Method for evaluating DNA-encoded library

Abstract

Provided is a method in which a DNA-encoded library (DEL) that contains a cleavable site in the DNA strand thereof is induced to a cross-linker-modified double-stranded DEL to evaluate the DEL.　According to the present invention, both of the advantage of a hairpin-stranded DEL and the advantage of a double-strand DEL are achieved by introducing a cleavable site such as deoxyuridine into a DNA strand. The present invention provides a compound screening technique having both of a "convenient DEL synthesis technology" and the "expansion / improvement of a DEL evaluation technology" by introducing into a cross-linker-modified DEL conveniently.

Description

Methods for evaluating DNA-encoded libraries

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

[0002] A compound library is a systematic collection of compound derivatives that may have specific activity, such as pharmaceutical candidate compounds. These compound libraries are often synthesized using the synthesis techniques and methodologies of combinatorial chemistry. Combinatorial chemistry is an experimental technique for efficiently synthesizing a wide variety of compounds through systematic synthetic routes from a series of compound libraries enumerated and designed based on combinatorial theory, and a field of research related to this technique. One type of compound library based on combinatorial chemistry is the DNA-encoded library. Hereinafter, DNA-encoded library will be abbreviated as DEL. In DEL, a DNA tag is attached to each compound in the library. The DNA tag has a sequence designed to identify the structure of each compound and functions as a label for the compound (Patent Documents 1 to 3).

[0003] To date, screening using DEL has led to the discovery of several promising compounds for pharmaceutical development. 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 away DEL with low affinity to the target. 4) Denaturing the target protein and eluting DEL with high affinity. 5) Amplifying the DNA sequence contained in the eluted DEL and identifying the sequence. However, the above-mentioned screening method has several problems. First, since the target protein must be immobilized, depending on the protein, the three-dimensional structure may change after the immobilization procedure. In such cases, the compounds obtained by screening do not bind to the target protein that is not immobilized, limiting the target application range of DEL (Patent Documents 4 and 5, Non-Patent Documents 4 and 5). Furthermore, while the above screening method can recover high-affinity binders, moderate-affinity binders (e.g., those with a Kd value on the order of μM) have low kinetic stability of the complex with the target protein, making them difficult to recover after washing. As is well known to those skilled in the art, such binding agents with moderate affinity are also useful as hit compounds that serve as starting points for drug discovery, and are also useful as information on structure-activity relationships (Non-Patent Document 6).

[0004] In an effort to solve the above-mentioned problems, several screening methods using crosslinker-modified DEL have been reported recently. As will be shown later, several different methods have been reported, but all have in common that a target in proximity to an affinity library molecule is reacted with a crosslinker to form a covalent bond. This method is useful because it enables DEL screening without (or before) immobilizing the target protein (Patent Documents 4 and 5, Non-Patent Documents 4 and 5, 7 to 9).

[0005] Here, we will briefly explain the DNA strand structure of DEL. Conventionally known DEL DNA strand structures are typically hairpin strands and double-stranded. Below, we will outline the overview and advantages and disadvantages of double-stranded DEL and hairpin strand DEL. (1) Hairpin strand DEL. DEL using hairpin strand DNA has a single-stranded structure in which two complementary DNA strands are connected. It is synthesized using hairpin-shaped DNA as a starting material (headpiece) that has functional groups for introducing various building blocks (Patent Document 3, Non-Patent Documents 1 and 2). (A) Advantages: (a) Short DNA tags can be used. In this method, relatively short double-stranded DNA tags of approximately 9-13 mers with 2-mer sticky ends are often used, and the 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 duplex within the molecule, and DNA sites 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 the low cost of DNA tag synthesis. Another advantage is that the use of shorter DNA tags reduces the overall length of DEL when encoding the same number of reaction cycles. In other words, even if a larger number of cycles is encoded, the overall length of DEL can be reduced to a range that allows efficient DNA sequence reading by a next-generation sequencer. In fact, in Non-Patent Document 3, by using hairpin strand DNA, the construction of DEL using hairpin strand DNA encoding as many as six reaction cycles was achieved. (b) High chemical stability: Unlike double-stranded DNA, even if the double-stranded structure of a hairpin strand melts during a heating reaction, the original intramolecular double-strand is reformed under subsequent reannealing 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 Document 2). In addition, in general, when nucleic acid strands have the same chain length, hairpin strands form duplexes more strongly than double-stranded strands (higher Tm value). Therefore, under various chemical conditions when introducing building blocks, each chemical structure of the hairpin strand DNA, particularly the structure of the base portion, should be resistant to structural change compared to the double strand.(B) Disadvantages: Hairpin strand DNA forms a double strand within the molecule, making it difficult to form a new double strand with another oligonucleotide strand. Therefore, it is difficult to convert it into a crosslinker-modified double-stranded DEL by adding a new crosslinker-modified oligonucleotide. (2) Double-Stranded DEL: DEL using double-stranded DNA is synthesized using single-stranded DNA (non-hairpin single-stranded DNA) or double-stranded DNA as the starting material (headpiece) that has functional groups for introducing various building blocks. (A) Disadvantages: In contrast to DEL using hairpin strand DNA, relatively long single-stranded or double-stranded DNA tags of approximately 20-30 mers with 4-10 mer sticky ends are often used (Patent Document 2, Non-Patent Document 10), and DEL encoding approximately three cycles of reaction is common. (B) Advantages: Double-stranded DNA can be denatured to single-stranded DNA or subjected to strand exchange reactions, which is advantageous in that it can be converted into DNA structures suitable for various applications. Therefore, it is also possible to convert it into a crosslinker-modified double-stranded DEL by adding a new crosslinker-modified single-stranded oligonucleotide (Non-Patent Documents 7, 8 and 11).

[0006] Thus, hairpin strand DNA and double-strand DNA each have advantages in synthesizing and evaluating DEL, but no technology is known that combines these advantages.

[0007] The following is a known example of a report on screening using crosslinker-modified DEL.

[0008] In Non-Patent Documents 7 and 8, single-stranded DEL having a library molecule at the 5' end is synthesized, and then double-stranded with DNA having a photoreactive crosslinker at the 3' end is formed, followed by screening to obtain a binder with moderate affinity. On the other hand, in this method, since the DNA linked to the photoreactive crosslinker does not contain a coding sequence, if it is subjected to strong separation or elution conditions to remove nonspecific binders, the double strands may dissociate, making it impossible to obtain a sequence encoding the desired structure. Furthermore, because single-stranded DEL is used, the advantages of hairpin-stranded DEL cannot be utilized in 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, single-stranded DEL having a library molecule at the 3' end is synthesized, and then a double strand is formed with a short-stranded DNA having a photoreactive crosslinker at the 5' end, resulting in a photoreactive crosslinker-modified DEL. An advantage of this method is that, because the photoreactive crosslinker is present at the 5' end, the coding sequence is covalently linked to the target by an extension reaction using DNA polymerase. This makes it possible to include strong separation and elution conditions in the screening. However, no method has been reported for easily synthesizing single-stranded DEL having a library molecule at the 3' end. Similarly, this method does not utilize the advantages of hairpin-stranded DEL in synthesis.

[0010] Patent Document 4 describes a hairpin chain DEL having a linking site with a crosslinker. Although this method can solve the problems of Non-Patent Document 5, it has another problem that the library synthesis must be carried out without damaging the functional group for crosslinker linkage, which imposes restrictions on usable reactions and / or library molecular structures.

[0011] Patent Document 6 describes the synthesis of a double-stranded DEL crosslinked by a reversible covalent bond (considered to have properties equivalent to those of a hairpin-stranded DEL in terms of duplex formation ability, etc.), and its conversion to a 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, the photocyclized pyrimidine base loses its 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, this method limits the reactions that can be used during DEL synthesis, and also limits the library molecular structures that can be constructed.

[0012] As described above, there is no known technique that has the same level of advantages in terms of synthesis as conventional hairpin chain DEL and that can be easily applied to screening with crosslinker-modified DEL.

[0013] International Publication No. WO 93 / 20243, International Publication No. 2004 / 039825, International Publication No. 2005 / 058479, International Publication No. 2019 / 149198, International Publication No. 2019 / 157692, CN112760720

[0014] Nature Chemical Biology, 2009, Vol. 5, pp. 647-654. A Handbook for DNA-Encoded Chemistry, edited by Robert A. Goodnow, Jr., John Wiley & Sons, Inc. Progress in Medicinal Chemistry, 2020, Vol. 59, pp. 181-249, Chapter 4 - An overview of DNA-encoded libraries: A versatile tool for drug discovery. discovery Angewandte Chemie International Edition, 2014, Vol. 53, pp. 10056-10059 ACS Combinatorial Science, 2019, Vol. 21, pp. 345-349 ChemBioChem, 2019, Vol. 20, pp. 955-962 ACS Combinatorial Science, 2020, Vol. 22, pp. 204-212 Chemistry: A European Journal, 2021, 27, pp. 7160-7167 Accounts of Chemical Research, 2021, vol. 54, pp. 3491-3503 Nature Chemistry, 2018, vol. 10, pp. 441-448 Annual Review of Biochemistry, 2018, vol. 87, pp. 479-502 Bioconjugate Chemistry, 2017, vol. 28, pp. 2293-2301 Journal of the American Chemical Society, 2014, vol. 136, pp. 12938-12946

[0015] The present invention provides a method for converting a DEL containing a cleavable site in a DNA strand into a crosslinker-modified double-stranded DEL and for evaluating the same.

[0016] One area of ​​nucleic acid chemistry, such as DNA, is nucleic acid cleavage technology. For example, by introducing deoxyuridine into a DNA strand, it can be selectively cleaved by the USER® enzyme. As a result of extensive research, the present inventors have discovered that by introducing a cleavable site, such as deoxyuridine, into a DNA strand, it is possible to combine the advantages of hairpin strand DNA and double-stranded DNA, and have further solved the above-mentioned problem by simply deriving it into a crosslinker-modified DEL. Therefore, the present invention is as follows.

[0017] [1] A method for evaluating a crosslinker-modified double-stranded DEL derived from a hairpin-type DNA-encoded library (DEL) having a "selectively cleavable site," comprising the following steps: (1) contacting the DEL 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 the library molecule bound to the biological target to the biological target, (3) separating the complex of the crosslinked library molecule and the biological target from the uncrosslinked library molecule, (4) identifying the sequence of the oligonucleotide contained in the library molecule in the recovered complex, and (5) using the sequence determined in (4) to identify the structure of one or more compounds that bind to the biological target. [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 bound directly to the 5'-end of the oligonucleotide or via a bifunctional spacer. [4] The method according to any of [1] to [3], wherein the derivation into a crosslinker-modified double-stranded DEL comprises the following steps: (i) cleaving at least one "selectively cleavable site" of a hairpin-type DEL having a "selectively cleavable site" to convert it into a double-stranded DEL; (ii) removing oligonucleotides not bound to library molecules from the double-stranded DEL obtained in (i), thereby converting it into a single-stranded DEL; (iii) forming a duplex between the single-stranded DEL obtained in (ii) and a crosslinker-modified DNA to derive a crosslinker-modified double-stranded DEL. [5] The method according to any of [1] to [3], wherein the derivation into a crosslinker-modified double-stranded DEL comprises the following steps: (i) At least one "selectively cleavable site" of a hairpin-type DEL having a "selectively cleavable site" is cleaved to convert it into a double-stranded DEL. (ii) From the double-stranded DEL obtained in (i), oligonucleotides to which no library molecules are bound are removed to convert it into a single-stranded DEL.(iii) forming a double strand with the single-stranded DEL obtained in (ii) and DNA having a reactive group for crosslinker modification; and (iv) reacting the reactive group for crosslinker modification with a crosslinker unit to derive a crosslinker-modified double-stranded DEL. [6] The method according to any one of [1] to [3], wherein the derivation into a crosslinker-modified double-stranded DEL comprises the following steps: (i) cleaving at least one "selectively cleavable site" of a hairpin-type DEL having a "selectively cleavable site" to convert it into a double-stranded DEL. (ii) removing oligonucleotides not bound to library molecules from the double-stranded DEL obtained in (i), and converting it into a single-stranded DEL. (iii) attaching a crosslinker-modified primer to the single-stranded DEL obtained in (ii), and extending the attached primer to derive a crosslinker-modified double-stranded DEL. [7] The method according to any one of [1] to [3], wherein the derivation into a crosslinker-modified double-stranded DEL comprises the following steps: (i) cleaving at least one "selectively cleavable site" of a hairpin-type DEL having a "selectively cleavable site" to convert it into a double-stranded DEL; (ii) removing oligonucleotides not bound to library molecules from the double-stranded DEL obtained in (i), thereby converting it into a single-stranded DEL; (iii) attaching a modified primer having a reactive group for crosslinker modification to the single-stranded DEL obtained in (ii), and extending the attached primer to derivatize it into a double-stranded DEL having a reactive group for crosslinker modification; and (iv) reacting the reactive group for crosslinker modification with a crosslinker unit to derivatize it into a crosslinker-modified double-stranded DEL. [8] The method according to any one of [1] to [3], wherein the derivation into a crosslinker-modified double-stranded DEL comprises the following steps: (i) At least one "selectively cleavable site" of a hairpin-type DEL having a "selectively cleavable site" is cleaved to convert it into a double-stranded DEL. (ii) A crosslinker-modified primer is attached to the double-stranded DEL obtained in (i), and the attached primer is extended to derive a crosslinker-modified double-stranded DEL.[9] The method according to any one of [1] to [3], wherein the derivation into a crosslinker-modified double-stranded DEL comprises the following steps: (i) cleaving at least one "selectively cleavable site" of a hairpin-type DEL having a "selectively cleavable site" to convert it into a double-stranded DEL; (ii) attaching a modified primer having a reactive group for crosslinker modification to the double-stranded DEL obtained in (i), extending the attached primer, and reacting the reactive group for crosslinker modification with the crosslinker unit to derive the crosslinker-modified double-stranded DEL.

