza, catalog number 50330) Lane 2: SS-AAZ-DEL3 Lane 3: Sample after primer extension reaction of SS-AAZ-DEL3 (PXL-DS-AAZ-DEL3) Lane 4: SS-SABA-DEL3 Lane 5: Sample after primer extension reaction of SS-SABA-DEL3 (PXL-DS-SABA-DEL3) Lane 6: SS-ClSABA-DEL3 Lane 7: Sample after primer extension reaction of SS-ClSABA-DEL3 (PXL-DS-ClSABA-DEL3) Lane 8: SS-mSABA-DEL3 Lane 9: Sample after primer extension reaction of SS-mSABA-DEL3 (PXL-DS-mSABA-DEL3) Lane 10: SS-Amino-DEL3 Lane 11: Sample after primer extension reaction of SS-Amino-DEL3 (PXL-DS-Amino-DEL3) Lane 12: 20 bp DNA Ladder (Lonza, catalog number 50330)

[0380] Example 11 [Comparison of the efficiency of binder recovery with or without the photocrosslinking reaction of the photoreactive crosslinker-modified double-stranded DEL synthesized in Example 10] <Preparation of DEL samples> The four types of photoreactive crosslinker-modified double-stranded DELs (PXL-DS-mSABA-DEL2, PXL-DS-Amino-DEL2, PXL-DS-mSABA-DEL3, and PXL-DS-Amino-DEL3) obtained in Example 10 were each diluted with deionized water to prepare 50 nM DEL samples.

[0381] <Photocrosslinking reaction> Equipment: CL-1000 Ultraviolet Crosslinker (manufactured by UVP, INC) Microtube: 1.5 mL siliconized microtube round bottom (manufactured by Waterson Co., Ltd., catalog number 131-615CH) Reaction tube: 96-well bottom vial (manufactured by Technolas Boshi Co., Ltd., catalog number 96-V050FB) Photocrosslinking reaction solution: ·Salmon Sperm DNA, sheared (Invitrogen, catalog number AM9680): 1.6 μL ·1 M NaCl (manufactured by FUJIFILM, catalog number 191-01665): 5.0 μL ·D-PBS(-) (manufactured by FUJIFILM, catalog number 045―29795): 32.4 μL ·Carbonic Anhydrase IX / CA9 (manufactured by Sino Biologocal, catalog number 10107-H08H): 10.0 μL ·Aqueous solution of various DEL samples (50 nM): 1.0 μL Reaction conditions: The CA9 protein and the DEL solution with the above composition were mixed in a microtube and reacted on ice for 1 hour. The total volume was dispensed into the reaction tube, maintained on ice, and irradiated with UV light at 365 nm for 20 minutes.

[0382] <Recovery of DEL crosslinked with protein> • Dynabeads Histag Isolation & Pulldown (Invitrogen, catalog number 10104D): 10.0 μL • Tween20 (manufactured by Sigma, catalog number P7949 - 100ML) • 10% SDS (Manufactured by NIPPON GENE, catalog number 311-90271) • Wash buffer (D-PBS(-) diluted with Tween20 and 10% SDS to prepare a 0.2% solution) • Imidazole (manufactured by FUJIFILM, catalog number 097-05391) • Elution buffer (dissolve imidazole in D-PBS(-) to a concentration of 200 mM and adjust to pH 7.4)

[0383] D-PBS(-) was added to the reaction solution after UV irradiation. The remaining solution was then mixed with Dynabeads his-tag pulldown and incubated at room temperature for 30 minutes. The Dynabeads were fixed to a magnetic stand, allowed to stand for 2 minutes, the supernatant was removed, and 200 μL of wash buffer was added to suspend the Dynabeads. This procedure was repeated five more times. 100 μL of emulsion buffer was added to the washed Dynabeads and allowed to stand at room temperature for 10 minutes. After the reaction, the Dynabeads were placed on a magnetic stand, and the supernatant was collected as a sample after 2 minutes.

[0384] <Preparation of samples that do not undergo photocrosslinking> The above series of operations were performed without UV irradiation, and samples were collected from each.

[0385] <Measurement of Ct value by real-time PCR> The Ct values ​​of the various DEL samples obtained above were measured by real-time PCR, as in Example 8. The results of comparing the ΔCt values ​​(difference from the Ct value of the negative control) are shown in Figure 25.

[0386] As described in Example 8, the affinity strength between the CA9 protein and "PXL-DS-mSABA-DEL2" and "PXL-DS-mSABA-DEL3" is expected to be moderate (Non-Patent Documents 6 and 7).

[0387] As shown in the graph in Figure 25, the ΔCt values ​​were small in all samples without UV irradiation, suggesting that, similar to the results in Example 8, it is difficult to obtain binders with moderate affinity in DEL screening if photocrosslinking is not performed.

[0388] On the other hand, in the samples subjected to UV irradiation, the ΔCt value increased compared to the samples without UV irradiation. This suggests that the photoreactive crosslinker-modified double-stranded DEL used in this example (which has a different linker structure from the photoreactive crosslinker-modified double-stranded DEL used in Example 8) can also be expected to yield binders with moderate affinity.

[0389] These results suggest that photoreactive crosslinker-modified double-stranded delta-electrolytes (DELs) with various linker structures, derived from hairpin-type delta-electrolytes (DELs) possessing "selectively cleavable sites," are useful in DEL screening using photocrosslinking reactions.

[0390] Example 12 [Comparison of binder recovery efficiency (DNA detection sensitivity) between "photoreactive crosslinker-modified double-stranded DEL with a covalent bond between the crosslinker and the coding sequence" and "photoreactive crosslinker-modified double-stranded DEL without a covalent bond between the crosslinker and the coding sequence"]

[0391] <Synthesis of photoreactive crosslinker-modified double-stranded DEL without covalent bonds between the crosslinker and coding sequence> Using the DEL compounds having single-stranded DNA obtained in Example 10 ("SS-SABA-DEL3", "SS-ClSABA-DEL3", "SS-mSABA-DEL3", and "SS-Amino-DEL3"), annealing with "PXL-Pr3" was performed according to the following procedure to synthesize four photo-reactive cross-linker-modified double-stranded DEL compounds ("PXL-DS-SABA-DEL4", "PXL-DS-ClSABA-DEL4", "PXL-DS-mSABA-DEL4", and "PXL-DS-Amino-DEL4") of the sequences shown in Table 40. The sequence listings in Table 40 are the same as those in Tables 34, 36, and 37, and it means that each of the four compounds is formed by the double strands of the oligonucleotide strands of SEQ ID NO: 148 and SEQ ID NO: 145, SEQ ID NO: 148 and SEQ ID NO: 146, SEQ ID NO: 148 and SEQ ID NO: 147, and SEQ ID NO: 148 and SEQ ID NO: 143. [Table 40]

[0392] To a PCR tube, a 10 μM aqueous solution of 30 μL of DEL compounds having various single-stranded DNAs; 3.77 μL of a 159 μM aqueous solution of "PXL-Pr3" were added. Deionized water was added to the resulting aqueous solution to make the total liquid volume 60 μL. Then, after incubation at 90 °C for 2 minutes, it was cooled to room temperature over 30 minutes.

[0393] <Preparation of DEL Sample> Four types of "photoreactive crosslinker-modified double-stranded DEL without covalent bonds between the crosslinker and coding sequence" obtained above ("PXL-DS-SABA-DEL4", "PXL-DS-ClSABA-DEL4", "PXL-DS-mSABA-DEL4", and "PXL-DS-Amino-DEL4"), and four types of "photoreactive crosslinker-modified double-stranded DEL with covalent bonds between the crosslinker and coding sequence" obtained in Example 10 ("PXL-DS-SABA-DEL3", "PXL-DS-ClSABA-DEL3", "PXL-DS-mSABA-DEL3", and "PXL-DS-Amino-DEL3") were each diluted with deionized water to prepare 50 nM DEL samples.

[0394] <Photocrosslinking reaction> Equipment: CL-1000 Ultraviolet Crosslinker (manufactured by UVP, INC.) Microtube: 1.5 mL Siliconized Microtube, Round Bottom (Watson Co., Ltd., Catalog No. 131-615CH) Reaction tube: 96-well bottom vial (manufactured by Technolaboss Co., Ltd., catalog number 96-V050FB) Photocrosslinking reaction solution: Salmon sperm DNA, sheared (Invitrogen, catalog number AM9680): 1.6 μL • 1M NaCl (FUJIFILM, catalog number 191-01665): 5.0 μL • D-PBS(-) (FUJIFILM, catalog number 045-29795): 32.4 μL • Carbonic Anhydrase IX / CA9 (Sino Biologocal, catalog number 10107-H08H): 10.0 μL • Aqueous solutions of various DEL samples (50 nM): 1.0 μL Reaction conditions: A mixture of CA9 protein and DEL solution with the above composition was incubated on ice for 2 hours. Afterward, it was kept on ice and irradiated with 365nm UV light for 20 minutes.