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

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

[12] The method of any of [4] to [7], wherein in step (ii), oligonucleotides to which no library molecule is bound have a functional molecule and are removed by a treatment according to the function of the functional molecule.

[13] The method of

[12] , wherein the functional molecule is biotin.

[14] The method of any of [4] to [7], wherein in step (ii), oligonucleotides to which no library molecule is bound are removed by degradation with an exonuclease.

[15] The method of

[14] , wherein the exonuclease is lambda exonuclease.

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

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

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

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

[18] The crosslinker is represented by formula (AA) to (AE): (wherein * represents the 5' end of the double-stranded DEL or the binding position of the bifunctional spacer bound to the 5' end)

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

[15] , wherein the crosslinker is represented by any one of the structures of formulae (AA) to (AE): (wherein * represents the 5' end of the double-stranded DEL or the binding position of the bifunctional spacer bound to the 5' end)

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

[15] , wherein the crosslinker has a structure represented by formula (BA) or (BB): (wherein * represents the 5' end of the double-stranded DEL or the binding position of the bifunctional spacer bound to the 5' end)

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

[15] , wherein the crosslinker is represented by formula (BA) or (BB): (wherein * represents the 5'-end of the double-stranded DEL or the bonding position with the bifunctional spacer bonded to the 5'-end)

[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 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 tetrazinyl group.

[24] The reactive group for crosslinker modification is represented by any one of formulae (CA) to (CL): (wherein * represents the 5'-end of the double-stranded DEL or the binding position to the bifunctional spacer bound to the 5'-end).

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

[19] , wherein the step of "crosslinking the crosslinker of the library molecule bound to the biological target with the biological target" in (2) 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

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

[27] The method according to

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

[28] The method according to any one of

[25] to

[27] , wherein the light irradiation is performed under conditions of light irradiation for 10 seconds to 180 minutes.

[29] The method according to any one of

[25] to

[27] , wherein the light irradiation is performed under conditions of light irradiation for 30 seconds to 30 minutes.

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

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

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

[30] , wherein the step (3) of "separating the complexes of the crosslinked library molecule and the biological target from the non-crosslinked library molecules" is a step of "separating the complexes of the crosslinked library molecule and the biological target from the non-crosslinked 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 one of [1] to

[30] , wherein the step (3) of "separating the complex of the cross-linked library molecule and the biological target from the non-cross-linked library molecule" is "separating the complex of the cross-linked library molecule and the biological target by immobilizing the biological target on a carrier for immobilizing the biological target and removing the non-cross-linked library molecule by washing."

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

[30] , wherein the hairpin-type DEL having a "selectively cleavable site" is a compound represented by the formula (I): (wherein X and Y are oligonucleotide chains, E and F are each independently an oligomer composed of a nucleotide or a nucleic acid analog, and E and F comprise complementary base sequences to form a double-stranded oligonucleotide, LP is a loop moiety, L is a linker, D is a divalent group derived from a reactive functional group, Sp is a bond or a bifunctional spacer, and An is a partial structure composed of at least one building block), X and Y have a sequence capable of at least partially forming a double strand, X is bonded to E at the 5' end, and Y is bonded to F at the 3' end, and at least one selectively cleavable site is present in at least one site of E, F, or LP.

[36] A hairpin-type DEL having a "selectively cleavable site" is represented by the formula (III): An-Sp-C-Bn (III) (wherein An and Sp have the same meanings as in

[35] , Bn represents a double-stranded oligonucleotide tag formed by an oligonucleotide strand X and an oligonucleotide strand Y, and C represents a double-stranded oligonucleotide tag represented by the formula (I) (wherein E, LP, L, D, and F have the same meaning as in

[35] , with the proviso that D is bonded to An directly or via a bifunctional spacer, and E and F are bonded to the corresponding ends of the double-stranded oligonucleotide tag Bn.)

[37] The method according to

[35] or

[36] , wherein An is the same as in

[35] and is a partial structure constructed from n building blocks α1 to αn (n is an integer of 1 to 10), and Bn is a double-stranded oligonucleotide tag formed from the oligonucleotide strand X and the oligonucleotide strand Y, and is a partial structure comprising an oligonucleotide containing a base sequence that can identify the structure of An.

[38] The method according to any one of

[35] to

[37] , wherein LP is a loop moiety represented by (LP1)p-LS-(LP2)q, and LS is a partial structure selected from the group of compounds described in (A) to (C) below: (A) a nucleotide (B) a nucleic acid analog (C) a C1-14 trivalent group which may have a substituent. LP1 is a partial structure selected, either singly or differently, in p numbers from the group of compounds described in (1) and (2) below: (1) a nucleotide (2) a nucleic acid analog. LP2 is a partial structure selected, either singly or differently, in q numbers from the group of compounds described in (1) and (2) below: (1) a nucleotide (2) a nucleic acid analog. The method according to any one of

[35] to

[37] , wherein the total number of p and q is 0 to 40.

[39] The method according to

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

[40] The method according to

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

[41] The method according to

[38] , wherein the sum of p and q is 2 to 7.

[42] The method according to

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

[43] The method according to any one of

[38] to

[42] , wherein LP1, LP2 and LS each have a structure selected from the following structures: (A) a nucleotide; or (B) a nucleic acid analog satisfying the following requirements (B11) to (B15): (B11) having a phosphate group (or an equivalent site) and a hydroxyl group (or an equivalent site), (B12) being composed of carbon, hydrogen, oxygen, nitrogen, phosphorus or sulfur, (B13) having a molecular weight of 142 to 1500, (B14) having 3 to 30 atoms between residues, and (B15) the bonding mode of atoms between residues is either all single bonds or contains 1 or 2 double bonds and the rest are single bonds.

[44] The method according to any one of

[38] to

[43] , wherein LP1, LP2, and LS each have a structure selected from the following structures: (A) a nucleotide; or (B) a nucleic acid analog satisfying the following requirements (B21) to (B25): (B21) having a phosphate and a hydroxyl group; (B22) being composed of carbon, hydrogen, oxygen, nitrogen, or phosphorus; (B23) having a molecular weight of 142 to 1000; (B24) having 3 to 15 atoms between residues; and (B25) all atoms bonded to each other by a single bond.

[45] The method of any of

[38] to

[44] , wherein LP1, LP2, and LS each have a structure selected from the following structures: (A) a nucleotide, or (B) a nucleic acid analog satisfying the following requirements (B31) to (B35): (B31) having a phosphate and a hydroxyl group, (B32) being composed of carbon, hydrogen, oxygen, nitrogen, or phosphorus, (B33) having a molecular weight of 142 to 700, (B34) having 4 to 7 atoms between residues, and (B35) all bonds between residues being single bonds.

[46] The method of any of

[38] to

[45] , wherein LP1 and LP2 each have the following structure: (B41) a d-Spacer, or (B5) a polyalkylene glycol phosphate ester.

[47] The method according to any one of

[38] to

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

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

[38] to

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

[49] The method according to any one of

[38] to

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

[50] The method according to any one of

[38] to

[45] , wherein LP1 and LP2 are each nucleotide.

[51] LS is represented by formula (a) to formula (g): (wherein * represents the bonding position with the linker, ** represents the bonding position with LP1 or LP2, and R represents a hydrogen atom or a methyl group).

[52] The method according to any of

[38] to

[50] , wherein LS is any one of the following: (wherein * represents the bonding position to the linker, and ** represents the bonding position to LP1 or LP2).

[53] The method according to any of

[38] to

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

[54] The method according to any of

[38] to

[50] , wherein LS is represented by formula (i) to formula (k): (wherein n1, m1, p1, and q1 each independently represent an integer of 1 to 20, * represents a bonding position to the linker, and ** represents a bonding position to LP1 or LP2).

[55] The method according to any of

[38] to

[50] , wherein LS is represented by formula (l): (wherein * denotes the position of binding to the linker, and ** denotes the position of binding to LP1 or LP2).

[56] The method according to any of

[38] to

[50] , wherein LS is any one of (B42), (B43) or (B44): (B42) Amino C6 dT (B43) mdC (TEG-Amino) (B44) Uni-Link (registered trademark) Amino Modifier.

[57] The method according to any of

[38] to

[50] , wherein LS is a nucleotide.

[58] The method according to any one of

[38] to

[42] and

[46] to

[50] , wherein L S is (C) an optionally substituted C1-14 trivalent group, and (C) is any one of the following structures: (1) a C1-10 aliphatic hydrocarbon which may have a substituent and which may be substituted with 1 to 3 heteroatoms, (2) a C6-14 aromatic hydrocarbon which may have a substituent, (3) a C2-9 aromatic heterocycle which may have a substituent, or (4) a C2-9 non-aromatic heterocycle which may have a substituent.

[59] The method according to any one of

[38] to

[42] and

[46] to

[50] , wherein L S is (C) an optionally substituted C1-14 trivalent group, and (C) is any one of the following structures: (1) a C1-6 aliphatic hydrocarbon which may have a substituent, (2) a C6-10 aromatic hydrocarbon which may have a substituent, or (3) a C2-5 aromatic heterocycle which may have a substituent.

[60] The method according to any one of

[38] to

[42] and

[46] to

[50] , wherein LS is (C) a C1-14 trivalent group which may have a substituent, and (C) is any one of the following structures: (1) a C1-6 aliphatic hydrocarbon, (2) benzene, or (3) a C2-5 nitrogen-containing aromatic heterocycle, wherein (1) to (3) may be unsubstituted or substituted with 1 to 3 substituents selected, either alone or differently, from substituent group ST1, and substituent group ST1 is a group consisting of C1-6 alkyl groups, C1-6 alkoxy groups, fluorine atoms, and chlorine atoms, with the proviso that when substituent group ST1 substitutes an aliphatic hydrocarbon, alkyl groups are not selected from substituent group ST1.

[61] The method of any of

[38] to

[42] and

[46] to

[50] , wherein LS is (C) an optionally substituted C1-14 trivalent group, and (C) is either one of the following structures: (1) a C1-6 alkyl group, or (2) a benzene that is unsubstituted or substituted with one or two C1-3 alkyl groups or C1-3 alkoxy groups.

[62] The method of any of

[38] to

[42] and

[46] to

[50] , wherein LS is (C) an optionally substituted C1-14 trivalent group, and (C) is either one of the following structures: (1) a C1-6 alkyl group.

[63] The method of any of

[35] to

[62] , wherein E and F are each independently an oligomer composed of a nucleotide or a nucleic acid analog, and the chain lengths of E and F are each 3 to 40.

[64] The method of any of

[35] to

[63] , wherein E and F are each independently an oligomer composed of nucleotides or a nucleic acid analog, and the chain lengths of E and F are each 4 to 30.

[65] The method of any of

[35] to

[64] , wherein E and F are each independently an oligomer composed of nucleotides or a nucleic acid analog, and the chain lengths of E and F are each 6 to 25.

[66] The method of any of

[35] to

[65] , wherein E and F are each independently an oligomer composed of nucleotides or a nucleic acid analog, and E and F contain complementary base sequences to form a double-stranded oligonucleotide, and the double-stranded oligonucleotide of E and F has a cohesive end.

[67] The method of

[66] , wherein the overhang of the cohesive end is 2 or more bases in length.

[68] The method according to any one of

[35] to

[65] , wherein E and F are each independently an oligomer composed of a nucleotide or a nucleic acid analog, E and F contain complementary base sequences to form a double-stranded oligonucleotide, and the double-stranded oligonucleotide of E and F is blunt-ended.

[69] The method according to any one of

[35] to

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

[70] The method of any of

[35] to

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

[71] The method of any of

[35] to

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

[72] The method of any of

[35] to

[71] , wherein E and F are each independently an oligomer composed of nucleotides.

[73] The method of any of

[35] to

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

[74] The method of any of

[35] to

[73] , wherein the nucleotide is a deoxyribonucleotide.

[75] The method of any of

[35] to

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

[76] The method of any of

[35] to

[71] , wherein E and F are each independently an oligomer composed of a nucleic acid analog.

[77] The method according to any of

[35] to

[76] , wherein L is (1) a C1-20 aliphatic hydrocarbon which may have a substituent and which may be substituted with 1 to 3 heteroatoms, or (2) a C6-14 aromatic hydrocarbon which may have a substituent.

[78] The method according to any of

[35] to

[77] , wherein L is a C1-6 aliphatic hydrocarbon which may have a substituent, a C1-6 aliphatic hydrocarbon which may be substituted with 1 or 2 oxygen atoms, or a C6-10 aromatic hydrocarbon which may have a substituent.

[79] The method according to any of

[35] to

[78] , wherein L is a C1-6 aliphatic hydrocarbon which can be substituted with substituent group ST1, or a benzene which can be substituted with substituent group ST1, wherein substituent group ST1 is a group consisting of a C1-6 alkyl group, a C1-6 alkoxy group, a fluorine atom, and a chlorine atom (however, when substituent group ST1 substitutes an aliphatic hydrocarbon, alkyl groups are not selected from substituent group ST1).

[80] The method according to any one of

[35] to

[79] , wherein L is a C1-6 alkyl group, or 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

[35] to

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

[82] The method according to any one of

[35] to

[81] , wherein the reactive functional group of D is a C-C, amino, ether, carbonyl, amide, ester, urea, sulfide, disulfide, sulfoxide, sulfonamide, or 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-based 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 , -CH ２ OSO ２ CF ３, an amino group, a hydroxyl group, or a carboxy group.

[86] The method according to any of

[35] to

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

[87] The method according to any of

[35] to

[86] , wherein the selectively cleavable site is a deoxyribonucleoside other than deoxyadenosine, deoxyguanosine, thymidine, and deoxycytidine.

[88] The method according to any of

[35] to

[87] , wherein the selectively cleavable site is 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 of any of

[35] to

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

[90] The method of any of

[35] to

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

[91] The method of any of

[35] to

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

[92] The method of any of

[35] to

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

[93] The method of any of

[35] to

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

[94] The method of any of

[35] to

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

[95] The method according to any of

[35] to

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

[96] The method according to

[95] , wherein the cleavable site contained in E or (LP1)p and the cleavable site contained in F or (LP2)q are cleavable under different conditions.