[0395] <Recovery of DEL cross-linked with protein> • Dynabeads Histag Isolation & Pulldown (Invitrogen, catalog number 10104D): 10.0 μL • Tween20 (manufactured by Sigma, catalog number P7949 - 100ML) • 10% SDS (Manufactured by NIPPON GENE, catalog number 311-90271) • Wash buffer (D-PBS(-) diluted with Tween20 and 10% SDS to prepare a 0.2% solution) • Imidazole (manufactured by FUJIFILM, catalog number 097-05391) • Elution buffer (dissolve imidazole in D-PBS(-) to a concentration of 200 mM and adjust to pH 7.4)

[0396] The reaction solution after UV irradiation was mixed with Dynabeads his-tag pulldown and incubated at room temperature for 30 minutes. The mixture was then fixed to a magnetic stand, allowed to stand for 2 minutes, the supernatant was removed, and 200 μL of wash buffer was added to suspend the Dynabeads. This procedure was repeated five more times. 100 μL of emulsion buffer was added to the washed Dynabeads, and the mixture was reacted at room temperature for 10 minutes. After the reaction, the Dynabeads were placed on the magnetic stand, and the supernatant was collected as a sample after 2 minutes.

[0397] <Preparation of samples that do not undergo photocrosslinking> The above series of operations were performed without UV irradiation, and samples were collected from each.

[0398] <Measurement of Ct value by real-time PCR> The Ct values ​​of the various DEL samples obtained above were measured by real-time PCR, as in Example 8. The results of comparing the ΔCt values ​​(difference from the Ct value of the negative control) are shown in Figure 26.

[0399] Similar to Example 8, it is assumed that the strength of the affinity as a binder between CA9 protein and each compound is in the following order (Non-Patent Documents 6 and 7). "PXL-DS-SABA-DEL3", "PXL-DS-SABA-DEL4"> "PXL-DS-ClSABA-DEL3", "PXL-DS-ClSABA-DEL4"> "PXL-DS-mSABA-DEL3", "PXL-DS-mSABA-DEL4"> "PXL-DS-Amino-DEL3 (negative control)", "PXL-DS-Amino-DEL4 (negative control)" As shown in the graph of Fig. 26, in the samples subjected to UV irradiation, for any of the binders, the "photoreactive crosslinker-modified double-stranded DEL (PXL-DS-DEL3) having a covalent bond between the crosslinker and the coding sequence" has a significantly higher ΔCt value than the "photoreactive crosslinker-modified double-stranded DEL (PXL-DS-DEL4) having no covalent bond between the crosslinker and the coding sequence". Even when the type of binder is the same, due to the difference in the structure of the photoreactive crosslinker-modified double-stranded DEL, the ΔCt values are different, suggesting that the "photoreactive crosslinker-modified double-stranded DEL having a covalent bond between the crosslinker and the coding sequence" has a higher binder recovery efficiency (DNA detection sensitivity).

[0400] This result means that the "photoreactive crosslinker-modified double-stranded DEL having a covalent bond between the crosslinker and the coding sequence" derived from the hairpin-type DEL having a "selectively cleavable site" is more useful than the "photoreactive crosslinker-modified double-stranded DEL having no covalent bond between the crosslinker and the coding sequence" in the DEL screening using the photocrosslinking reaction.

[0401] Example 13 [Verification of binder recovery efficiency in the photocrosslinking reaction of photoreactive crosslinker-modified double-stranded DEL applied to strong separation and elution conditions]

[0402] <Preparation of DEL sample> Similar to Example 12, four types of "photoreactive crosslinker-modified double-stranded DEL without covalent bonds between the crosslinker and coding sequence" ("PXL-DS-SABA-DEL4", "PXL-DS-ClSABA-DEL4", "PXL-DS-mSABA-DEL4", and "PXL-DS-Amino-DEL4") and four types of "photoreactive crosslinker-modified double-stranded DEL with covalent bonds between the crosslinker and coding sequence" ("PXL-DS-SABA-DEL3", "PXL-DS-ClSABA-DEL3", "PXL-DS-mSABA-DEL3", and "PXL-DS-Amino-DEL3") were diluted with deionized water to prepare 50 nM DEL samples.

[0403] <Photocrosslinking reaction> Equipment: CL-1000 Ultraviolet Crosslinker (manufactured by UVP, INC.) Reaction tube: 96-well bottom vial (manufactured by Technolaboss Co., Ltd., catalog number 96-V050FB) Photocrosslinking reaction solution: Salmon sperm DNA, sheared (Invitrogen, catalog number AM9680): 1.6 μL • 1M NaCl (FUJIFILM, catalog number 191-01665): 5.0 μL • D-PBS(-) (FUJIFILM, catalog number 045-29795): 39.4 μL • Carbonic Anhydrase IX / CA9 (Sino Biologocal, catalog number 10107-H08H): 3.0 μL • Aqueous solutions of various DEL samples (50 nM): 1.0 μL Reaction conditions: A mixture of CA9 protein and DEL solution with the above composition was incubated on ice for 2 hours. Afterward, it was kept on ice and irradiated with 365nm UV light for 20 minutes.

[0404] <Recovery of DEL cross-linked with protein> • Dynabeads Histag Isolation & Pulldown (Invitrogen, catalog number 10104D): 10.0 μL • Tween20 (manufactured by Sigma, catalog number P7949 - 100ML) • Wash buffer (D-PBS(-) diluted with Tween20 to prepare a 0.2% solution) • 200 mM imidazole solution (manufactured by FUJIFILM, catalog number 097-05391)

[0405] The reaction solution after UV irradiation was mixed with Dynabeads his-tag pulldown and incubated at room temperature for 30 minutes. The mixture was then fixed to a magnetic stand, allowed to stand for 2 minutes, the supernatant was removed, and 200 μL of wash buffer was added to suspend the Dynabeads. The mixture was then reacted at 90°C for 10 minutes. This procedure was repeated three more times. After washing, 30 μL of 200 mM imidazole solution was added to the Dynabeads and the mixture was reacted at room temperature for 10 minutes. After the reaction, the Dynabeads were placed on the magnetic stand, and the supernatant was collected as a sample after 2 minutes.

[0406] <Preparation of samples that do not undergo photocrosslinking> The above series of operations were performed without UV irradiation, and samples were collected from each.

[0407] As described above in "<Recovery of Protein-Crosslinked DEL>", the recovery of protein-crosslinked DEL is subject to heating conditions, resulting in strong separation and elution conditions.

[0408] <Measurement of Ct value by real-time PCR> The Ct values ​​of the various DEL samples obtained above were measured by real-time PCR, as in Example 8. The results of comparing the ΔCt values ​​(difference from the Ct value of the negative control) are shown in Figure 27.

[0409] As shown in the graph of Fig. 27, in the samples subjected to UV irradiation, for any of the binders, the "photo-responsive cross-linker-modified double-stranded DEL (PXL-DS-DEL3) having a covalent bond between the cross-linker and the coding sequence" has a significantly higher ΔCt value than the "photo-responsive cross-linker-modified double-stranded DEL (PXL-DS-DEL4) having no covalent bond between the cross-linker and the coding sequence". Even though the types of binders are the same, due to the difference in the structure of the photo-responsive cross-linker-modified double-stranded DEL, the ΔCt values are different, suggesting that the "photo-responsive cross-linker-modified double-stranded DEL having a covalent bond between the cross-linker and the coding sequence" has a higher binder recovery efficiency (DNA detection sensitivity).

[0410] These results mean that the "photo-responsive cross-linker-modified double-stranded DEL having a covalent bond between the cross-linker and the coding sequence", which is derived from the hairpin-type DEL having a "selectively cleavable site", is more useful than the "photo-responsive cross-linker-modified double-stranded DEL having no covalent bond between the cross-linker and the coding sequence" in the DEL screening using a photocrosslinking reaction.

[0411] Also, these results suggest that the "photo-responsive cross-linker-modified double-stranded DEL having a covalent bond between the cross-linker and the coding sequence", which is derived from the hairpin-type DEL having a "selectively cleavable site", is also applicable to the DEL screening under strong separation conditions and elution conditions for the purpose of removing non-specific binders, etc.