[97] The method according to any of

[35] to

[96] , wherein An is a partial structure constructed from n building blocks α1 to αn (n is an integer of 1 to 10).

[98] The method according to any one of

[35] to

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

[99] The method according to any one of

[35] to

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

[100] The method according to any one of

[35] to

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

[101] The method according to any one of

[35] to

[100] , wherein a building block of An is a compound having a molecular weight of 150 or less.

[102] The method according to any one of

[35] to

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

[103] The method according to any one of

[35] to

[102] , wherein An is a low molecular weight organic compound having substituents, either alone or differently, selected from the group 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

[35] to

[103] , wherein An has a molecular weight of 5,000 or less.

[105] The method according to any one of

[35] to

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

[106] The method according to any one of

[35] to

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

[107] The method according to any one of

[35] to

[97] , wherein An is a polypeptide.

[108] The method according to any one of

[35] to

[107] , wherein Sp is a bifunctional spacer.

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

[107] , wherein the bifunctional spacers are SpD-SpL-SpX, respectively, wherein SpD is a divalent group derived from a reactive group capable of forming a C-C, amino, ether, carbonyl, amide, ester, urea, sulfide, disulfide, sulfoxide, sulfonamide, or sulfonyl bond, SpL is polyalkylene glycol, polyethylene, C1-20 aliphatic hydrocarbon optionally substituted with a heteroatom, peptide, oligonucleotide, or a combination thereof, and SpX is a divalent group derived from a reactive group forming an amino, carbonyl, amide, ester, urea, or sulfonamide bond.

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

[107] , wherein the bifunctional spacers are SpD-SpL-SpX, SpD is a divalent group derived from a primary amino group, SpL is polyethylene glycol or polyethylene, and SpX is a divalent group derived from a carboxy group.

[111] The method of any one of

[35] to

[110] , wherein the oligonucleotide chain X and the oligonucleotide chain Y have a sequence capable of forming a double strand.

[112] The method of any one of

[35] to

[111] , wherein the oligonucleotide chain X and the oligonucleotide chain Y comprise complementary base sequences.

[113] The method of any one of

[35] to

[112] , wherein the oligonucleotide chain X and the oligonucleotide chain Y each have a length of 1 to 200 bases.

[114] The method of any one of

[35] to

[113] , wherein the oligonucleotide chain X and the oligonucleotide chain Y each have a length of 3 to 150 bases.

[115] The method according to any one of

[35] to

[114] , wherein the oligonucleotide strand X and the oligonucleotide strand Y each have a length of 30 to 150 bases.

[116] The method according to any one of

[35] to

[115] , wherein the oligonucleotide strand X and the oligonucleotide strand Y have blunt ends.

[117] The method according to any one of

[35] to

[115] , wherein the oligonucleotide strand X and the oligonucleotide strand Y have protruding ends.

[118] The method according to

[117] , wherein the protruding portion of the protruding end has a length of 1 to 30 bases.

[119] The method according to

[117] or

[118] , wherein the protruding portion of the protruding end has a length of 2 to 5 bases.

[120] The method according to any one of

[117] to

[119] , wherein the oligonucleotide strand X and the oligonucleotide strand Y each have a protruding end, and a specific molecule recognition sequence is further bound to the protruding end.

[121] The method according to any one of

[35] to

[120] , wherein a functional molecule is bound to either X or Y.

[122] The method according to any one of

[35] to

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

[123] The method according to any one of

[35] to

[107] , wherein Sp is a bond.

[0018] The present invention provides a method for converting DEL containing a cleavable site in the DNA strand into a crosslinker-modified double-stranded DEL and evaluating the resulting product. This provides a compound screening technology that combines a "simple DEL synthesis method" with an "expanded and improved DEL evaluation method" compared to conventional methods. Therefore, the present invention expands the opportunities for obtaining hit compounds useful in the development of pharmaceuticals, agricultural chemicals, and medical materials.

[0019] An exemplary method for producing DEL in Mode 1 is shown. Starting from a headpiece containing a first oligonucleotide strand containing a cleavable site in the DNA strand, a loop site, and a second oligonucleotide strand, DEL is produced by repeatedly binding building blocks and double-stranded ligation of oligonucleotide tags corresponding to the building blocks (three times in FIG. 1 ), and optionally double-stranded ligation of an oligonucleotide tag containing a primer region. An exemplary method for using DEL in Mode 1 is shown. For DEL containing a cleavable site in the first oligonucleotide strand of the headpiece, the cleavable site can be cleaved using a cleavage means such as an enzyme to induce double-stranded oligonucleotides that are not linked at the loop site, thereby enabling highly efficient PCR. An exemplary method for using DEL in Mode 2 is shown. For DEL containing a cleavable site in the second oligonucleotide strand of the headpiece, the cleavable site can be cleaved using a cleavage means such as an enzyme to induce double-stranded oligonucleotides that are not linked at the loop site, thereby enabling highly efficient PCR. An exemplary method for using DEL in Mode 3 is shown. For DELs containing cleavable sites in both the first and second oligonucleotide strands of the headpiece, a cleavage means such as an enzyme can be used to cleave both cleavable sites, resulting in a double-stranded oligonucleotide without a loop site, enabling highly efficient PCR. An exemplary use of DELs in Mode 4 is shown. For DELs containing two different cleavable sites in the first and second oligonucleotide strands of the headpiece, the cleavage conditions can be selected to selectively cleave either the first or second oligonucleotide strand. An exemplary use of DELs in Mode 5 is shown. By providing a cleavable site near the end of a DNA tag and cleaving the site as desired, a new protruding end can be generated. The protruding end can be used as a sticky end to ligate a desired nucleic acid sequence, such as a UMI (unique molecular identifier), and thereby confer new functionality. An exemplary use of DELs in Mode 6 is shown.In the present invention, a cleavable site can be used in combination with a modifying group or a functional molecule. For example, it is possible to prepare DEL by converting hairpin-stranded DNA into single-stranded DNA. For example, a double-stranded oligonucleotide chain having a functional molecule (e.g., biotin) at the 3' end is ligated to a synthesized DEL compound (A), the cleavable site is cleaved (B), and a treatment according to the function of the functional molecule is performed (C). For example, when 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. An exemplary use of DEL obtained in Mode 6 is shown below. DEL having single-stranded DNA obtained in Mode 6 can be conferred new functions by forming a duplex with a modified oligonucleotide having a desired functional site (e.g., crosslinker-modified DNA such as a photoreactive crosslinker). An exemplary use of DEL in Mode 7 is shown below. In the present invention, a crosslinker can be introduced using the cleavable site. The synthesized DEL compound is cleaved at a cleavable site (A), a modified primer is added (B), and a crosslinker-modified double-stranded DEL compound can be synthesized based on the added primer (C). Crosslinker-modified double-stranded DEL compounds can significantly improve detection sensitivity in DEL library screening (see Non-Patent Documents 7 and 11, etc.). In Example 1, a graph showing the conversion rate of the cleavage reaction at each incubation time was performed to verify the cleavage reaction of 10 hairpin-type DEL partial structures 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) using USER® enzyme.

[0033] Figure 1 is a schematic diagram showing the synthesis procedure 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) in Examples 2, 3, 4, 5, and 7. Using the corresponding headpiece as a starting material, hairpin DEL synthesis is achieved by two-step double-stranded ligation with the double-stranded oligonucleotides Pr_TAG and CP. 1 is a graph showing Ct values ​​measured by real-time PCR for eight types of hairpin DEL (U-DEL1, U-DEL2, U-DEL4, U-DEL7, U-DEL8, U-DEL9, U-DEL10, and H-DEL) and double-stranded DEL (DS-DEL) for each sample amount in Example 2. Samples in which various DELs were treated with USER® enzyme are indicated as "USER(+)," and untreated samples are indicated as "USER(-)." The cleavable hairpin DELs containing deoxyuridine (U-DEL1, U-DEL2, U-DEL4, U-DEL7, U-DEL8, U-DEL9, and U-DEL10) showed Ct values ​​equivalent to those of double-stranded DEL (DS-DEL) after treatment with USER® enzyme. In Example 3, this is a gel image obtained by denaturing polyacrylamide gel electrophoresis showing the progress of the cleavage reaction of hairpin DEL containing six types of deoxyuridine (U-DEL5, U-DEL7, U-DEL9, U-DEL11, U-DEL12, and U-DEL13) by USER® enzyme. The numbers in the figure indicate the numbers of each lane. In Example 4, this is a gel image obtained by denaturing polyacrylamide gel electrophoresis showing the progress of the cleavage reaction of hairpin DEL containing four types of deoxyinosine (I-DEL1, I-DEL2, I-DEL3, and I-DEL4) by endonuclease V. The numbers in the figure indicate the numbers of each lane. In Example 5, this is a gel image obtained by denaturing polyacrylamide gel electrophoresis showing the progress of the cleavage reaction of hairpin DEL containing ribonucleoside (R-DEL1) by RNase HII. The numbers in the figure indicate the numbers of each lane.This is a schematic diagram showing the synthesis procedure for a model library containing 3x3x3 (27) compound species using U-DEL9-HP as a starting material. In Example 6, U-DEL9-HP was used as a starting material, and the synthesis of the model library was achieved through three split-and-pool steps (cycles A, B, and C). Each cycle included a ligation reaction of a double-stranded oligonucleotide tag and a chemical reaction for introducing a building block. This is a gel image obtained by agarose gel electrophoresis showing the progress of the ligation reaction in each cycle in the model library synthesis of Example 6. The numbers in the figure indicate the lane numbers. Figure 18A is a chromatograph obtained from a sample after completion of cycle C in the model library synthesis of Example 6. Figure 18B is a deconvolution result of the MS spectrum obtained from a sample after completion of cycle C in the model library synthesis of Example 6. In Example 6, the image of the gel obtained by denatured polyacrylamide gel electrophoresis shows the progress of the cleavage reaction by the USER (registered trademark) enzyme of the model library. The numbers in the figure indicate the number of each lane. In Example 7, the image of the gel obtained by denatured polyacrylamide gel electrophoresis shows the progress of the cleavage reaction by the USER (registered trademark) enzyme of five DEL compounds having biotin at the 3' end ("AAZ-BIO-DEL", "SABA-BIO-DEL", "ClSABA-BIO-DEL", "mSABA-BIO-DEL", and "Amino-BIO-DEL"). The numbers in the figure indicate the number of each lane. In Example 7, a DEL compound having single-stranded DNA ("SS-AAZ-DEL", "SS-SABA-DEL", "SS-ClSABA-DEL", "SS-mSABA-DEL", and "SS-Amino-DEL") and a photoreactive crosslinker-modified primer "PXL-Pr" were used to carry out a primer extension reaction, and the results are shown in the image of a gel obtained by polyacrylamide gel electrophoresis. The numbers in the figure indicate the number of each lane.In Example 8, to compare the binder recovery efficiency of various photoreactive crosslinker-modified double-stranded DELs with and without photocrosslinking reaction, this graph shows the Ct values ​​and ΔCt values ​​(difference from the Ct value of the negative control) measured by real-time PCR for the amount recovered. Samples without UV irradiation are labeled "UV(-)", and samples with UV irradiation are labeled "UV(+)". Furthermore, "Solution S" is labeled "S", and "Solution E" is labeled "E". Note that each notation in the graph corresponds to each sample as follows: Notation in the 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" In Example 10, a DEL compound having single-stranded DNA ("SS-AAZ-DEL", "SS-SABA-DEL", "SS-ClSABA-DEL", "SS-mSABA-DEL", and "SS-Amino-DEL"), and a photoreactive crosslinker-modified primer "PXL-Pr2" were used to carry out a primer extension reaction, showing the results of the gel image obtained by polyacrylamide gel electrophoresis. The numbers in the figure indicate the number of each lane. In Example 10, a DEL compound having single-stranded DNA ("SS-AAZ-DEL3", "SS-SABA-DEL3", "SS-ClSABA-DEL3", "SS-mSABA-DEL3", and "SS-Amino-DEL3") and a photoreactive crosslinker-modified primer "PXL-Pr3" were used to carry out a primer extension reaction. This is an image of a gel obtained by polyacrylamide gel electrophoresis. The numbers in the figure indicate the number of each lane. In Example 11, in order to compare the binder recovery efficiency of various photoreactive crosslinker-modified double-stranded DELs with different linker structures with or without photocrosslinking reaction, this is a graph showing the ΔCt value (difference from the negative control) calculated using the Ct value measured by real-time PCR. Samples without UV irradiation are labeled "UV(-)", and samples with UV irradiation are labeled "UV(+)".In addition, each notation in the graph corresponds to each sample as follows. Notation in the graph: Sample mSABA-DEL2: "PXL-DS-mSABA-DEL2" mSABA-DEL3: "PXL-DS-mSABA-DEL3" In Example 12, various "photoreactive crosslinker-modified double-stranded DEL having a covalent bond between the crosslinker and the coding sequence" and various "photoreactive crosslinker-modified double-stranded DEL without a covalent bond between the crosslinker and the coding sequence" were compared in binder recovery efficiency, and the ΔCt value (difference from the negative control) calculated using the Ct value measured by real-time PCR is shown in the graph. Note that each bar in the graph corresponds to each sample as follows, from left to right. 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, the 10th bar from the left: "PXL-DS-SABA-DEL4" subjected to UV irradiation, the 11th bar from the left: "PXL-DS-ClSABA-DEL4" subjected to UV irradiation, the rightmost bar: "PXL-DS-mSABA-DEL4" subjected to UV irradiation. In Example 13, various "photoreactive crosslinker-modified double-stranded DEL having a covalent bond between the crosslinker and the coding sequence" and various "photoreactive crosslinker-modified double-stranded DEL without a covalent bond between the crosslinker and the coding sequence" were measured by real-time PCR to compare the binder recovery efficiency, and the ΔCt value (difference from the negative control) calculated using the Ct value is a graph showing. Note that each bar in the graph corresponds to each sample as follows, from left to right.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, rightmost bar: "PXL-DS-mSABA-DEL4" with UV irradiation. In Example 14, the hairpin DEL compound ("mSABA-DEL5") is shown to be undergoing cleavage reaction with the USER (registered trademark) enzyme, showing the gel image obtained by denaturing polyacrylamide gel electrophoresis. The numbers in the figure indicate the number of each lane. In Example 14, a DEL compound having single-stranded DNA ("SS-mSABA-DEL5") and a photoreactive crosslinker-modified primer "PXL-Pr5" were used to carry out a primer extension reaction, and this is an image of a gel obtained by polyacrylamide gel electrophoresis. The numbers in the figure indicate the number of each lane. In Example 15, a DEL compound having single-stranded DNA "SS-mSABA-DEL" and a crosslinker-modified primer "TPD-Pr", and "SS-ClSABA-DEL" and "ACA-Pr" were used to carry out a primer extension reaction, and this is an image of a gel obtained by polyacrylamide gel electrophoresis. The numbers in the figure indicate the number of each lane.In Example 16, a DEL compound having single-stranded DNA ("SS-mSABA-DEL"), and a reactive group-modified primer for crosslinker modification "BCN-Pr" were used to carry out a primer extension reaction, which shows the results of the gel obtained by polyacrylamide gel electrophoresis. The numbers in the figure indicate the numbers of each lane. In Example 17, a single-stranded DEL-modified model library, a photoreactive crosslinker-modified primer "PXL-Pr" and a reactive group-modified primer for crosslinker modification "BCN-Pr" were used to carry out a primer extension reaction, which shows the results of the gel obtained by polyacrylamide gel electrophoresis. The numbers in the figure indicate the numbers of each lane.