[0412] Example 14 [Conversion from hairpin DNA to single-stranded DNA using U-DEL-13-HP as a raw material and imparting new functions] [Synthesis of the raw material headpiece ("mSABA-DEL-HP5") of the DEL compound] The "mSABA-DEL-HP5" compound with the sequence shown in Table 41 was synthesized using "U-DEL13-HP" as the starting material, following the same procedure as in Example 10 <Synthesis of three single-stranded DNA DEL compounds ("SS-SABA-DEL3", "SS-ClSABA-DEL3", and "SS-mSABA-DEL3")>. Note that the notation in Table 41 is the same as in Table 36. [Table 41]

[0413] A portion of the obtained solution was sampled, diluted with deionized water, and then subjected to mass spectrometry by ESI-MS under the conditions of analytical condition 3 in Example 3 to identify the target compound "mSABA-DEL-HP5" (theoretical molecular weight of each compound and the detected molecular weight are shown in Table 41).

[0414] <Synthesis of hairpin DEL compound ("mSABA-DEL5")> The hairpin DEL compound ("mSABA-DEL5") with the sequence shown in Table 42 was synthesized by double-strand ligation of the raw material headpiece "mSABA-DEL-HP5" with Pr_TAG2_CP, in the same manner as in Example 10. Note that the sequence notation in Table 42 is the same as in Table 36. [Table 42]

[0415] A portion of the obtained solution was sampled, diluted with deionized water, and then subjected to mass spectrometry by ESI-MS under the conditions of analytical condition 3 in Example 3 to identify the target compound "mSABA-DEL5" (theoretical molecular weight of each compound and the detected molecular weight are shown in Table 42).

[0416] <Cutting of hairpin DEL compound ("mSABA-DEL5") by USER® enzyme> The hairpin DEL compound "mSABA-DEL5" obtained above was cleaved by USER® enzyme using the same procedure as in Example 7 <Cleavage of 5 types of DEL compounds with biotin at the 3' end (AAZ-BIO-DEL, SABA-BIO-DEL, ClSABA-BIO-DEL, mSABA-BIO-DEL, Amino-BIO-DEL) by USER® enzyme>, and converted into the DEL compound "DS-mSABA-DEL5" having a double-stranded nucleic acid sequence shown in Table 43. Note that the sequence notation in Table 43 is the same as in Table 36, meaning that it is formed by a double-stranded oligonucleotide chain of SEQ ID NO: 153 and SEQ ID NO: 154. [Table 43]

[0417] A portion of the obtained solution was sampled, diluted with deionized water, and then subjected to mass spectrometry by ESI-MS under the conditions of analytical condition 3 of Example 3 to identify the target double-stranded nucleic acid DEL compound "DS-mSABA-DEL5" (theoretical molecular weight and detected molecular weight of each compound are shown in Table 43).

[0418] Furthermore, a portion of the obtained reaction solution was sampled and analyzed by modified polyacrylamide gel electrophoresis under the same conditions as in Example 3. The results shown in Figure 28 confirm that "mSABA-DEL5" was cleaved in high yield and converted to "DS-mSABA-DEL5". The samples in each lane of Figure 28 are as follows. Lane 1: 20 bp DNA Ladder (Lonza, catalog number 50330) Lane 2: mSABA-DEL5 Lane 3: Sample after cleavage reaction with mSABA-DEL5 using USER® enzyme. Lane 4: 20 bp DNA Ladder (Lonza, catalog number 50330)

[0419] <Conversion of the double-stranded nucleic acid DEL compound "DS-mSABA-DEL5" to single-stranded DEL by Lambda Exonuclease> The double-stranded nucleic acid-containing DEL compound "DS-mSABA-DEL5" obtained above was treated with Lambda Exonuclease in the same manner as in Example 9 to prepare the single-stranded DNA-containing DEL compound "SS-mSABA-DEL5". "SS-mSABA-DEL5" is the oligonucleotide chain of Sequence ID No. 154 in Table 43.

[0420] A portion of the obtained supernatant was sampled, diluted with deionized water, and then subjected to mass spectrometry by ESI-MS under the conditions of analysis condition 3 in Example 3. A molecular weight of 23825.8 was observed, and the target single-stranded DNA-containing DEL compound "SS-mSABA-DEL5" was identified.

[0421] <Synthesis of photoreactive crosslinker-modified primer "PXL-Pr5"> The photoreactive crosslinker-modified primer "PXL-Pr5" with the sequence shown in Table 44 was synthesized using the same procedure as in Example 10 <Synthesis of photoreactive crosslinker-modified primer "PXL-Pr2">. However, instead of "L-Pr" as a starting material, "L-Pr5" (synthesized in the same way as in Example 1, with the sequence shown in Table 45) was used. The sequence notation in Table 44 is the same as in Table 32. Also, the sequence notation in Table 45 is the same as in Table 8. [Table 44]

[0422] [Table 45]

[0423] A portion of the obtained solution was sampled, diluted with deionized water, and then subjected to mass spectrometry by ESI-MS under the conditions of analytical condition 3 in Example 3 to identify the target photoreactive crosslinker-modified primer "PXL-Pr5" (theoretical molecular weight of each compound and the detected molecular weight are shown in Table 44).

[0424] <Synthesis of a photoreactive crosslinker-modified double-stranded DEL compound ("PXL-DS-mSABA-DEL5")> The single-stranded DNA-containing DEL compound "SS-mSABA-DEL5" obtained above Using the template DNA, a primer extension reaction using "PXL-Pr5" was performed in the same procedure as in Example 7 to synthesize the photoreactive crosslinker-modified double-stranded DEL compound "PXL-DS-mSABA-DEL5" with the sequence shown in Table 46. Note that the sequence notation in Table 46 is the same as in Tables 36 and 44, meaning that it is formed by a double strand of oligonucleotide chains of SEQ ID NO: 157 and SEQ ID NO: 154. [Table 46]

[0425] A portion of the obtained solution was sampled, diluted with deionized water, and then subjected to mass spectrometry by ESI-MS under the conditions of analytical condition 3 in Example 3 to identify the target photoreactive crosslinker-modified double-stranded DEL "PXL-DS-mSABA-DEL5" (the theoretical molecular weight of the compound and the detected molecular weight are shown in Table 46).

[0426] Furthermore, a portion of the obtained reaction solution was sampled and analyzed by polyacrylamide gel electrophoresis under the conditions shown in Example 7. The results shown in Figure 29 confirm that the primer extension reaction converted the solution to "PXL-DS-mSABA-DEL5" in high yield. The samples for each lane in Figure 29 are as follows. The samples were prepared so that each DEL compound was loaded at approximately 40 ng. Lane 1: 20 bp DNA Ladder (Lonza, catalog number 50330) Lane 2: DS-mSABA-DEL5 Lane 3: SS-mSABA-DEL5 Lane 4: Sample after primer extension reaction of SS-mSABA-DEL5 (PXL-DS-mSABA-DEL5) Lane 5: 20 bp DNA Ladder (Lonza, catalog number 50330)

[0427] Example 15 [Synthesis of cross-linker modified double-stranded DEL] <Synthesis of three types of crosslinker-modified primers (PA-Pr, TPD-Pr, ACA-Pr)> Various crosslinker-modified primers with the sequences shown in Table 47 were synthesized using the following procedure. Note that in the sequence notation in Table 47, "(PA)" represents the following formula (22). [ka] It means the base represented by "(TPD)" is in the following formula (23) [ka] It means the base represented by "(ACA)" is in the following formula (24). [ka] This refers to the base represented by , and other notations are the same as in Table 2. [Table 47] The active ester raw materials required to synthesize each compound are as follows: Compound: Active ester raw material PA-Pr: N-succinimidyl 4-azidobenzoate TPD-Pr: 4-(3-(trifluoromethyl)-3H-diazilin-3-yl)benzoate N-succinimidyl ACA-Pr: N-succinimidyl acrylate

[0428] A 1 mM solution of L-Pr (described in Example 7) in sodium borate buffer (250 mM, pH 9.4) cooled to 10°C was added to a PCR tube. 50 equivalents of the active ester starter (25 μL, 0.2 M dimethyl sulfoxide solution) were added to the tube, and the resulting mixture was shaken at 10°C for 30 minutes.

[0429] The reaction mixture was treated with 12 μL of 5 M aqueous sodium chloride solution and 396 μL of cooled (-20°C) ethanol, and allowed to stand at -78°C for 30 minutes. After centrifugation, the supernatant was removed, and the resulting pellet was air-dried.

[0430] The obtained pellet was dissolved in 50 mM triethylammonium acetate buffer (pH 7.5) and purified by reverse-phase HPLC using a Phenomenex Gemini C18 column. The target product was eluted using 50 mM triethylammonium acetate buffer (pH 7.5) and acetonitrile / 500 mM triethylammonium acetate buffer (9:1, v / v) using a two-way mobile phase gradient profile. The fraction containing the target product was collected, mixed, and concentrated. The resulting solution was desalted using an Amicon® Ultra Centrifugal filter (3kD cutoff), precipitated with ethanol, and then deionized water was added to the pellet to form a solution.