[0020] As described above and a concept well known to those skilled in the art, in the present invention, a compound library refers to a group of compound derivatives systematically collected from compounds that may have a specific activity, such as drug candidate compounds. These compound libraries are often synthesized based on the synthesis techniques and methodologies of combinatorial chemistry. Combinatorial chemistry is an experimental technique and a field of research related to the efficient synthesis of a wide variety of compounds through systematic synthetic routes from a series of compound libraries enumerated and designed based on combinatorial theory.

[0021] As mentioned above and well known to those skilled in the art, one type of compound library based on combinatorial chemistry is a DNA-encoded library. DNA-encoded library is appropriately abbreviated as DEL. DEL is also essentially synonymous with DNA-encoded compound library. In the present invention, a DNA-encoded library refers to a library in which a DNA tag is attached to each compound in the library. The sequence of the DNA tag is designed so that each structure of each compound can be identified, and it functions as a label for the compound.

[0022] A nucleotide is generally understood as a substance in which a phosphate group is bound to a nucleoside. Nucleotide and nucleoside are terms well known to those skilled in the art, and in one general embodiment, a nucleoside is understood as a nucleic acid base, such as a purine base or a pyrimidine base, bound via a glycosidic bond to the 1-position of a sugar, such as a pentose sugar. Nucleosides and nucleotides are also units that constitute nucleic acids such as DNA and RNA. Nucleic acids are also a well-known concept to those skilled in the art, and in one general embodiment, they are 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 below.

[0023] Furthermore, in this specification, in addition to nucleic acid polymers composed of nucleotides or nucleic acid analogs, nucleic acid monomers such as nucleotides or nucleic acid analogs may also be referred to simply 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] Nucleotides in the broad sense include not only natural nucleotides (original nucleotides) but also artificial nucleotides (various nucleic acid analogs). Nucleotides in the broad sense of the present invention include the following aspects: (A) Natural nucleoside nucleotides (examples of such nucleosides include adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxyuridine, deoxyguanosine, deoxycytidine, inosine, and diaminopurine deoxyriboside). (B) Nucleoside nucleotides having a nucleic acid base analog. (Examples of nucleosides having such nucleobase analogs 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, and 2-thiocytidine.) (C) Nucleotides having an intercalated nucleobase; (D) Unnatural nucleotides having ribose or 2'-deoxyribose; and (E) Nucleotides having a modified sugar in the sugar moiety. (Examples of such modified sugars include modified ribose, modified 2'-deoxyribose, 2'-O-methylribose, 2'-fluororibose, D-threoninol, arabinose, hexose, anhydrohexitol, altritol, and 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), and nucleic acids in which the oxygen in the ribose has been replaced.) Each nucleic acid analog is described in detail below. (F1) PMO PMO is a nucleic acid analog having a morpholine ring in the sugar moiety and an uncharged phosphorodiamidate structure in the phosphate diester moiety.(F2) LNA LNA is a nucleic acid analogue with a bridged structure at the sugar moiety. Most typically, the 2'-hydroxyl of ribose is bridged to the 4'-carbon of the same ribose sugar by a C1-6 alkylene or C1-6 heteroalkylene. Examples of bridged structures include methylene, propylene, ether, or amino bridges. A typical LNA is 2',4'-BNA (2'-O,4'-C-methano-bridged nucleic acid). (F3) GNA Glycol nucleic acid is also called GNA. Examples include R-GNA and S-GNA. In this case, the ribose is replaced by a glycol unit linked to a phosphodiester bond. (F4) TNA Threose nucleic acid is also called TNA. In this case, the ribose is replaced by α-L-threofuranosyl-(3'→2'). (F5) SNA Serinol nucleic acid is also called SNA. In this case, the ribose is replaced by a serinol unit linked to a phosphodiester bond. (F6) aTNA Acyclic threoninol nucleic acids are also called aTNA. Examples include D-aTNA and L-aTNA. In this case, the ribose is replaced with a threoninol unit linked to a phosphodiester bond. (F7) Sugars in which the oxygen in ribose is replaced. Specific examples include replacement of oxygen with S, Se, or alkylene (e.g., methylene or ethylene). (G) Backbone-modified nucleotides (Examples of backbone-modified nucleotides include peptide nucleic acids (also called PNA, in which a 2-aminoethyl-glycine linkage replaces the ribose and phosphodiester backbone).) (H) Phosphate-modified nucleotides (Examples of phosphate-modified nucleotides include phosphorothioates, 5'-N-phosphoramidites, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, phosphotriesters, bridged phosphoramidates, bridged phosphorothioates, and bridged 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 any particular limitation, it refers to a natural nucleotide. The term "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 essentially exists in nature. In one embodiment, the natural nucleotide of the present invention is the nucleotide described in (A) above. (Nucleic Acid Analog) The term "nucleic acid analog" is a term well known to those skilled in the art, and the structure of the nucleic acid analog of the present invention is not limited as long as it has the effects of the present invention. In one embodiment, the nucleic acid analog is a compound described in any of (B) to (H) above. In one embodiment, the nucleic acid analog of the present invention is a compound having a phosphate moiety and a hydroxyl group moiety in a nucleic acid monomer. The nucleic acid analog is more preferably a compound having a phosphate moiety and a hydroxyl group. In one embodiment, the nucleic acid analog of 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 phosphoramidizing the phosphate (or equivalent moiety) of a nucleic acid analog and using the resulting monomer in which the hydroxyl group (or equivalent moiety) is protected with a protecting group. Furthermore, the partial structure of a nucleic acid analog other than the phosphate site (or equivalent site) and the hydroxyl group (or equivalent site) can be referred to as a nucleic acid analog residue. The structure of the nucleic acid analog residue is not limited as long as it has the effects of the present invention. For reference, the structural characteristics of natural nucleic acids (deoxyadenosine, thymidine, deoxycytidine, deoxyguanosine) are as follows: the molecular weight is approximately 322 (thymidine monophosphate) to 347 (deoxyguanosine monophosphate), and the number of atoms between the hydroxyl oxygen atom at the 3' position and the phosphorus atom at the 5' position constituting the nucleic acid chain (including oxygen atoms and phosphorus atoms; hereinafter also referred to as the number of atoms between residues) is 6. Furthermore, the following nucleic acid analogs are known as those that can be used 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 (registered trademark) Amino Modifier Molecular weight: 227, number of residue atoms: 6 (see literature: Nucleic Acid Research, 1992, Vol. 20, pp. 6253-6259) d-Spacer Molecular weight: 198, number of residue atoms: 6 Triethylene glycol phosphate ester (Spacer9) Molecular weight: 230, number of residue atoms: 11.

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

[0027] Therefore, in one embodiment, the nucleic acid analog is compound (B1) characterized as follows: (B11) It has a phosphate group (or an equivalent site) and a hydroxyl group (or an equivalent site). (B12) It is composed of carbon, hydrogen, oxygen, nitrogen, phosphorus, or sulfur. (B13) It has a molecular weight of 142 to 1500. (B14) The number of atoms between residues is 5 to 30. (B15) The bonds between the atoms between residues are all single bonds, or contain one or two double bonds and the rest are single bonds.

[0028] In one embodiment, the nucleic acid analog is compound (B2) characterized as follows: (B21) It has a phosphate and a hydroxyl group. (B22) It is composed of carbon, hydrogen, oxygen, nitrogen, or phosphorus. (B23) It has a molecular weight of 142 to 1000. (B24) The number of atoms between residues is 5 to 20. (B25) The bonds between the atoms between residues are all single bonds.

[0029] In one embodiment, the nucleic acid analog is compound (B3) characterized as follows: (B31) It has a phosphate and a hydroxyl group. (B32) It is composed of carbon, hydrogen, oxygen, nitrogen, or phosphorus. (B33) It has a molecular weight of 142 to 700. (B34) The number of atoms between residues is 5 to 12. (B35) The bonds between the atoms between residues are all single bonds.

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

[0031] In the present invention, an oligonucleotide and an oligonucleotide chain refer to a polymer of nucleotides having one or more nucleotides at the 5'-end, the 3'-end, and an internal position between the 5'-end and the 3'-end.

[0032] Mutually complementary base sequences refer to nucleotide sequences that can form a so-called complementary base pair between two nucleic acid oligonucleotides, in which adenine and thymine (or uracil) or guanine and cytosine are paired in a fixed combination and connected by hydrogen bonds. The formation of a complementary base pair 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, Hoogsteen base pairs, or base pairs formed by 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 limitations on the sequence of "mutually complementary base sequences," nor on the homology between the two sequences, as long as they are sequences that can form a double-stranded chain of two oligonucleotides and can be used for the purposes of the present invention. The homology is preferably 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 preference.

[0033] To reiterate, in the present invention, hybridization means the act of forming a double strand between oligonucleotides or oligonucleotide chains containing complementary base sequences, and the phenomenon in which oligonucleotides or oligonucleotide chains containing complementary sequences form a double strand.

[0034] In the present invention, a double strand refers to a state in which two nucleic acid strands form complementary base pairs (hybridized). The two nucleic acid strands may be derived from two nucleic acid chains or may be derived from two nucleic acid sequences within a single nucleic acid chain molecule.

[0035] In the present invention, the term "double-stranded oligonucleotide" and "double-stranded oligonucleotide chain" refer to a secondary structure formed by hybridization of two or more different oligonucleotide chains. The chain lengths of the two oligonucleotides may be different, and they may have an unhybridized region. The region where the two strands hybridize is a double strand.

[0036] In the present invention, double-stranded DNA refers to a secondary structure formed by hybridization of two different DNA strands. The strand lengths of the DNA strands may be different and may have unhybridized regions. The DNA strand is not limited to naturally occurring deoxyribonucleotides, but refers to any oligonucleotide strand that can be amplified by DNA polymerase.

[0037] In the present invention, "forming a duplex" means forming a duplex 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 duplex is not formed in a particular solvent or condition, the nucleic acid is a duplex-forming nucleic acid if it forms a duplex under standard conditions.

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

[0039] In the present invention, blunt ends means that the ends of a double-stranded oligonucleotide are paired with no overhanging ends.

[0040] In the present invention, a "cohesive end" refers to a double-stranded oligonucleotide having an overhang at one of its ends. The overhang at the cohesive 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 a specific embodiment, the overhang can be used as a hybridization region when performing sticky end ligation.

[0041] PCR refers to polymerase chain reaction. PCR is a means of amplifying oligonucleotide chains and is a technique well known to those skilled in the art. The PCR process can be summarized as follows: (1) a double-stranded oligonucleotide chain to be amplified is dissociated into two single strands by heat treatment or the like; (2) the temperature is adjusted to a suitable temperature for enzymatic reaction, and then a complementary strand is synthesized to each single strand using an enzyme (e.g., DNA polymerase) present in the reaction system. In other words, one double-stranded oligonucleotide can be amplified into two. PCR can amplify oligonucleotide chains with high efficiency by repeating the processes (1) and (2) by adjusting the temperature.

[0042] In the present invention, a primer means an oligonucleotide that can be annealed to a template oligonucleotide strand and extended by a polymerase in a template-dependent manner.

[0043] In the present invention, the primer sequence for PCR means the sequence of the portion of the oligonucleotide chain to which the primer anneals, and is preferably a sequence suitable for PCR as known in the art, and is preferably present at the end of the oligonucleotide chain.

[0044] In the present invention, a nick refers to a portion of a double-stranded oligonucleotide chain where an internucleotide bond is missing, resulting in a break in the oligonucleotide chain. The 5' side of this missing portion may or may not have a phosphate group.

[0045] In the present invention, a gap refers to a portion in a double-stranded oligonucleotide chain where one or more consecutive nucleotides are deleted, resulting in separation of the oligonucleotide chains. The 5' side of the deleted portion may or may not have a phosphate group.

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

[0047] In the present invention, nucleic acid linking reaction and ligation refer to a reaction for linking the ends of nucleic acids together.

[0048] Enzymatic nucleic acid joining reaction and enzymatic ligation refer to a reaction in which the ends of nucleic acids are joined together using an enzyme.