[0431] A portion of the obtained solution was sampled, diluted with deionized water, and then subjected to mass spectrometry by ESI-MS under the conditions of analytical condition 3 in Example 3 to identify the target compounds (the theoretical molecular weight and detected molecular weight of the compounds are shown in Table 47).

[0432] <Synthesis of the compound "AOP-L-Pr"> The compound "AOP-L-Pr" with the sequence shown in Table 48 was synthesized using the following procedure. Note that in the sequence notation in Table 48, "(AOP-aminoC6-L)" is represented by the following formula (25). [ka] This refers to the base represented by , and other notations are the same as in Table 2. [Table 48]

[0433] A solution of L-Pr (synthesized in the same manner as in Example 1, sequence shown in Table 28) in sodium borate buffer (150 mM, pH 9.4) cooled to 10°C (200 μL, 1 mM) was added to a PCR tube. 40 equivalents of N-Fmoc-15-amino-4,7,10,13-tetraoxaoctadecanoic acid (20 μL, 0.4 M N-dimethylacetamide solution) were added to the tube, followed by 40 equivalents of 4-(4,6-dimethoxy[1.3.5]triazine-2-yl)-4-methylmorpholinium chloride hydrate (DMTMM) (16 μL, 0.5 M aqueous solution). The resulting mixture was shaken at 10°C for 4 hours.

[0434] The reaction mixture was treated with 23.6 μL of 5 M aqueous sodium chloride solution and 778.8 μL of cooled (-20°C) ethanol, and allowed to stand overnight at -78°C. After centrifugation, the supernatant was removed, and the resulting pellet was air-dried. 180 μL of deionized water was added to the pellet to make a solution, then 20 μL of piperidine was added, and the mixture was shaken at 10°C for 3 hours.

[0435] The obtained solution was treated with 20 μL of 5 M aqueous sodium chloride solution and 660 μL of cooled (-20°C) ethanol, and allowed to stand at -78°C for 30 minutes. After centrifugation, the supernatant was removed, and 200 μL of deionized water was added to the resulting pellet to make a 1 mM solution.

[0436] A portion of the obtained solution was sampled, diluted with deionized water, and then mass spectrometry by ESI-MS was performed under the conditions of analytical condition 2 in Example 1 to identify the target product (theoretical molecular weight of each sequence and the detected molecular weight are shown in Table 48).

[0437] <Synthesis of Crosslinker-Modified Primers (BMP-Pr)> Crosslinker-modified primers (BMP-Pr) with the sequences shown in Table 49 were synthesized using the following procedure. Note that in the sequence notation in Table 49, "(BMP)" is represented by the following formula (26). [ka] This refers to the base represented by , and other notations are the same as in Table 2. [Table 49]

[0438] A solution of AOP-L-Pr (40 μL, 0.5 mM) in sodium phosphate buffer (125 mM, pH 9.4) cooled to 10°C was added to a PCR tube. 50 equivalents of N-succinimidyl 3-maleimidopropionic acid (5 μL, 0.2 M dimethyl sulfoxide solution) were added to the tube, and the resulting mixture was shaken at 10°C for 40 minutes. Then, 20 μL of dimethyl sulfoxide was added, and the resulting mixture was shaken for a further 25 minutes at 10°C.

[0439] The reaction mixture was treated with 5 μL of 5 M aqueous sodium chloride solution and 215 μL of cooled (-20°C) ethanol, and allowed to stand at -78°C for 30 minutes. After centrifugation, the supernatant was removed, and the resulting pellet was air-dried. The resulting pellet was dissolved in deionized water, and the solution was desalted using an Amicon® Ultra Centrifugal filter (3 kD cutoff).

[0440] A portion of the obtained supernatant was sampled, diluted with deionized water, and then subjected to mass spectrometry by ESI-MS under the conditions of analytical condition 3 in Example 3 to identify the target crosslinker-modified primer "BMP-Pr" (theoretical molecular weight of each compound and the detected molecular weight are shown in Table 49).

[0441] <Synthesis of cross-linker modified double-stranded DEL ("TPD-DS-mSABA-DEL")> Using the single-stranded DNA DEL compound "SS-mSABA-DEL" obtained in Example 10 as the template DNA, a primer extension reaction was carried out using the cross-linker modified primer "TPD-Pr" obtained in the above <Synthesis of 3 types of cross-linker modified primers> in the following procedure to synthesize the cross-linker modified double-stranded DEL compound "TPD-DS-mSABA-DEL" with the sequence shown in Table 50. Note that the sequence notation in Table 50 is the same as in Tables 26 and 47, indicating that "TPD-DS-mSABA-DEL" is formed by the double strands of oligonucleotide chains of SEQ ID NO: 163 and SEQ ID NO: 131. [Table 50]

[0442] A portion of the obtained solution was sampled, diluted with deionized water, and then subjected to mass spectrometry by ESI-MS under the conditions of analytical condition 3 in Example 3 to identify the target crosslinker-modified double-stranded DEL "TPD-DS-mSABA-DEL" (the theoretical molecular weight of the compound and the detected molecular weight are shown in Table 50).

[0443] <Synthesis of cross-linker modified double-stranded DEL ("ACA-DS-ClSABA-DEL")> Using the single-stranded DNA-containing DEL compound "SS-ClSABA-DEL" obtained in Example 10 as the template DNA, a primer extension reaction using the cross-linker-modified primer "ACA-Pr)" obtained above was performed in the same procedure as in Example 7 to synthesize the cross-linker-modified double-stranded DEL compound "ACA-DS-ClSABA-DEL" with the sequence shown in Table 51. Note that the sequence notation in Table 51 is the same as in Tables 26 and 47, indicating that "ACA-DS-ClSABA-DEL" is formed by a double strand of oligonucleotide chains of SEQ ID NO: 164 and SEQ ID NO: 129. [Table 51]

[0444] A portion of the obtained solution was sampled, diluted with deionized water, and then subjected to mass spectrometry by ESI-MS under the conditions of analytical condition 3 of Example 3 to identify the target crosslinker-modified double-stranded DEL "ACA-DS-ClSABA-DEL" (the theoretical molecular weight and detected molecular weight of the compound are shown in Table 51).

[0445] <Confirmation of primer extension reaction by gel electrophoresis> A portion of the solutions obtained in the above-mentioned synthesis of crosslinker-modified double-stranded DEL ("TPD-DS-mSABA-DEL") and synthesis of crosslinker-modified double-stranded DEL ("ACA-DS-ClSABA-DEL") were sampled and analyzed by polyacrylamide gel electrophoresis under the conditions shown in Example 7. As shown in Figure 30, it was confirmed that the primer extension reaction converted the compounds to "TPD-DS-mSABA-DEL" and "ACA-DS-ClSABA-DEL" in high yield. The samples for each lane in Figure 30 are as follows. The samples were prepared so that the loading amount of each DEL compound was approximately 40 ng. Lane 1: 20 bp DNA Ladder (Lonza, catalog number 50330) Lane 2: SS-mSABA-DEL Lane 3: Sample after primer extension reaction of SS-mSABA-DEL (TPD-DS-mSABA-DEL) Lane 4: SS-ClSABA-DEL Lane 5: Sample after primer extension reaction of SS-ClSABA-DEL (ACA-DS-ClSABA-DEL) Lane 6: 20 bp DNA Ladder (Lonza, catalog number 50330)

[0446] Example 16 [Synthesis of crosslinker-modified double-stranded DEL using double-stranded DEL containing reactive groups for crosslinker modification] <Synthesis of primers ("BCN-Pr") with reactive groups for crosslinker modification> The primer "BCN-Pr" having a reactive group for crosslinker modification of the sequence shown in Table 52 was synthesized by the following procedure. In the sequence notation in Table 52, "(BCN)" is represented by the following formula (27) [ka] This refers to the base represented by , and other notations are the same as in Table 2.

[0447] [Table 52]

[0448] Similar to Example 15, "BCN-Pr" was synthesized using "AOP-L-Pr" as the starting material and succinimidyl(1R,8S,9s)-bicyclo[6.1.0]nona-4-in-9-ylmethyl carbonate as the active ester of the starting material.

[0449] A portion of the obtained solution was sampled, diluted with deionized water, and then subjected to mass spectrometry by ESI-MS under the conditions of analytical condition 3 in Example 3 to identify the target compound "BCN-Pr" (the theoretical molecular weight of the compound and the detected molecular weight are shown in Table 52).