[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 embodiment, DNA ligase is an enzyme that joins the ends of DNA strands together via a phosphodiester bond. In one embodiment, DNA ligase is understood to be a ligase belonging to EC number 6.5.1.1 or 6.5.1.2. DNA ligase is also called polydeoxyribonucleotide synthase or polynucleotide ligase. Examples of DNA ligase include DNA ligase I, II, III, IV, and T4 DNA ligase.

[0051] In one embodiment, RNA ligase is an enzyme that joins the ends of RNA strands via a phosphodiester bond. In one embodiment, RNA ligase is understood to be a ligase belonging to EC number 6.5.1.3. In another embodiment, RNA ligase belongs to the family of poly(ribonucleotide):poly(ribonucleotide) ligase. RNA ligase is also called polyribonucleotide synthase or polyribonucleotide ligase.

[0052] In the present invention, chemical ligation refers to a reaction that joins the ends of nucleic acids without using an enzyme.

[0053] In chemical ligation, the ends of nucleic acids having chemically reactive functional groups react with each other to form a linking moiety. Examples of functional groups that can undergo a chemical reaction include a pair of an optionally substituted alkynyl group and an optionally substituted azide group, a pair of an optionally substituted diene having a 4π-electron system (for example, an optionally substituted 1,3-unsaturated compound such as an optionally substituted 1,3-butadiene, 1-methoxy-3-trimethylsilyloxy-1,3-butadiene, cyclopentadiene, cyclohexadiene, or furan) and an optionally substituted dienophile or an optionally substituted heterodienophile having a 2π-electron system (for example, an optionally substituted alkenyl group or an optionally substituted alkynyl group), a pair of an optionally substituted amino group and a carboxylic acid group, a pair of a phosphorothioate group and an iodo group (for example, a phosphorothioate group at the 3'-terminus and an iodo group at the 5'-terminus), or a pair of a phosphate group and a hydroxy group (for example, a pair of a phosphate group at the 5'-terminus and a hydroxy group at the 3'-terminus, or a pair of a hydroxy group at the 5'-terminus and a phosphate group at the 3'-terminus). Chemical ligation is a concept well known to those skilled in the art, and those skilled in the art can appropriately achieve chemical ligation based on their common technical knowledge. In addition to the above, see Artificial DNA; PNA & XNA, 2014, Vol. 5, e27896, Current Opinion in Chemical Biology, 2015, Vol. 26, pp. 80-88, etc.

[0054] In the present invention, "selectively cleavable" means that only a specific site in a compound can be selectively cleaved under predetermined conditions without causing any change in the other molecular structures of the compound.

[0055] In the present invention, the term "selectively cleavable site" refers to a site in a compound that can be selectively cleaved under predetermined conditions.

[0056] In one embodiment, a preferred structure of the "selectively cleavable site" in the present invention is a "selectively cleavable nucleic acid." The site may be a site composed of multiple nucleic acids and cleaved in response to a specific sequence, or may be a site composed of a single nucleic acid. When the cleavable site is a nucleic acid, this is preferable from the viewpoints of (1) the efficiency of production, which can be achieved by using established production methods such as a nucleic acid synthesizer, and (2) since the reaction conditions for constructing the DEL building blocks require that the nucleic acid in the DNA tag portion is not decomposed, if the cleavable site is a nucleic acid, it is also not decomposed.

[0057] A more preferred structure of the "selectively cleavable nucleic acid" is a nucleic acid containing a nucleotide not contained in the sequence of the DEL DNA tag. If the cleavable site is a nucleotide not contained in the sequence of the DNA tag, it can be used without limiting the sequence of the DNA tag in order to avoid cleavage of the DNA tag portion.

[0058] The nucleic acids used in the sequence of the DNA tag are preferably deoxyadenosine, deoxyguanosine, thymidine, and deoxycytidine. Therefore, the preferred structure of 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 having a cleavable base." For example, in the case of a "nucleotide having a cleavable base" in DEL, the N-glycosidic bond between the base moiety and the sugar moiety is cleaved by the action of a DNA glycosylase, leaving an abasic site. The phosphodiester bond adjacent to the abasic site is cleaved by a change in chemical conditions (e.g., increased temperature, basic hydrolysis, etc.) or by an enzyme having apurinic / apyrimidinic (AP) endonuclease activity or AP lyase activity (e.g., endonuclease III, endonuclease IV, endonuclease V, endonuclease VI, endonuclease VII, endonuclease VIII, APE1 (human AP endonuclease), Fpg (formamidopyridine-DNA glycosylase), etc.), forming a one-base gap or nick.

[0060] Examples of "nucleotides having a cleavable base" 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 having a cleavable base will be apparent to those skilled in the art. By incorporating these "nucleotides having a cleavable base" into DEL and using a DNA glycosylase that specifically recognizes its structure, the DEL can be selectively debased.

[0061] In the present invention, the term "DNA glycosylase" refers to any enzyme having glycosylase activity that recognizes any nucleic acid base in an oligonucleotide, cleaves the N-glycosidic bond between the base and sugar moiety, and creates an abasic site. Examples of such enzymes 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 (an abbreviation for single-strand-selective uracil DNA glycosylase, which recognizes deoxyuridine).

[0062] More preferred examples of the "selectively cleavable site" in the present invention include deoxyinosine and deoxyuridine.

[0063] A particularly preferred example of the "selectively cleavable site" in the present invention is deoxyuridine.

[0064] In one embodiment, the "selectively cleavable site" of the present invention is preferably cleaved using an enzyme. Enzymes generally 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 rather recognize and act only on the "selectively cleavable site." Cleavage using the enzyme may also be achieved by changing the chemical conditions after structurally altering the "selectively cleavable site" with the enzyme. Examples of such enzymes include glycosylases and nucleases.

[0065] In the present invention, glycosylase refers to 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 them, DNA glycosylase, as described above, is an enzyme that recognizes the nucleic acid base moiety in an oligonucleotide and hydrolyzes the glycosidic bond.

[0066] In the present invention, a nuclease is an enzyme that has the function of hydrolyzing the phosphodiester bond between the sugar and phosphate of a nucleic acid. Examples of nucleases include AP endonucleases, nicking endonucleases, and ribonucleases.

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

[0068] Nicking endonucleases (e.g., Nb.BbvCI, Nb.BsmI, Nb.BsrDI, etc.) recognize specific DNA sequences and generate a nick by cleaving the phosphodiester bond in only one of the two strands. Endonuclease V can generate a nick by cleaving the second phosphodiester bond in the 3' direction from deoxyinosine, and is useful in carrying out the present invention.

[0069] Ribonucleases are enzymes that degrade RNA. In the present invention, ribonucleosides are used as "selectively cleavable sites" and can be used by allowing ribonucleases to act on them. RNase HII, a type of ribonuclease, can generate nicks by cleaving the phosphodiester bond at the 5' end of ribonucleotides incorporated into DNA sequences, and is useful in carrying out the present invention.

[0070] In the present invention, USER® refers to "Uracil-Specific Excision Reagent" Enzyme. USER® is an endonuclease cocktail containing uracil DNA glycosylase (UDG) and endonuclease VIII that removes uracil. USER® removes uracil from double-stranded DNA, creating a one-base gap and cleaving the DNA strand. In the USER® process, UDG first removes the uracil base, creating an abasic site. The endonuclease then decomposes the phosphodiester bond, releasing the base-free deoxyribose, creating a one-base gap. In the description herein, USER® enzyme and USER® enzyme refer to USER® as defined above.

[0071] In the present invention, an exonuclease is an enzyme that has the function of sequentially hydrolyzing phosphodiester bonds from the 5'-end 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 whose 5'-end is phosphorylated, and is useful in the present invention when converting double-stranded DEL into single-stranded DEL.

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

[0074] In the present invention, the base sequence capable of identifying each building block refers to a specific base sequence designed to correspond to the structure of each building block. Designing a sequence means assigning a nucleic acid base sequence to each structure, for example, assigning the nucleic acid base sequence AAA to building block structure A, the nucleic acid base sequence TTT to structure B, and the nucleic acid base sequence CGC to structure C. The sequence can be freely designed as long as the object of the present invention is achieved. For example, any number of base sequences can be assigned to one building block.

[0075] In the present invention, an oligonucleotide tag refers to a partial structure comprising an oligonucleotide containing a base sequence that allows identification of the structure of a partial structure constructed from building blocks. In the present invention, an oligonucleotide tag may be an oligonucleotide corresponding to each building block, or may be a longer 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; however, in terms of ease of PCR amplification and sequencer analysis, it is desirable for the nucleotides to be suitable for these procedures. Examples of preferred nucleotides include nucleotides having the above-described natural nucleic acid base as the base moiety and the above-described ribose or 2'-deoxyribose as the sugar moiety, and more preferred examples include deoxyadenosine, thymidine, deoxycytidine, and deoxyguanosine.

[0076] (Headpiece) In the present invention, the headpiece refers to a starting compound for producing a compound library such as DEL. The structure of the headpiece of the present invention is not limited as long as it achieves the object of the present invention, but in the most typical embodiment, it has at least one site to which a building block can be linked and at least one site to which an oligonucleotide tag can be linked, and further contains 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 to which the oligonucleotide tag can be linked.

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

[0078] In one embodiment, the headpiece is desirably chemically stable. In another embodiment, the headpiece preferably has a structure that allows the DNA tag and the building block to be appropriately arranged in space. In one embodiment, the headpiece preferably has moderate flexibility. Here, moderate spatial arrangement and flexibility (structural characteristics of the headpiece) will be further described. The structural characteristics of the headpiece described here may be achieved by the headpiece alone, or by binding the headpiece to a bifunctional spacer. In one embodiment, the structural characteristics of the headpiece are preferably such that the headpiece or DNA tag does not inhibit the building block formation reaction, or conversely, the headpiece or building block does not inhibit the DNA tag extension reaction. In one embodiment, the structural characteristics of the headpiece are preferably such that the headpiece or DNA tag portion does not affect the interaction between the building block compound (library compound) and the target (e.g., target protein). In one embodiment, the structural characteristics of the headpiece are preferably such that the DNA tag and the building block portion are oriented on opposite sides (e.g., at an angle of 90 degrees or more). In one embodiment, the preferred structural characteristics of the headpiece are those that separate the loop portion of the headpiece and the building block by several to a dozen atoms in terms of the organic compound skeleton. In one embodiment, the headpiece preferably has a suitable affinity with the DNA tag portion and the building block portion. Suitable affinity means, for example, chemical reactivity and stability that allow the bond to be formed, maintained, and cleaved under the desired conditions to carry out the present invention. In the present invention, a bifunctional spacer refers to a spacer portion having at least two reactive groups that enable the bonding between the building block portion and the headpiece.

[0079] In the description of the present invention, the terms "headpiece," "headpiece compound," and "compound for a headpiece" are terms that refer to compounds of the same concept. In the description of the present invention, a "compound used as a headpiece" can be understood essentially in the same way as "use of a compound as a headpiece" from the viewpoint of use, and can be understood essentially in the same way as "method of using a compound as a headpiece" from the viewpoint of method. The same applies to compound libraries.

[0080] A preferred headpiece structure will be described below, but the headpiece structure is not limited as long as it achieves the effects of the present invention.

[0081] In one embodiment, the headpiece is composed of: (D) a reactive functional group having at least one site that can be linked directly to a building block or indirectly via a bifunctional spacer; (L) a linker extending from the reactive functional group; (E) a first oligonucleotide strand having one binding site that can be linked to one strand of an oligonucleotide tag; (F) a second oligonucleotide strand having one binding site that can be linked to the other strand of an oligonucleotide tag; and (LP) a loop site that connects the linker and the two oligonucleotide strands; and at least one of E, F, or LP has at least one selectively cleavable site.

[0082] In one embodiment, the headpiece is a compound represented by formula (I): (wherein E and F each independently represent an oligomer composed of a nucleotide or a nucleic acid analog, and E and F comprise complementary base sequences to form a double-stranded oligonucleotide, LP represents a loop moiety, L represents a linker, and D represents a reactive functional group), and the compound has at least one selectively cleavable site in at least one of E, F, or LP.

[0083] In the present invention, the partial structure of the loop region that binds to the linker may be referred to as a linking region (LS). Furthermore, in the present invention, E-LP-F may be collectively referred to as a hairpin region.

[0084] (First and Second Oligonucleotide Strands) Preferred embodiments of the first oligonucleotide strand (E) and the second oligonucleotide strand (F) are described below.

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

[0086] The site to which the oligonucleotide tag is ligated preferably has a structure suitable for enzymatic ligation or chemical ligation. In one embodiment, the headpiece and the oligonucleotide tag are ligated by double-stranded ligation using an enzyme. In this case, it is preferable that the first and second oligonucleotide strands 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, it is preferable that one of the first and second oligonucleotide strands is longer than the other strand by the chain length of the overhanging end. Furthermore, for ligation using DNA ligase, it is preferable that the 5' end of the strand containing the 5' end of the headpiece of the first and second oligonucleotide strands is phosphorylated.

[0087] The first and second oligonucleotide strands may also contain a part or the whole of a primer binding sequence for PCR. The primer binding sequence preferably has a chain length of 17 to 25 bases.

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

[0089] In one embodiment, the linker is (L1) a C1-20 aliphatic hydrocarbon which may have a substituent and which may be substituted with 1 to 3 heteroatoms, or (2) a C6-14 aromatic hydrocarbon which may have a substituent.

[0090] In other embodiments, L is the following embodiment (L2), (L3), (L4), or (L5). (L2) A C1-6 aliphatic hydrocarbon which may have a substituent, a C1-6 aliphatic hydrocarbon which may be substituted with one or two oxygen atoms, or a C6-10 aromatic hydrocarbon which may have a substituent. (L3) A C1-6 aliphatic hydrocarbon which can be substituted with substituent group ST1, or a benzene which can be substituted with substituent group ST1. Here, substituent group ST1 is a group consisting of a C1-6 alkyl group, a C1-6 alkoxy group, a fluorine atom, and a chlorine atom. However, when substituent group ST1 substitutes an aliphatic hydrocarbon, the alkyl group is not selected from substituent group ST1. (L4) A C1-6 alkyl, or a benzene which is unsubstituted or substituted with one or two C1-3 alkyl groups or C1-3 alkoxy groups. (L5) A C1-6 alkyl.