[0450] <Synthesis of double-stranded DEL containing reactive groups for crosslinker modification> Using the "SS-mSABA-DEL" obtained in Example 10 as the template DNA, a primer extension reaction using "BCN-Pr" was performed in the same procedure as in Example 7 to synthesize the double-stranded DEL compound "BCN-DS-mSABA-DEL" having reactive groups for crosslinker modification of the sequence shown in Table 53. Note that the sequence notation in Table 53 is the same as in Tables 26 and 52, indicating that "BCN-DS-mSABA-DEL" is formed by a double strand of oligonucleotide chains of SEQ ID NO: 166 and SEQ ID NO: 131. [Table 53]

[0451] A portion of the obtained solution was sampled, diluted with deionized water, and then subjected to mass spectrometry by ESI-MS under the conditions of analytical condition 3 of Example 3. The double-chain DEL "BCN-DS-mSABA-DEL" containing the reactive group for the desired crosslinker modification was identified (the theoretical molecular weight of the compound and the detected molecular weight are shown in Table 53).

[0452] Furthermore, a portion of the obtained reaction solution was sampled and analyzed by polyacrylamide gel electrophoresis under the conditions shown in Example 7. The results shown in Figure 31 confirm that the primer extension reaction resulted in a high yield conversion to "BCN-DS-mSABA-DEL". The samples for each lane in Figure 31 are as follows. The samples were prepared so that the loading amount of each DEL compound was approximately 40 ng. Lane 1: 20 bp DNA Ladder (Lonza, catalog number 50330) Lane 2: SS-mSABA-DEL Lane 3: Sample after primer extension reaction of SS-mSABA-DEL (BCN-DS-mSABA-DEL) Lane 4: 20 bp DNA Ladder (Lonza, catalog number 50330)

[0453] <Synthesis of crosslinker-modified double-stranded DEL ("PSF-DS-mSABA-DEL") by click reaction with double-stranded DEL containing reactive groups for crosslinker modification> A crosslinker was introduced to the "BCN-DS-mSABA-DEL" obtained above by a click reaction to synthesize the crosslinker-modified double-stranded DEL compound "PSF-DS-mSABA-DEL" with the sequence shown in Table 54. The synthesis procedure is shown below. Note that in the sequence notation in Table 54, "(PSF-t)" is represented by the following formula (28). [ka] This refers to the group represented by , and other notations are the same as in Table 26, indicating that "PSF-DS-mSABA-DEL" is formed by a double strand of oligonucleotide chains from SEQ ID NO: 131 and SEQ ID NO: 167. [Table 54]

[0454] N-succinimidyl 2-azidoacetate (125 μL, 0.2 M dimethyl sulfoxide solution) was added to a PCR tube. N,N-diisopropylethylamine (5.2 μL), followed by 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (30 mg), was added to the tube, and the resulting mixture was shaken at 10°C for 1 hour.

[0455] Dimethyl sulfoxide was added to the resulting mixture and diluted it 10-fold (diluted solution).

[0456] In a PCR tube, 2 μL (0.1 mM) of BCN-DS-mSABA-DEL solution in sodium phosphate buffer (250 mM, pH 7.0) was added, followed by 1.8 μL of dimethyl sulfoxide. Then, 0.2 μL of the resulting dilution was added, and the resulting solution was shaken at 25°C for 2 hours.

[0457] One μL of the obtained solution was sampled, diluted with deionized water, and then subjected to mass spectrometry by ESI-MS under the conditions of analytical condition 3 in Example 3 to identify the target click-modified double-chain DEL "PSF-DS-mSABA-DEL" (the theoretical molecular weight of the compound and the detected molecular weight are shown in Table 54).

[0458] <Synthesis of crosslinker-modified double-stranded DEL ("BMP-DS-mSABA-DEL") by click reaction with double-stranded DEL containing reactive groups for crosslinker modification> The "BCN-DS-mSABA-DEL" obtained above was modified by introducing a crosslinker via a click reaction to synthesize the crosslinker-modified double-stranded DEL compound "BMP-DS-mSABA-DEL" with the sequence shown in Table 55. The synthesis procedure is shown below. In the sequence notation in Table 55, "(BMP-t)" is represented by the following formula (29). [ka] This refers to the group represented by , and the other notations are the same as in Table 26, indicating that "BMP-DS-mSABA-DEL" is formed by two strands of oligonucleotide chains from SEQ ID NO: 131 and SEQ ID NO: 168. [Table 55]

[0459] 3-azidopropylamine (4.8 mg, 0.2 M dimethyl sulfoxide solution) was added to a PCR tube. N-succinimidyl 3-maleimidopropionic acid (30 mg) was added to the tube, and the resulting mixture was shaken at 10°C for 1 hour.

[0460] Dimethyl sulfoxide was added to the resulting mixture and diluted 10-fold (diluted solution).

[0461] To a PCR tube, a BCN-DS-mSABA-DEL solution (2 μL, 0.1 mM) of sodium phosphate buffer (250 mM, pH 7.0) was added, and then 1.8 μL of dimethyl sulfoxide was added. Subsequently, 0.2 μL of the diluted solution obtained above was added, and the resulting solution was shaken at 25 °C for 2 hours.

[0462] 1 μL of the resulting solution was sampled, diluted with deionized water, and then mass spectrometry by ESI-MS was performed under the conditions of Analysis Condition 3 in Example 3 to identify the target click-modified double-stranded DEL "BMP-DS-mSABA-DEL" (the theoretical molecular weight and the detected molecular weight of the compound are shown in Table 55).

[0463] Example 17 [Conversion from a model library using U-DEL9-HP as a raw material to single-stranded DNA and imparting new functions] [Conversion of the model library having single-stranded DNA to DEL compounds using Lambda Exonuclease]< Using the sample after the cleavage reaction by the USER (registered trademark) enzyme of the model library (synthesized in Example 6), the conversion of the model library having single-stranded DNA to DEL compounds was carried out in the same procedure as in Example 9. And the resulting solution was used as the starting material for the next step.

[0464] [Conversion of the model library converted to DEL having single-stranded DNA to cross-linker-modified double-stranded DEL]< Using the model library converted to DEL having single-stranded DNA obtained above as the template DNA, primer extension reactions using "PXL-Pr" (synthesized in Example 7) and "BCN-Pr" (synthesized in Example 16) were carried out in the same procedure as in Example 7.

[0465] [Results]< A portion of the reaction solutions obtained from the two primer extension reactions described above was sampled and analyzed by polyacrylamide gel electrophoresis under the conditions shown in Example 7. As shown in Figure 32, it was confirmed that the primer extension reaction converted the single-stranded DEL model library into photoreactive crosslinker-modified double-stranded DEL and double-stranded DEL containing reactive groups for crosslinker modification in high yield. The samples for each lane in Figure 32 are as follows. The samples were prepared so that the loading amount of each DEL compound was approximately 40 ng. Lane 1: 20 bp DNA Ladder (Lonza, catalog number 50330) Lane 2: Sample after cleavage reaction using USER(registered trademark)enzyme from the model library. Lane 3: Sample after single-strand DEL reaction using Lambda Exonuclease from the model library. Lane 4: Sample after primer extension reaction with PXL-Pr using a single-stranded DEL-modified model library. Lane 5: Sample after primer extension reaction with BCN-Pr using a single-stranded DEL model library. Lane 6: 20 bp DNA Ladder (Lonza, catalog number 50330) [Industrial applicability]

[0466] This invention provides a method for utilizing nucleic acid compounds containing selectively cleavable sites. Furthermore, this invention provides a method for inducing and evaluating DELs containing cleavable sites in a DNA strand into crosslinker-modified double-stranded DELs, enabling compound screening that combines a "simpler DEL synthesis method" and "expanded and improved DEL evaluation methods" compared to conventional methods.

Claims

1. A method for evaluating crosslinker-modified double-stranded DNA-coding libraries (DELs) derived from hairpin-type DNA-coding libraries (DELs) having "selectively cleavable sites," comprising the following steps: (1) The DEL is brought into contact with a biological target under conditions suitable for at least one library molecule of the DEL to bind to the biological target. (2) Crosslinking the crosslinker of a library molecule bound to a biological target with the biological target, (3) Separating the complex of crosslinked library molecules and biological targets from the uncrosslinked library molecules. (4) Identify the sequences of oligonucleotides present in the library molecules within the recovered complex. (5) Using the sequences determined in (4), identify the structure of one or more compounds that bind to a biological target. A method consisting of the following.

2. The method according to claim 1, wherein the crosslinker of the crosslinker-modified double-stranded DEL is covalently linked to an oligonucleotide having a coding sequence.

3. The method according to claim 1 or 2, wherein the crosslinker of the crosslinker-modified double-stranded DEL is directly bound to the 5' end of the oligonucleotide or is bound via a bifunctional spacer.

4. The method according to any one of claims 1 to 3, wherein the induction of crosslinker-modified double-stranded DEL includes the following steps: (i) Cut at least one "selectively cleavable region" of a hairpin-type DEL having a "selectively cleavable region" and convert it into a double-stranded DEL. (ii) From the double-stranded DEL obtained in (i), remove the oligonucleotides that are not bound to the library molecule and convert them to single-stranded DEL. By forming a double strand with the single-stranded DEL obtained in (iii)(iii) and the cross-linker modified DNA, we induce cross-linker modified double-stranded DEL.