[0091] (Reactive Functional Group) Preferred embodiments of the reactive functional group (D) are described below. As described above, the reactive functional group has at least one site that can be directly linked to a building block or indirectly linked via a bifunctional spacer, and is the site that bonds to a linker group. Typically, the reactive functional group is a monovalent group (D-) in the headpiece, and is a "divalent group derived from a reactive functional group" (-D-) based on the (D-) in DEL. For example, when D is an amino group, the specific structure of (D-) is (R-HN-) (R is a substituent described below). For example, it reacts with an activated carboxy 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-) is (-NR-). R is not limited as long 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 1 to 3 substituents selected, either singly or differently, from the group 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, when (D-) is a methylene group having a leaving group (X-), the specific structure of (D-) is (X-CH ２ -), which reacts with a nucleophilic reagent 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. ２ For example, when (D-) is an aldehyde group, the specific structure of (D-) is (HOC-). The aldehyde group forms a carbon-nitrogen bond by, for example, a reductive amination reaction with an amino group, and in this case, (-D-) becomes -CH ２ -, which forms a carbon-carbon double bond by reaction with, for example, a phosphorus ylide group, in which case (-D-) becomes -CH=, and which forms a carbon-carbon triple bond by reaction with, for example, an α-diazophosphonate group, in which case (-D-) becomes -C≡.

[0092] In one embodiment, the moiety (D-) is the following embodiment (D1): (D1) A functional group capable of forming a C-C, amino, ether, carbonyl, amide, ester, urea, sulfide, disulfide, sulfoxide, sulfonamide, or sulfonyl bond. (This is taken literally, but in this case, (-D-) is a C-C, amino, ether, carbonyl, amide, ester, urea, sulfide, disulfide, sulfoxide, sulfonamide, or sulfonyl bond.)

[0093] In other embodiments, (D-) is the following embodiment (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-) is not limited to -(C1 hydrocarbon)-, -NR-, -O-, -(C=O)-, -S-, or -CH ２ (D3) A C1 hydrocarbon having a halogen atom, a C1 hydrocarbon having a sulfonic acid leaving group, an amino group, a hydroxyl group, a carboxy group, a halogenated carboxy group, a thiol group, or an aldehyde group. In this case, (-D-) can be -(C1 hydrocarbon)-, -NR-, -O-, -(C=O)-, -S-, -CH ２ It can be -, -CH=, or -C≡, etc. (D4) -CH ２ Cl, —CH ２ Br, —CH ２ OSO ２ CH ３ , -CH ２ OSO ２ CF ３ , an amino group, a hydroxyl group, or a carboxyl group. In this case, (-D-) represents -CH ２ (D5) A primary amino group. In this case, (-D-) becomes -NH-.

[0094] A preferred embodiment of the loop site (LP) will be described below. The loop site (LP) is preferably designed so that the first oligonucleotide strand (E) and the second oligonucleotide strand (F) form a duplex within the molecule, and the headpiece can form a hairpin structure. That is, the loop site (LP) preferably has a chain length and bond flexibility that make the loop structure thermodynamically stable. Therefore, in one embodiment, the loop site (LP) is as follows: LP is a loop moiety represented by (LP1)p-LS-(LP2)q, LS is a partial structure selected from the group of compounds described in (A) to (C) below: (A) a nucleotide (B) a nucleic acid analog (C) a C1-14 trivalent group which may have a substituent; LP1 is a partial structure selected, either singly or differently, in p numbers from the group of compounds described in (1) and (2) below: (1) a nucleotide (2) a nucleic acid analog; LP2 is a partial structure selected, either singly or differently, in q numbers from the group of compounds described in (1) and (2) below: (1) a nucleotide (2) a nucleic acid analog; and the total number of p and q is 0 to 40.

[0095] More preferred embodiments of the loop region are as explained above. The structure of the loop region will be further explained below.

[0096] Here, the nucleotides are the natural nucleotides explained above, and the nucleic acid analogs are as explained above.

[0097] Here, LP1 is p partial structures selected, either singly or differently, from the group of compounds described in (1) and (2) below, and LP2 is q partial structures selected, either singly or differently, from the group of compounds described in (1) and (2) below. (1) Nucleotide (2) Nucleic Acid Analog "p partial structures selected, either singly or differently," means that, for example, when p is 4, LP1 can be selected, either singly 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] The loop region may also contain a part or the whole of a 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 site becomes a nucleic acid oligomer. The nucleic acid oligomer of the present invention is an oligomer in which nucleotides or nucleic acid analogs are linked as monomers. An oligomer can also be called a linear compound. Therefore, the nucleic acid oligomer of the present invention is either an oligonucleotide chain, a nucleic acid analog chain, or a mixed chain of nucleotides and nucleic acid analogs.

[0100] When LS is (A) a nucleotide or (B) a nucleic acid analog, the loop portion is a nucleic acid oligomer. In this case, the headpiece can be produced 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, one embodiment of the headpiece manufacturing method involves preparing a nucleic acid synthesis monomer in which the linker portion (L) and the reactive functional group portion (D) are linked to the LS, and then synthesizing a nucleic acid oligomer. Examples of such nucleic acid synthesis monomers include the aforementioned Amino C6 dT, mdC (TEG-Amino), and Uni-Link (registered trademark) Amino Modifier. In this embodiment, for example, in the structure of the monomer mdC (TEG-Amino), the nucleotide portion corresponds to the linking portion (LS), and the side chain portion extending from the base corresponds to the linker portion (L) and the reactive functional group portion (D). During preparation, the reactive functional group (D) may be protected with a protecting group.

[0102] In this case, in one embodiment, the nucleic acid analog is the following compound (B6): (B6) A compound in which the above-mentioned (-LD) is bound to the base moiety of a nucleotide.

[0103] In one embodiment, the nucleic acid analog is the following compound (B61), (B62), (B63), (B64), or (B65). (B61) (-L-D) is (-L1-D1) (B6) (B62) (-L-D) is (-L2-D2) (B6). (B63) (-L-D) is (-L3-D3) (B6). (B64) (-L-D) is (-L4-D4) (B6). (B65) The compound according to any one of (B61) to (B64) wherein (-D) is (-D5).

[0104] When the LS is (A) a nucleotide or (B) a nucleic acid analog, in one embodiment of the headpiece production, a nucleic acid oligomer can be synthesized first, and then the linker site (L) and the reactive functional group site (D) can be attached. In this case, it is preferable to incorporate the "specific nucleic acid analog" to which the linker site is attached into the hairpin site (nucleic acid analog oligomer) as the linking site (LS). Examples of the "specific nucleic acid analog" include the aforementioned Amino C6 dT, mdC (TEG-Amino), and Uni-Link (registered trademark) Amino Modifier. In this embodiment, for example, mdC (TEG-Amino) itself corresponds to the linking site (LS), and the additional site further attached from the base side chain corresponds 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 strand (E) and the second oligonucleotide strand (F) form a duplex within the molecule, and the headpiece forms a hairpin structure. In one embodiment, the total number of p and q is 1 to 40. In one embodiment, the total number of p and q is 2 to 20. In one embodiment, the total number of p and q is 2 to 10. In one embodiment, the total number of p and q is 2 to 7.

[0106] In one embodiment, the loop site of the present invention is composed of (A) a nucleotide and 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 (registered trademark) Amino Modifier (B52) Triethylene glycol phosphate ester

[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 site is a nucleic acid oligomer having the sequences shown 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 headpiece, the number of cleavable portions is preferably five or less, and more preferably one or two.

[0110] In the headpiece, when there are two or more cleavable sites, it is preferred that at least one cleavable site is in the first oligonucleotide strand or between the first oligonucleotide strand and the linker binding site, and at least one cleavable site is in the second oligonucleotide strand or between the second oligonucleotide strand and the linker binding site.

[0111] In one embodiment, in the headpiece, the position of the cleavable site is preferably within 20 bases, more preferably within 10 bases, and even more preferably within 3 bases, from the binding point between the loop site and the first oligonucleotide strand or the second oligonucleotide strand.

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

[0113] Just to be clear, the preferred embodiment of the "selectively cleavable site" and the preferred embodiments of, for example, E, F, or LP are separate concepts. In other words, even if the position of the "selectively cleavable site" is included in E, the preferred embodiment of E does not necessarily apply to the "selectively cleavable site."

[0114] In one embodiment, the compound constituting the DEL of the present invention is a compound represented by the following formula (II): (wherein X and Y are oligonucleotide chains, E and F are each independently an oligomer composed of a nucleotide or a nucleic acid analog, with the proviso that E and F have complementary base sequences and form a double-stranded oligonucleotide, LP is a loop moiety, L is a linker, D is a divalent group derived from a reactive functional group, Sp is a bond or a bifunctional spacer, and An is a partial structure composed of at least one building block), wherein X and Y have a sequence capable of at least partially forming a double strand, X is bonded to E at the 5' end, and Y is bonded to F at the 3' end, and the compound has at least one selectively cleavable site in at least one site of E, F, or LP.

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

[0116] (Bifunctional Spacer) As described above, the bifunctional spacer is a spacer moiety having at least two reactive groups that enable bonding between the partial structure An of the compound library and 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 partial structure An of the compound library. SpL is a chemically inert spacing moiety. Note that, like the reactive functional group (D), the reactive group (SpX) becomes a monovalent group (-SpX) in the bifunctional spacer alone (in the state of a reagent before bonding with the headpiece), and becomes a "divalent group derived from a reactive group" (-SpX-) based on the (-SpX) in DEL (in the state bonded to the headpiece). Similarly, the reactive group (SpD) is a monovalent group (SpD-) before being bonded to An, and in DEL (a state bonded to An), it becomes a "divalent group derived from the reactive group" (-SpD-) based on the (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 has the following structure (SpX1), (SpX2), or (SpX3), which is a reactive group suitable when the reactive functional group of the headpiece is an amino group. (SpX1): carboxy group, halogenated carboxy group, aldehyde group, or halogenated sulfonyl group (SpX2): carboxy group or halogenated sulfonyl group (SpX3): carboxy group

[0118] Preferred embodiments of SpD are the same as the aforementioned D. In one embodiment, SpD is the aforementioned (D1), (D2), (D3), (D4) or (D5).

[0119] Preferred embodiments of SpL are as follows. In one embodiment, SpL is the above-mentioned (L1), (L2), (L3), (L4) or (L5). In one embodiment, SpL is the following (SpL1), (SpL2) or (SpL3). (SpL1) polyalkylene glycol, polyethylene, C1-20 aliphatic hydrocarbon optionally substituted with a heteroatom, peptide, oligonucleotide, or combinations thereof. (SpL2) polyalkylene glycol, polyethylene, C1-10 aliphatic hydrocarbon, or peptide. (SpL3) polyethylene glycol or polyethylene.

[0120] In one embodiment, the 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 means a bond.

[0122] In carrying out the present invention, it is advantageous if the headpiece can be synthesized using a nucleic acid synthesizer. In this embodiment, as described above, in one embodiment, a nucleic acid synthesis monomer is prepared in which a linker portion (L) and a reactive functional group portion (D) are linked to an LS, 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 (registered trademark) Amino Modifier. On the other hand, when using commercially available nucleic acid synthesis monomers or nucleic acid analogs that can be used in nucleic acid synthesizers, the length of the linker portion may be limited. In such cases, in one embodiment, the introduction of an appropriate bifunctional spacer makes it possible to adjust the distance between the headpiece and An, which is advantageous in carrying out the invention.

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

[0124] In the present invention, "C1-6 alkyl" means a straight or branched alkyl group having 1 to 6 carbon atoms. Specific examples include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, etc.

[0125] In the present invention, "C1-3 alkyl" means a straight or branched chain alkyl group having 1 to 3 carbon atoms. Specific examples include methyl, ethyl, propyl, and isopropyl.

[0126] In the present invention, "C1-6 alkoxy" means a straight or branched chain alkoxy having 1 to 6 carbon atoms. Specific examples include methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentyloxy, hexyloxy, etc.

[0127] In the present invention, "C1-3 alkoxy" means a straight or branched chain alkoxy having 1 to 3 carbon atoms. Specific examples include methoxy, ethoxy, propoxy, and isopropoxy.

[0128] In the present invention, the term "hydrocarbon" refers to a linear, branched or cyclic, saturated or unsaturated compound composed solely of carbon and hydrogen atoms.

[0129] In the present invention, "aliphatic hydrocarbon" refers to a non-aromatic hydrocarbon. "Aliphatic hydrocarbon" may be linear, branched, or cyclic, and may be saturated or unsaturated. Specific examples of the structure include alkyl, alkenyl, alkynyl, cycloalkyl, or cycloalkenyl, or a structure formed by a combination thereof. In the present invention, "C1-20 aliphatic hydrocarbon" refers to an aliphatic hydrocarbon having 1 to 20 carbon atoms. In the present invention, "C1-10 aliphatic hydrocarbon" refers to an aliphatic hydrocarbon having 1 to 10 carbon atoms. In the present invention, "C1-6 aliphatic hydrocarbon" refers to an aliphatic hydrocarbon having 1 to 6 carbon atoms.