5. The method according to any one of claims 1 to 3, wherein the induction of crosslinker-modified double-stranded DEL includes the following steps: (i) Cut at least one "selectively cleavable region" of a hairpin-type DEL having a "selectively cleavable region" and convert it into a double-stranded DEL. (ii) From the double-stranded DEL obtained in (i), remove the oligonucleotides that are not bound to the library molecule and convert them to single-stranded DEL. (iii) The single-stranded DEL obtained in (iii) and DNA having a reactive group for crosslinker modification are double-stranded. (iv) The reaction group for crosslinker modification is reacted with the crosslinker unit to induce the crosslinker-modified double-stranded DEL.

6. The method according to any one of claims 1 to 3, wherein the induction of crosslinker-modified double-stranded DEL includes the following steps: (i) Cut at least one "selectively cleavable region" of a hairpin-type DEL having a "selectively cleavable region" and convert it into a double-stranded DEL. (ii) From the double-stranded DEL obtained in (i), remove the oligonucleotides that are not bound to the library molecule and convert them to single-stranded DEL. The single-stranded DEL obtained in (iii)(iii) is coated with a crosslinker-modified primer, and the coated primer is extended to induce crosslinker-modified double-stranded DEL.

7. The method according to any one of claims 1 to 3, wherein the induction of crosslinker-modified double-stranded DEL includes the following steps: (i) Cut at least one "selectively cleavable region" of a hairpin-type DEL having a "selectively cleavable region" and convert it into a double-stranded DEL. (ii) From the double-stranded DEL obtained in (i), remove the oligonucleotides that are not bound to the library molecule and convert them to single-stranded DEL. (iii) The single-stranded DEL obtained in (iii) is given a modification primer having a reactive group for crosslinker modification, the given primer is extended, and it is converted into a double-stranded DEL having a reactive group for crosslinker modification. (iv) The reaction group for crosslinker modification is reacted with the crosslinker unit to induce the crosslinker-modified double-stranded DEL.

8. The method according to any one of claims 1 to 3, wherein the induction of crosslinker-modified double-stranded DEL includes the following steps: (i) Cut at least one "selectively cleavable region" of a hairpin-type DEL having a "selectively cleavable region" and convert it into a double-stranded DEL. (ii) The double-stranded DEL obtained in (i) is coated with a crosslinker-modified primer, and the coated primer is extended to induce crosslinker-modified double-stranded DEL.

9. The method according to any one of claims 1 to 3, wherein the induction of crosslinker-modified double-stranded DEL includes the following steps: (i) Cut at least one "selectively cleavable region" of a hairpin-type DEL having a "selectively cleavable region" and convert it into a double-stranded DEL. (ii) The double-stranded DEL obtained in (i) is given a modification primer having a reactive group for crosslinker modification, the given primer is extended, and the reactive group for crosslinker modification reacts with the crosslinker unit to induce crosslinker-modified double-stranded DEL.

10. The method according to claim 6 or claim 8, characterized by the following: (I) Use a hairpin-type DEL in which at least one "selectively cleavable site" is located 3' from the site to which the library molecule is bound. (II) Use a crosslinker-modified primer in which the crosslinker is either directly bound to the 5' end of the oligonucleotide or bound via a bifunctional spacer.

11. The method according to claim 7 or claim 9, characterized by the following: (I) Use a hairpin-type DEL in which at least one "selectively cleavable site" is located 3' from the site to which the library molecule is bound. (II) Use a modification primer having a reaction group for crosslinker modification, in which the reaction group for crosslinker modification is directly bound to the 5' end of the oligonucleotide or bound via a bifunctional spacer.

12. The method according to any one of claims 4 to 7, wherein in step (ii) above, the oligonucleotide to which the library molecule is not bound has a functional molecule and is removed by a treatment corresponding to the function of the functional molecule.

13. The method according to claim 12, wherein the functional molecule is biotin.

14. The method according to any one of claims 4 to 7, wherein in step (ii), the removal of oligonucleotides to which library molecules are not bound is by degradation by an exonuclease.

15. The method according to claim 14, wherein the exonuclease is lambda exonuclease.

16. The method according to any one of claims 1 to 15, wherein the crosslinker comprises at least one azide group, diazirine group, sulfonyl fluoride group, diazo group, cinnamoyl group, or acrylate group.

17. The method according to any one of claims 1 to 15, wherein the crosslinker comprises at least one azide group, a diazirine group, or a sulfonyl fluoride group.

18. The crosslinker is given by equations (AA) to (AE): 【Chemistry 53】 (In the formula, * represents the 5' end of the double-stranded DEL, or the binding position to the bifunctional spacer that is bound to the 5' end.) The method according to any one of claims 1 to 15, comprising any of the structures.

19. The crosslinker is given by equations (AA) to (AE): 【Chemistry 54】 (In the formula, * represents the 5' end of the double-stranded DEL, or the binding position to the bifunctional spacer that is bound to the 5' end.) The method according to any one of claims 1 to 15, wherein the structure is one of the following.

20. The crosslinker is given by formula (BA) or (BB): 【Transformation 55】 (In the formula, * represents the 5' end of the double-stranded DEL, or the binding position to the bifunctional spacer that is bound to the 5' end.) The method according to any one of claims 1 to 15, comprising any of the structures.

21. The crosslinker is given by formula (BA) or (BB): 【Transformation 56】 (In the formula, * represents the 5' end of the double-stranded DEL, or the binding position to the bifunctional spacer that is bound to the 5' end.) The method according to any one of claims 1 to 15, wherein the structure is one of the following.

22. The method according to any one of claims 5, 7, 9, or 11, wherein the reactive group for crosslinker modification is a reactive group for a click reaction.

23. The method according to any one of claims 5, 7, 9, or 11, wherein the reactive group for crosslinker modification is an alkynyl group, an alkenyl group, an azide group, or a tetradinyl group.

24. The reactive groups for crosslinker modification are given by formula (CA) to (CL): 【Chemistry 57】 (In the formula, * indicates the 5' end of the double-stranded DEL, or the binding site to the bifunctional spacer that is bound to the 5' end.) The method according to any one of claims 5, 7, 9, or 11, wherein the structure is one of the following.

25. The method according to any one of claims 1 to 19, wherein the step of (2) "crosslinking the crosslinker of the library molecule bound to the biological target with the biological target" is the step of "crosslinking the crosslinker of the library molecule bound to the biological target with the biological target by light irradiation".

26. The method according to claim 25, wherein the light irradiation condition is light irradiation with a wavelength of 250 to 500 nm.

27. The method according to claim 25, wherein the light irradiation condition is light irradiation with a wavelength of 365 nm.

28. The method according to any one of claims 25 to 27, wherein the light irradiation conditions are light irradiation for 10 seconds to 180 minutes.

29. The method according to any one of claims 25 to 27, wherein the light irradiation condition is light irradiation for 30 seconds to 30 minutes.

30. The method according to any one of claims 1 to 24, wherein the step of (2) "crosslinking the crosslinker of a library molecule bound to a biological target with the biological target" is the step of "crosslinking the crosslinker of a library molecule bound to a biological target with the biological target by incubation."

31. The method according to any one of claims 1 to 30, wherein the step of (3) "separating the complex of crosslinked library molecules and a biological target from the uncrosslinked library molecules" is the step of "separating the complex of crosslinked library molecules and a biological target from the uncrosslinked library molecules by electrophoresis."

32. The method according to claim 31, wherein the electrophoresis is gel electrophoresis.

33. The method according to claim 31, wherein the electrophoresis is capillary electrophoresis.

34. The method according to any one of claims 1 to 30, wherein the step of (3) "separating the complex of crosslinked library molecules and a biological target from uncrosslinked library molecules" is "separating the complex of crosslinked library molecules and a biological target by immobilizing the biological target onto an immobilization carrier and washing away the uncrosslinked library molecules."

35. A hairpin-type DEL having a "part that can be selectively cut" is given by formula (I) 【Chemistry 58】 (In the formula, X and Y are oligonucleotide chains, E and F are independent of each other. It is an oligomer composed of nucleotides or nucleic acid analogs, However, E and F contain complementary base sequences and form a double-stranded oligonucleotide. LP is the loop section, L is a linker, D is a divalent group derived from a reactive functional group, Sp is a bond or a bifunctional spacer, An is a substructure composed of at least one building block. It is a compound represented by the following: X and Y have sequences that can form a double helix in at least part of their structure. X is attached to E at its 5' end. Y binds to F at its 3' end. (E, F, or LP has at least one selectively cleavable portion.) This is DEL, represented by The method according to any one of claims 1 to 34.