[0130] In the present invention, "aromatic hydrocarbon" refers to aromatic hydrocarbons. In the present invention, "C6-14 aromatic hydrocarbon" refers to aromatic hydrocarbons having 6 to 14 carbon atoms. Specific examples include benzene, naphthalene, and anthracene. In the present invention, "C6-10 aromatic hydrocarbon" 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 a heteroatom in the ring structure, an element selected, either singly or differently, from the group consisting of nitrogen, oxygen, and sulfur. In one embodiment, the aromatic heterocycle is a "C1-9 aromatic heterocycle" having 1 to 9 carbon atoms, and in another embodiment, the "C1-9 aromatic heterocycle" is a 5- to 10-membered aromatic heterocycle. In one embodiment, the aromatic heterocycle is a "C1-5 aromatic heterocycle" having 1 to 5 carbon atoms, and in another embodiment, the "C1-5 aromatic heterocycle" is a 5- to 6-membered aromatic heterocycle. In one embodiment, the aromatic heterocycle is a "C2-9 aromatic heterocycle" having 2 to 9 carbon atoms, and in another embodiment, the "C2-9 aromatic heterocycle" is a 5- to 10-membered aromatic heterocycle. In one embodiment, the aromatic heterocycle is a "C2-5 aromatic heterocycle" having 2 to 5 carbon atoms, and in another embodiment, the "C2-5 aromatic heterocycle" is a 5- or 6-membered aromatic heterocycle.

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

[0133] The non-aromatic heterocycle of the present invention is a non-aromatic heterocycle having, as a heteroatom in the ring structure, an element selected, either singly or differently, from the group consisting of nitrogen, oxygen, and sulfur. The non-aromatic heterocycle may contain a partially unsaturated bond. In one embodiment, the non-aromatic heterocycle is a "C2-9 non-aromatic heterocycle" having 2 to 9 carbon atoms, and in another embodiment, the "C2-9 non-aromatic heterocycle" is a 5- to 10-membered non-aromatic heterocycle.

[0134] In the present invention, the term "C1-14 trivalent group" refers to 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 the present invention, when it is stated that "may be replaced by a heteroatom", the 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, for example, propyl (-CH ２ -CH ２ -CH ３ ), "propyl which may be substituted with a heteroatom" refers to a methylene (-CH ２ -) is replaced by oxygen, ２ -O-CH ３ ) or (—O—CH ２ -CH ３ )) and nitrogen-substituted amines ((-CH ２ -NH-CH ３ ) or (—NH—CH ２ -CH ３ )) is a concept that contains structures such as

[0136] In the present invention, when it is stated that a group "may have a substituent," the substituent is not limited as long as the object of the present invention is achieved. The substituent is preferably a C1-6 alkyl group, a C1-6 alkoxy group, an amino group, a hydroxy 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 the present invention, polypeptides and peptides refer to compounds or partial structures formed by linking amino acids. Amino acids are a general term for organic compounds having both amino and carboxy functional groups. The amino acids that make up the polypeptides and peptides of the present invention are not particularly limited and include modified amino acids. In accordance with common usage in the field of life science, proline (classified as an imino acid) is also included in the amino acids of the present invention. The amino acids that make up the polypeptides and peptides of the present invention are preferably alpha amino acids, and more preferably "proteinogenic amino acids."

[0138] In the present invention, the halogen atom includes a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.

[0139] C-C, amino, ether, carbonyl, amide, ester, urea, sulfide, disulfide, sulfoxide, sulfonamide, and sulfonyl bonds are chemical bonds having a chemical structure that can be understood by their respective names. Those skilled in the art will understand that, for example, an ether bond is a bond that can generally be represented by "-O-", and a carbonyl bond is a bond that can generally be represented by "-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 the effects of the present invention are achieved. The substituent on the nitrogen atom is preferably a C1-6 alkyl group or a hydrogen atom, and more preferably a hydrogen atom. Needless to say, a C-C bond refers to a carbon-carbon bond. C-C bonds include single bonds, double bonds, and triple bonds. In one embodiment, a bond appropriately selected from the above 11 types is constructed in steps a and / or c of the production method of the present invention. These 11 types of bonds are particularly fundamental bond patterns in organic chemistry, and 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 11 types of bonds in appropriate combinations.

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

[0141] In one embodiment, the partial structure An of the compound library of the present invention is constructed from the above-mentioned 12 elements. These 12 elements are particularly basic 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 combination.

[0142] A low molecular weight organic compound having a substituent selected, either singly or differently, from the group consisting of an aryl group, a non-aromatic cyclyl group, a heteroaryl group, and a non-aromatic heterocyclyl group is a low molecular weight organic compound having a chemical structure that can be understood from the name of each of these groups. The concept of a low molecular weight compound is 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, more preferably a phenyl group.

[0144] The non-aromatic cyclyl groups of the present invention are preferably 5- to 8-membered, more preferably 5- or 6-membered non-aromatic cyclyl groups, which may contain partially unsaturated bonds.

[0145] The heteroaryl group and non-aromatic heterocyclyl group of the present invention are groups having, in the ring structure, heteroatoms selected, either singly 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- to 8-membered groups, more preferably 5- 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 above four types of groups. These four types of groups are particularly basic partial structures in organic compounds, and 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 types of groups in appropriate combinations.

[0147] Those skilled in the art will appreciate that the compound libraries constructed in the above-described preferred embodiments, i.e., 11 types of bonds, 12 types of elements, and / or 4 types of groups, are of particular value, and that compound libraries constructed outside of these preferred embodiments will generally have limited applications and, in many cases, limited commercial value.

[0148] The synthetic history of An refers to a record of all operations performed until An is synthesized, and in particular refers to the structure and order of the building blocks used until An is synthesized. For example, when reactions are carried out in two or more separate reaction vessels using different building blocks and / or under different reaction conditions, the synthetic history is imparted as sequence information of the oligonucleotide by linking an oligonucleotide chain of a predetermined sequence to the product in each reaction vessel before or after the reaction. By repeating such operations until An is constructed, an oligonucleotide Bn having the synthetic history of An is constructed.

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

[0150] Following the above process, let us take the synthesis of a peptide library using solid-phase synthesis as an example. In split-and-pool synthesis, at each step of peptide amplification, N types of supports are first mixed and homogenized, without cutting out the sample from the solid-phase supports to which the amino acids are peptide-bonded, and then the mixture is divided into equal parts to amplify the next N types of amino acids.

[0151] In other words, one type of peptide chain is produced for each carrier, and if all 20 natural amino acids are applied at each stage, a peptide library of all possible combinations for peptides of a specific length will be constructed.

[0152] If this peptide library is to be screened for antigen presentation or receptor binding, assays can be performed using peptides on a solid support using methods such as ELISA. This means that there is no need to excise the sample peptides from the support; instead, carrier particles that react to the assay can be picked up (for example, fluorescently labeled carrier particles approximately 0.1 mm in size can be picked up using an optical microscope). The peptides on the particles can then be analyzed using an analytical instrument (such as a peptide analyzer) to determine the target peptide sequence, or the candidate peptide sequences can be indirectly determined using other combinatorial chemical identification methods (such as tagging).

[0153] Furthermore, in the production method of the present invention, an example will be described in which v types of structures are synthesized by split-and-pool synthesis when m is 2, and w types of structures are synthesized when m is 3. In this explanation, the steps are repeated in the order of (c) and (d). (m=2) In the step of m=2, α2 is added to A1-Sp-C-B1 in step (c) and β2 is added in step (d), respectively, to produce A2-Sp-C-B2. Here, v types of α2 structures (α2(a-v)) and corresponding v types of β2 (β2(a-v)) 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(a-v)-Sp-C-B2(a-v)). In split-and-pool synthesis, v types of A2-Sp-C-B2 are mixed and then divided into w parts. Dividing, most specifically, means dividing the mixture into w reaction vessels. (m=3) In the step where m=3, α3 is added to A2-Sp-C-B2 in step (c) and β3 is added in step (d) to produce A3-Sp-C-B3. Here, w types of α3 structures (α3(a-w)) and w types of corresponding β3s (β2(a-w)) are prepared, and steps (c) and (d) are carried out for w (A2(a-v)-Sp-C-B2(a-v) mixtures). Then, through steps n=2 and 3, (v × w) types of A3-Sp-C-B3 can be efficiently synthesized in (v + w) syntheses.

[0154] (Biological Evaluation) Mixing the w products obtained yields a mixture of (v x w) types of A3-Sp-C-B3 compound library. For example, if a drug receptor binding test is performed on this mixture, screening for (v x w) types of compounds can be performed in a single run. By washing away compounds that do not bind to the drug receptor, only the bound compounds can be isolated. In DEL such as that of the present invention, the DNA of the isolated A3-Sp-C-B3 compound is amplified to an amount that can be sequenced, and the structure of A3 can be determined from the sequence information.

[0155] The terms "compound library," "building block," "split and pool," etc. are well known to those skilled in the art in the field of combinatorial chemistry, and can be used appropriately with reference to the following documents: (1) Takahashi Takashi and Doi Takayuki, "Combinatorial Chemistry," Journal of the Society of Organic Synthesis Chemistry, 2002, Vol. 60, pp. 426-433 (2) Combinatorial Chemistry Research Group (ed.), "Combinatorial Chemistry," Kagaku Dojin

[0156] A DNA-encoded library (or DEL) is a compound library consisting of a group of compounds (DNA-encoded compounds) labeled with DNA or oligonucleotides having substantially the same functions as DNA. By the split-and-pool synthesis described above, the structure or synthetic history of each compound is imparted to the labeled DNA as sequence information. Due to these characteristics, a DNA-encoded library can be used for 10 ２ ~10 ２０ The structure of the compound can be identified by screening a mixture of various compounds and identifying the DNA sequences contained in the obtained compounds using techniques known in the art (e.g., using a next-generation sequencer and / or a microarray). In one embodiment of the screening technique, a technique can be selected in which a target such as a protein is contacted with a DNA-encoded library and compounds that bind to the target are selected.

[0157] The term "biological target" is well known to those skilled in the art. In one embodiment, the term "biological target" in the present invention refers to a group of biological substances that can be targeted in the development of pharmaceuticals and agrochemicals. Examples of such substances include enzymes (e.g., kinases, phosphatases, methylases, demethylases, proteases, and DNA repair enzymes), proteins involved in protein:protein interactions (e.g., receptor ligands), receptor targets (e.g., GPCRs), ion channels, cells, bacteria, viruses, parasites, DNA, RNA, prions, and carbohydrates. The term "biological activity evaluation" is well known to those skilled in the art. In one embodiment, the term "biological activity evaluation" in the present invention refers to the evaluation of the presence or strength of the biological activity of a compound (e.g., the ability to bind to a biological target, the ability to inhibit enzymatic activity, the ability to promote enzymatic activity, etc.). For specific examples of biological activity evaluation, see Patent Documents 2 and 3, and Non-Patent Documents 1 to 6, cited above. "Functionality evaluation" is a term well known to those skilled in the art. In one embodiment, in the present invention, "functionality evaluation" refers to evaluating the presence or absence, or the strength of, a specific function of a compound (e.g., binding ability, biological activity, luminescence properties, etc.).

[0158] By using a DNA strand with a cleavable site, the present invention provides several advantageous approaches to DEL and methods for producing DEL. Modes 1 to 7 are described in detail below.

[0159] Mode 1 The present invention provides a DEL using the above-mentioned "hairpin-shaped headpiece having a cleavable site."

[0160] As illustrated in Figure 1, in Mode 1, starting from a first oligonucleotide strand containing a cleavable site in the DNA strand, a headpiece containing a loop site and a second oligonucleotide strand, the binding of building blocks and double-stranded ligation of oligonucleotide tags corresponding to the building blocks are repeated (three times in Figure 1), and if desired, double-stranded ligation of an oligonucleotide tag containing a primer region is further performed to achieve the production of DEL.

[0161] As illustrated in Figure 2, in mode 1, for a DEL containing a cleavable site in the first oligonucleotide strand of the headpiece, the cleavable site is cleaved using a cleavage means such as an enzyme, leading to a double-stranded oligonucleotide that is not linked at the loop site, thereby enabling highly efficient PCR.

[0162] Regarding Mode 2, in DEL using a "hairpin-shaped headpiece with a cleavable site," the cleavable site may be present in the second oligonucleotide strand, as illustrated in Figure 3. The characteristics of Mode 2 are similar to those of Mode 1, except for the cleavable site.

[0163] (Regarding Mode 3) As illustrated in Figure 4, in DEL using a "hairpin-shaped headpiece having a cleavable site," the cleavable site may be present in both the first and second oligonucleotide strands. In this mode, the loop site is cleaved from both oligonucleotide strands, which is expected to further improve PCR efficiency.

[0164] (Regarding Mode 4) As illustrated in Figure 5, in the present invention, cleavable sites may be present in both the first oligonucleotide strand (E) and the second oligonucleotide strand (F), and the structures of the cleavable sites may be different. In such cases, the difference in the properties of the two (or more) cleavable sites can be utilized to control the cleavage site. For example, deoxyuridine may be used as the cleavable site in the first oligonucleotide strand (E), and deoxyinosine may be used as the cleavable site in the second oligonucleotide strand (F). In this case, using the USER enzyme allows selective cleavage of the deoxyuridine in the first oligonucleotide strand (E). On the other hand, when alkyladenine DNA glycosylase and endonuclease VIII are used, the cleavage site starting from deoxyinosine in the second oligonucleotide strand (F) can be selectively cleaved. In this way, by selecting the cleavage site as desired, a wider range of DEL modifications can be made, and a wider range of techniques can be applied to the subsequent evaluation.

[0165] (Regarding Format 5) As illustrated in Figure 6, in the present invention, a cleavable site can also be provided in the DNA tag portion (for example, the oligonucleotide strand (Y)). By providing a cleavable site near the end of the DNA tag and cleaving the site as desired, a new protruding end can be generated. The protruding ends can be used as sticky ends to ligate desired nucleic acid sequences, such as UMIs (unique molecular identifiers). After biological evaluation, the selected DEL compounds are given a UMIs region as described above, and then subjected to DNA sequencing, enabling analysis with reduced PCR amplification bias. Thus, in the present invention, by having a selectively cleavable site in the nucleic acid sequence, it is possible to impart unprecedented performance to the DEL compounds in terms of their production and use.