36. A hairpin-type DEL having a "part that can be selectively cut" is given by formula (III) An-Sp-C-Bn (III) (In the formula, An and Sp have the same meanings as in claim 35. Bn represents a double-stranded oligonucleotide tag formed by oligonucleotide chain X and oligonucleotide chain Y. C is given by equation (I) 【Chemistry 59】 (In the formula, E, LP, L, D, and F have the same meanings as in claim 35, wherein D binds directly to An or via a bifunctional spacer, and E and F bind to the corresponding terminals of the double-stranded oligonucleotide tag Bn.) This is DEL, represented by The method according to claim 35.

37. An is the same as in claim 35 and is a substructure constructed of n building blocks α1 to αn (where n is an integer from 1 to 10). Bn is a double-stranded oligonucleotide tag formed by oligonucleotide chain X and oligonucleotide chain Y, and is a substructure containing an oligonucleotide with a base sequence that can identify the structure of An. The method according to claim 35 or 36.

38. LP, This is a loop region represented by (LP1)p-LS-(LP2)q, LS is a substructure selected from the group of compounds described in (A) to (C) below, (A) Nucleotides (B) Nucleic acid analogs (C) Trivalent C1-14 groups which may have substituents LP1 is a substructure selected individually or differently from the group of compounds described in (1) and (2) below, (1) Nucleotides (2) Nucleic acid analogs LP2 is a substructure in which q elements are selected individually or differently from the group of compounds described in (1) and (2) below. (1) Nucleotides (2) Nucleic acid analogs The total number of p and q is between 0 and 40. The method according to any one of claims 35 to 37.

39. The method according to claim 38, wherein the total number of p and q is 2 to 20.

40. The method according to claim 38, wherein the total number of p and q is 2 to 10.

41. The method according to claim 38, wherein the total number of p and q is 2 to 7.

42. The method according to claim 38, wherein the total number of p and q is 0.

43. LP1, LP2, and LS each have the following structure: (A) Nucleotides or (B) Nucleic acid analogs that meet the requirements of (B11) to (B15) below (B11) Having phosphoric acid (or equivalent part) and a hydroxyl group (or equivalent part), (B12) Composed of carbon, hydrogen, oxygen, nitrogen, phosphorus or sulfur, (B13) Molecular weight is between 142 and 1500. (B14) The number of atoms between residues is 3 to 30. (B15) The bonding pattern between atoms in residues is either all single bonds, or one or two double bonds with the remainder being single bonds. The method according to any one of claims 38 to 42, wherein the structure is selected individually or differently from those.

44. LP1, LP2, and LS each have the following structure: (A) Nucleotides or (B) Nucleic acid analogs that meet the requirements of (B21) to (B25) below (B21) Having phosphoric acid and hydroxyl groups, (B22) Composed of carbon, hydrogen, oxygen, nitrogen, or phosphorus, (B23) Molecular weight is between 142 and 1000. (B24) The number of atoms between residues is 3 to 15. (B25) The bonding mode between atoms in each residue is all single bonds. The method according to any one of claims 38 to 43, wherein the structure is selected individually or differently from those.

45. LP1, LP2, and LS each have the following structure: (A) Nucleotides or (B) Nucleic acid analogs that meet the requirements of (B31) to (B35) below (B31) Having phosphoric acid and hydroxyl groups, (B32) Composed of carbon, hydrogen, oxygen, nitrogen, or phosphorus, (B33) Molecular weight is between 142 and 700. (B34) The number of atoms between residues is 4 to 7. (B35) The bonding mode between atoms in each residue is all single bonds. The method according to any one of claims 38 to 44, wherein the structure is selected individually or differently from those.

46. LP1 and LP2 are as follows: (B41) d-Spacer, (B5) Polyalkylene glycol phosphate ester The method according to any one of claims 38 to 45, wherein the method is any one of the following:

47. The method according to any one of claims 38 to 46, wherein LP1 and LP2 are each diethylene glycol phosphate ester or triethylene glycol phosphate ester.

48. The method according to any one of claims 38 to 47, wherein LP1 and LP2 are each triethylene glycol phosphate esters.

49. The method according to any one of claims 38 to 46, wherein LP1 and LP2 are each d-Spacers.

50. LP1 and LP2 are nucleotides, The method according to any one of claims 38 to 45.

51. LS is given by equations (a) to (g): 【Transformation 60】 (In the formula, * indicates the bond position with the linker, ** indicates the bond position with LP1 or LP2, and R is a hydrogen atom or a methyl group.) The method according to any one of claims 38 to 50, wherein the method is any one of the following:

52. LS is given by equation (h): 【Chemistry 61】 (In the formula, * indicates the linker connection position, and ** indicates the linker connection position.) The method according to any one of claims 38 to 50.

53. The method according to any one of claims 38 to 50, wherein LS is a polyalkylene glycol phosphate ester.

54. LS is given by equations (i) to (k): 【Transformation 62】 (In the formula, n1, m1, p1, and q1 are each independent integers between 1 and 20, * indicates the linker connection position, and ** indicates the linker connection position with LP1 or LP2.) The method according to any one of claims 38 to 50.

55. LS is given by equation (l): 【Transformation 63】 (In the formula, * indicates the linker connection position, and ** indicates the linker connection position.) The method according to any one of claims 38 to 50.

56. LS is (B42), (B43), or (B44): (B42) Amino C6 dT (B43) mdC (TEG-Amino) (B44) Uni-Link (Registered Trademark) Amino Modifier The method according to any one of claims 38 to 50, wherein the method is any one of the following:

57. LS is a nucleotide. The method according to any one of claims 38 to 50.

58. LS is a trivalent C1-C14 group which may have a (C) substituent, and (C) has the following structure: (1) C1-10 aliphatic hydrocarbons which may have substituents and which may be replaced by 1-3 heteroatoms, (2) C6-14 aromatic hydrocarbons which may have substituents, (3) A C2- to C9 aromatic heterocycle which may have substituents, or (4) C2-9 non-aromatic heterocycles which may have substituents The method according to any one of claims 38 to 42 and 46 to 50, wherein the method is any one of the above.

59. LS is a trivalent C1-C14 group which may have a (C) substituent, and (C) has the following structure: (1) C1-6 aliphatic hydrocarbons which may have substituents, (2) C6-10 aromatic hydrocarbons which may have substituents, (3) C2-5 aromatic heterocycles which may have substituents The method according to any one of claims 38 to 42 and 46 to 50, wherein the method is one of the above.

60. LS is a trivalent C1-C14 group which may have a (C) substituent, and (C) has the following structure: (1) C1-6 aliphatic hydrocarbons, (2) Benzene, or (3) C2-5 nitrogen-containing aromatic heterocycle Here, (1) to (3) may be unsubstituted or substituted with one to three substituents selected individually or differently from substituent group ST1, wherein substituent group ST1 is composed of C1-6 alkyl groups, C1-6 alkoxy groups, fluorine atoms, and chlorine atoms; however, if substituent group ST1 is substituted with an aliphatic hydrocarbon, alkyl groups are not selected from substituent group ST1. The method according to any one of claims 38 to 42 and 46 to 50, wherein the method is one of the above.

61. LS is a trivalent C1-C14 group which may have a (C) substituent, and (C) has the following structure: (1) C1-6 alkyl groups, (2) Unsubstituted or benzenes substituted with one or two C1-3 alkyl groups or C1-3 alkoxy groups The method according to any one of claims 38 to 42 and 46 to 50, wherein the method is one of the above.

62. LS is a trivalent C1-C14 group which may have a (C) substituent, and (C) has the following structure: (1) C1-6 alkyl groups The method according to any one of claims 38 to 42 and 46 to 50.

63. E and F are oligomers composed independently of nucleotides or nucleic acid analogs. The chain lengths of E and F are 3 to 40, The method according to any one of claims 36 to 62.

64. E and F are oligomers composed independently of nucleotides or nucleic acid analogs. The chain lengths of E and F are 4 to 30, respectively. The method according to any one of claims 35 to 63.

65. E and F are oligomers composed independently of nucleotides or nucleic acid analogs. The chain lengths of E and F are 6 to 25, respectively. The method according to any one of claims 35 to 64.

66. E and F are oligomers composed independently of nucleotides or nucleic acid analogs. E and F contain complementary base sequences, forming a double-stranded oligonucleotide. The E and F double-stranded oligonucleotides are the overhanging ends. The method according to any one of claims 35 to 65.

67. The method according to claim 66, wherein the protruding portion of the protruding end has a length of two bases or more.

68. E and F are oligomers composed independently of nucleotides or nucleic acid analogs. E and F contain complementary base sequences, forming a double-stranded oligonucleotide. The E and F double-stranded oligonucleotides have blunt ends. The method according to any one of claims 35 to 65.

69. The chain lengths of the complementary base sequences contained in E and F are each three bases or longer. The method according to any one of claims 35 to 68.

70. The chain lengths of the complementary base sequences contained in E and F are each four bases or longer. The method according to any one of claims 35 to 69.

71. The chain lengths of the complementary base sequences contained in E and F are each 6 bases or longer. The method according to any one of claims 35 to 70.

72. E and F are oligomers composed of nucleotides, The method according to any one of claims 35 to 71.

73. The method according to any one of claims 35 to 72, wherein the nucleotide is a ribonucleotide or a deoxyribonucleotide.

74. The method according to any one of claims 35 to 73, wherein the nucleotide is a deoxyribonucleotide.

75. The method according to any one of claims 35 to 74, wherein the nucleotide is deoxyadenosine, deoxyguanosine, thymidine, or deoxycytidine.

76. The method according to any one of claims 35 to 71, wherein E and F are oligomers composed of nucleic acid analogs independently.

77. L, (1) C1-20 aliphatic hydrocarbons which may have substituents and which may be replaced by 1-3 heteroatoms, or (2) C6-14 aromatic hydrocarbons which may have substituents The method according to any one of claims 35 to 76.

78. The method according to any one of claims 35 to 77, wherein L is a C1-6 aliphatic hydrocarbon which may have substituents, a C1-6 aliphatic hydrocarbon which may be replaced by one or two oxygen atoms, or a C6-10 aromatic hydrocarbon which may have substituents.

79. The method according to any one of claims 35 to 78, wherein L is a C1-6 aliphatic hydrocarbon substituted with substituent group ST1, or a benzene substituted with substituent group ST1, where substituent group ST1 is a group consisting of C1-6 alkyl groups, C1-6 alkoxy groups, fluorine atoms, and chlorine atoms (however, when substituent group ST1 is substituted with an aliphatic hydrocarbon, alkyl groups are not selected from substituent group ST1).

80. The method according to any one of claims 35 to 79, wherein L is a C1-6 alkyl group, or a benzene that is unsubstituted or substituted with one or two C1-3 alkyl groups or C1-3 alkoxy groups.

81. The method according to any one of claims 35 to 80, wherein L is a C1-6 alkyl group.

82. The reactive functional group of D is The method according to any one of claims 35 to 81, wherein the functional group is C-C, amino, ether, carbonyl, amide, ester, urea, sulfide, disulfide, sulfoxide, sulfonamide, or a reactive functional group capable of forming a sulfonyl bond.

83. The method according to any one of claims 35 to 82, wherein the reactive functional group D is a C1 hydrocarbon having a leaving group, an amino group, a hydroxyl group, a precursor of a carbonyl group, a thiol group, or an aldehyde group.

84. The method according to any one of claims 35 to 83, wherein the reactive functional group of D is a C1 hydrocarbon having a halogen atom, a C1 hydrocarbon having a sulfonic acid leaving group, an amino group, a hydroxyl group, a carboxyl group, a halogenated carboxyl group, a thiol group, or an aldehyde group.

85. The reactive functional group of D is -CH ２ Cl, -CH ２ Br, -CH ２ OSO ２ CH ３ ien-CH ２ OSO ２ CF ３ The method according to any one of claims 35 to 84, wherein the amino group is a hydroxyl group or a carboxyl group.

86. The method according to any one of claims 35 to 85, wherein the reactive functional group of D is a primary amino group.

87. The selectively cleavable site is a deoxyribonucleoside that is neither deoxyadenosine, deoxyguanosine, thymidine, nor deoxycytidine. The method according to any one of claims 35 to 87.

88. The selectively cleavable sites are deoxyuridine, bromodeoxyuridine, deoxyinosine, 8-hydroxydeoxyguanosine, 3-methyl-2'-deoxyadenosine, N6-etheno-2'-deoxyadenosine, 7-methyl-2'-deoxyguanosine, 2'-deoxyxanthosine, or 5,6-dihydroxy-5,6-dihydrodeoxythymidine. The method according to any one of claims 35 to 87.

89. The selectively cleavable site is deoxyuridine or deoxyinosine. The method according to any one of claims 35 to 88.

90. The site that can be selectively cleaved is deoxyuridine. The method according to any one of claims 35 to 89.

91. The site that can be selectively cleaved is deoxyinosine. The method according to any one of claims 35 to 89.

92. The selectively cleavable site is the second phosphodiester bond in the 3' direction from deoxyinosine. The method according to any one of claims 35 to 86.

93. The site that can be selectively cleaved is the ribonucleoside. The method according to any one of claims 35 to 86.

94. There is only one part that can be selectively cut. The method according to any one of claims 35 to 93.

95. At least one cleavable portion is included in E or (LP1)p, and at least one cleavable portion is included in F or (LP2)q, The method according to any one of claims 35 to 93.

96. The cleavable portion included in E or (LP1)p and the cleavable portion included in F or (LP2)q are cleavable under different conditions. The method according to claim 95.

97. An is a substructure constructed from n building blocks α1 to αn (where n is an integer from 1 to 10). The method according to any one of claims 35 to 96.

98. The method according to any one of claims 35 to 97, wherein An is a low molecular weight organic compound.

99. The method according to any one of claims 35 to 98, wherein the building block of An is a compound with a molecular weight of 500 or less.

100. The method according to any one of claims 35 to 99, wherein the building block of An is a compound with a molecular weight of 300 or less.

101. The method according to any one of claims 35 to 100, wherein the building block of An is a compound with a molecular weight of 150 or less.

102. The method according to any one of claims 35 to 101, wherein An is an organic compound composed of elements selected individually or differently from the group of elements consisting of H, B, C, N, O, Si, P, S, F, Cl, Br, and I.

103. The method according to any one of claims 35 to 102, wherein An is a low molecular weight organic compound having substituents selected individually or differently from the group of substituents consisting of an aryl group, a non-aromatic cyclyl group, a heteroaryl group, and a non-aromatic heterocyclyl group.

104. The method according to any one of claims 35 to 103, wherein An has a molecular weight of 5000 or less.

105. The method according to any one of claims 35 to 104, wherein An has a molecular weight of 800 or less.

106. The method according to any one of claims 35 to 105, wherein An has a molecular weight of 500 or less.

107. An is a polypeptide. The method according to any one of claims 35 to 97.

108. The method according to any one of claims 35 to 107, wherein Sp is a bifunctional spacer.

109. The two functional spacers are SpD-SpL-SpX, respectively. SpD is a divalent group derived from a reactive group that can constitute a C-C, amino, ether, carbonyl, amide, ester, urea, sulfide, disulfide, sulfoxide, sulfonamide, or sulfonyl bond. SpL may be polyalkylene glycol, polyethylene, C1-20 aliphatic hydrocarbons which may optionally be replaced by heteroatoms, peptides, oligonucleotides, or combinations thereof. SpX is a divalent group derived from a reactive group that forms an amino, carbonyl, amide, ester, urea, or sulfonamide bond. The method according to any one of claims 1 to 107.

110. The two functional spacers are SpD-SpL-SpX, respectively. SpD is a divalent group derived from a primary amino group, SpL is polyethylene glycol or polyethylene. SpX is a divalent group derived from a carboxyl group. The method according to any one of claims 1 to 107.

111. The method according to any one of claims 35 to 110, wherein oligonucleotide chain X and oligonucleotide chain Y are sequences capable of forming a double helix.

112. The method according to any one of claims 35 to 111, wherein oligonucleotide chain X and oligonucleotide chain Y contain complementary base sequences.

113. The method according to any one of claims 35 to 112, wherein oligonucleotide chain X and oligonucleotide chain Y are each 1 to 200 bases in length.

114. The method according to any one of claims 35 to 113, wherein oligonucleotide chain X and oligonucleotide chain Y are each 3 to 150 bases in length.

115. The method according to any one of claims 35 to 114, wherein oligonucleotide chain X and oligonucleotide chain Y are each 30 to 150 bases in length.

116. The method according to any one of claims 35 to 115, wherein oligonucleotide chain X and oligonucleotide chain Y have blunt ends.

117. The method according to any one of claims 35 to 115, wherein oligonucleotide chain X and oligonucleotide chain Y have protruding ends.

118. The method according to claim 117, wherein the protruding portion of the protruding end is 1 to 30 bases in length.

119. The method according to claim 117 or 118, wherein the protrusion of the protruding end is 2 to 5 bases in length.

120. The method according to any one of claims 117 to 119, wherein oligonucleotide chain X and oligonucleotide chain Y each have a protruding end, and a specific molecular identification sequence is further bound to the protruding end.

121. The method according to any one of claims 35 to 120, wherein a functional molecule is bound to either X or Y.

122. The method according to any one of claims 35 to 120, wherein biotin is bound to either X or Y.

123. The method according to any one of claims 35 to 107, wherein Sp is a bond.

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