[0166] Here, UMIs (Uniform Molecular Identification Sequences) are molecular identifiers that are added to DNA contained in a sample to give each DNA molecule an individual DNA sequence (see Nature Methods, 2012, Vol. 9, pp. 72-74). By adding such molecular identifiers before PCR amplification, it becomes possible to distinguish PCR duplicates (sequences derived from the same molecule) when quantifying the number of DNA molecules having a specific sequence in a sample, enabling quantification with reduced PCR amplification bias.

[0167] (Regarding Format 6) As illustrated in Figure 7, the present invention allows the use of a cleavable site in combination with a modifying group or functional molecule. For example, it is possible to prepare DEL by converting hairpin-stranded DNA into single-stranded DNA. An example of a DEL compound using a headpiece with a cleavable site in the E portion is shown in Figure 7. (Step A) A double-stranded oligonucleotide chain having a solid-phase-supported removable modifying group (e.g., biotin) at the 3' end is ligated to the synthesized DEL compound. (Step B) The cleavable site is cleaved. (Step C) A treatment according to the function of the modifying group is applied. For example, in the case of biotin, streptavidin beads with biotin affinity are used to selectively remove the oligonucleotide chain bound to biotin from the system. This makes it possible to obtain DEL having single-stranded DNA.

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

[0169] Here, biotin refers to all biotins that bind to avidin, including vitamin B ７ but also, for example, desthiobiotin.

[0170] In one aspect, the present invention provides suitable conditions for a method of converting a DEL containing a cleavable site in a DNA strand into a crosslinker-modified double-stranded DEL and evaluating the resulting DEL. Another method for preparing a DEL by converting a hairpin strand DNA into single-stranded DNA includes the following method using an exonuclease: (Step A) A hairpin-type DNA having a "selectively cleavable site" is cleaved using an enzyme, such as USER® Enzyme, that cleaves the cleavage site in a state where the 5' end is phosphorylated after the cleavage reaction. (Step B) One of the oligonucleotide strands, the 5' end of which is phosphorylated, is degraded and removed, for example, by treatment with lambda exonuclease. This results in a DEL having single-stranded DNA (single-stranded DEL).

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

[0172] When obtaining the single-stranded DEL having the library molecule in the 3' direction, the "selectively cleavable site" of the starting hairpin-type DEL is located in the 3' direction from the site where the library molecule is bound.

[0173] As illustrated in Figure 8, DEL having single-stranded DNA can be conferred new functions by forming a double strand with a modified oligonucleotide having a desired functional moiety (e.g., a crosslinker-modified DNA such as a photoreactive crosslinker, or a crosslinker-modified primer such as a photoreactive crosslinker). Furthermore, when a crosslinker-modified primer is used, the optionally added primer may be extended to yield a crosslinker-modified double-stranded DEL compound. Such crosslinker-modified double-stranded DEL compounds are useful in the present invention because a covalent bond is formed between the biological target and the coding sequence after screening.

[0174] (Regarding Format 7) As illustrated in Figure 9, in the present invention, a crosslinker can be introduced using a cleavable site. As shown in Figure 9, a DEL compound using a headpiece with a cleavable site in the E portion is used as an example. (Step A) The cleavable site is cleaved from the synthesized DEL compound. (Step B) A modified primer (e.g., a crosslinker-modified primer such as a photoreactive crosslinker) having the desired functional site is added. (Step C) The added primer is extended to synthesize a crosslinker-modified double-stranded DEL compound. In the context of DEL evaluation, the crosslinker-modified double-stranded DEL compound can further bind the crosslinker to the target protein when the building block compound (library low molecular weight compound) binds to the target protein, significantly improving detection sensitivity (see Non-Patent Documents 7 and 11, etc.). In the practical application of DEL technology, which evaluates a large number of library compounds, it is very useful to enhance the affinity of the library compounds and improve detection sensitivity. The present invention provides a novel and highly efficient method for producing a crosslinker-modified double-stranded DEL compound, and is therefore extremely useful.

[0175] In one aspect, the present invention provides suitable conditions for the method of converting DEL containing a cleavable site in a DNA strand into a crosslinker-modified double-stranded DEL and evaluating it.In one embodiment, the crosslinker-modified double-stranded DEL compound is preferably linked to an oligonucleotide having a coding sequence via a covalent bond.After screening, such a "crosslinker-modified double-stranded DEL compound" forms a covalent bond between the target and the coding sequence, and is resistant to stronger separation and elution conditions than conventional ones, such as for removing non-specific binders, and is very useful.

[0176] (Crosslinker) In one embodiment, in the present invention, the term "crosslinker" refers to a reactive group capable of forming a covalent bond by reacting with a biological target such as a protein or a nucleic acid molecule. For example, crosslinkers such as those described in Thermo Scientific Crosslinking Technical Handbook are known.

[0177] The crosslinker used in the present invention is preferably a reactive group containing at least one of an 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 containing at least one of an azide group, a diazirine group, or a sulfonyl fluoride group.

[0178] In one embodiment, in the present invention, "having a crosslinker" and "modified with a crosslinker" mean having a partial structure containing a crosslinker as a substituent.

[0179] In one preferred embodiment, the "crosslinker-modified double-stranded DEL," "crosslinker-modified DNA," and "crosslinker-modified primer" have a crosslinker bound directly or via a bifunctional spacer to the 5'-end of the "double-stranded DEL," "DNA," and "primer," respectively. In this case, the crosslinker preferably has a structure represented by any of the following formulae (AA) to (AE), (BA), or (BB): (In the formula, * means the 5' end of the "double-stranded DEL," "DNA," or "primer," or the binding position of the bifunctional spacer 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 is converted into a highly reactive group (e.g., nitrene or carbene) upon irradiation with light and forms a covalent bond with a nearby biological target. For example, an azide group and a diazirine group are known, and structures represented by the above formulae (AA) to (AE) are known.

[0181] In one 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 a biological target protein to form a covalent bond. For example, structures represented by the above formulae (BA) to (BB) are known.

[0182] In the present invention, the crosslinking reaction between the crosslinker and the biological target is preferably carried out within a temperature range that does not significantly alter the desired conformation of the biological target, for example, a temperature range of 4 to 40°C.

[0183] (Bifunctional spacer of crosslinker) As described above, the bifunctional spacer is a spacer moiety having at least two reactive groups that enable binding between the partial structure An of the compound library and the headpiece. Furthermore, in the present invention, the bifunctional spacer is a spacer moiety having two reactive groups that enable binding between the crosslinker and the "double-stranded DEL", "DNA", or "primer". This bifunctional spacer may be referred to as the "bifunctional spacer of the crosslinker". In contrast, the bifunctional spacer that binds to the aforementioned compound library may be referred to as the "bifunctional spacer of the compound library". In one embodiment, the preferred embodiment of the "bifunctional spacer of the crosslinker" is the same as the preferred embodiment of the "bifunctional spacer of the compound library" described above. In another embodiment, the "bifunctional spacer of the crosslinker" preferably has 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 has a molecular chain length equivalent to that of the "bifunctional spacer of the compound library".

[0184] (Coding Sequence) In the present invention, the term "coding sequence" refers to a sequence portion of an oligonucleotide contained in a DEL, which has a sequence that allows identification of the structure of a library molecule.

[0185] The "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 below. In one embodiment, the "reactive group for crosslinker modification" is a reactive group that has reactivity selectivity with the crosslinker. By having reaction selectivity with the crosslinker, reaction conditions in which a crosslinker cannot be applied, such as when the crosslinker reacts first and undergoes structural transformation, can be applied to the present invention. In other words, by introducing a unit having a reactive group for crosslinker modification into the process of the present invention, using reaction conditions in which a crosslinker cannot be applied in the process of the present invention, and then reacting 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 a binding reaction. When two compounds each having this pair are to be bound, the pair reacts preferentially with high selectivity to form a bond, even if various other functional groups are present in the compounds.

[0187] An example of the above pair is the pair of functional groups in a click reaction. The "click reaction" is a concept well known to those skilled in the art. (See, for example, HC Kolb, MG Finn & KB Sharpless: Angew. Chem. Int. Ed., 40, 2004 (2001)). In one embodiment, the "click reaction" can be understood as follows: A "click reaction" refers to a reaction having at least the following characteristics: (1) functional orthogonality (i.e., a functional moiety reacts only with a reactive moiety complementary to that functional moiety, without reacting with other reactive moieties); and (2) the resulting bond is irreversible (i.e., once reactants react to form a product, it is difficult to decompose the product back into the reactants), or in some cases, the resulting bond can be reversible (i.e., under appropriate conditions, it reverts to the reactants). Optionally, "click" chemistry can further have one or more of the following characteristics: (1) stereospecificity; (2) reaction conditions without rigorous purification, atmospheric control, etc.; (3) readily available starting materials and reagents; (4) the use of innocuous solvents or the complete elimination of solvents; (5) product isolation by crystallization or distillation; (6) physiological stability; (7) large thermodynamic driving forces (e.g., 10-20 kcal / mol); (8) single reaction product; and (9) high chemical yields (e.g., greater than 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 reaction, more preferably an alkynyl group, an alkenyl group, an azide group, or a tetrazinyl group, and further preferably any of formulae (CA) to (CL).

[0189] Here, the pair of "reactive group for crosslinker modification" and "reactive group paired with the reactive group for crosslinker modification" preferably includes an azide group for an alkynyl group and a tetrazinyl group for an alkenyl group. These pairs are in a so-called bolt-and-nut relationship and are interchangeable. For example, when an alkynyl group is used as the "reactive group for crosslinker modification," an azide group can be used as the "reactive group paired with the reactive group for crosslinker modification." The selection thereof is well known to those skilled in the art. Preferred examples of the "reactive group for crosslinker modification" and the "reactive group pairing with the reactive group for crosslinker modification" include the above-mentioned (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), etc.

[0190] The "crosslinker unit" is not particularly limited as long as it is a unit having the above-mentioned "reactive group that pairs with the reactive group for crosslinker modification" and a crosslinker.

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

[0192] The "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, the "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] For the embodiment of the "modified primer having a reactive group for crosslinker modification," see the above-mentioned "crosslinker-modified primer," except that "crosslinker" is read as "reactive group for crosslinker modification."

[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. Nucleic acids of various sequences in the examples can be prepared, for example, by an automated nucleic acid synthesizer according to standard methods. An example of an automated nucleic acid synthesizer is the nS-8II (manufactured by Gene Design). Nucleic acids can also be prepared by contract synthesis or through a contract laboratory. Examples of contract laboratories well known to those skilled in the art include Gene Design and LGC Biosearch Technologies. Generally, these contract laboratories prepare nucleic acids of sequences designated by the client under a confidentiality agreement and deliver them to the client.

[0196] Example 1 [Verification of the cleavage reaction of a partial structure of hairpin-type DEL containing deoxyuridine by USER (registered trademark) enzyme] Compounds having the sequences shown in Table 1 were prepared using an automated nucleic acid synthesizer nS-8II (manufactured by Gene Design Co., Ltd.). As will be clear to those skilled in the art, in the sequence notation in Table 1, each sequence unit is bonded by a phosphodiester bond, and "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): "(amino-NC6-dT)" means a modified nucleic acid represented by the following formula (2): The modified nucleic acid represented by the formula (3) is represented by "(dSpacer)": "(aminoC7)" means a group represented by the following formula (4): The amino-NC6-dT is a group represented by the following formula (5), which was synthesized according to the method described in the Journal of the American Chemical Society, 1993, Vol. 115, pp. 7128-7134: The nucleic acid was introduced using a nucleic acid synthesis reagent.

[0197] In Table 1, "No." in the left column indicates the sequence number, and "Seq." in the right column indicates the sequence. The left side of the sequence represents the 5' side, and the right side represents the 3' side. 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]

[0199] A 0.1 mM aqueous solution of each compound having the sequence shown in Table 1 was prepared, and the cleavage reaction by USER (registered trademark) enzyme was examined according to the following procedure.

[0200] To a PCR tube, 1 μL of a 0.1 mM aqueous solution of the compound of 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. 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 of each reaction solution was sampled 1 hour and 3 hours after the start of incubation. 20 μL of 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 20 hours later. 20 μL of U-DEL8-HP and U-DEL9-HP were sampled after a further 1 hour of incubation at 90°C.

[0202] Of the sampled solutions, U-DEL1-sh, U-DEL2-sh, U-DEL3-sh, and U-DEL4-sh were analyzed under the following analytical condition 1, and 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 condition 2.

[0203] Analysis condition 1: Apparatus: 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°C Solvent: Solution A: Water (0.75% v / v hexafluoroisopropanol; 0.038% v / v triethylamine; 5 μM ethylenediaminetetraacetic acid) Solution B: 90% v / v aqueous methanol solution (0.75% v / v hexafluoroisopropanol; 0.038% v / v triethylamine; 5 μM ethylenediaminetetraacetic acid) Gradient conditions: Measurement was started at a flow rate of 0.36 mL / min with a fixed mixing ratio of solution A to solution B of 95 / 5 (v / v). After 0.56 minutes, the mixing ratio of solution A to solution B was linearly changed to 40 / 60 (v / v) over a period of 5.5 minutes. Detection wavelength: 260 nm

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

[0205] The sequences and theoretical molecular weights of the products (abasic forms of the deoxyuridine moiety and cleaved fragments) expected in each reaction solution, as well as the molecular weights detected in each reaction solution, are shown in Tables 2 and 3. The designations for each column in Tables 2 and 3 are as follows:

[0206] "Entry" (far left): indicates the experiment number, 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): represents the sequence number. Of the sequence numbers (No.), Nos. 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 are substrates for the respective reaction solutions, Nos. 11, 14, 17, 20, 22, 25, 29, 31, 33, and 35 are abasic forms of the deoxyuridine moiety of the respective substrates, and the remaining sequence numbers are fragments formed by cleavage of the respective substrates.

[0208] "Seq." (third from the left): represents a sequence, with the left side representing the 5' side and the right side representing the 3' side. In the sequence notation, "(B)" represents the following formula (6): The other notations are the same as in Table 1.

[0209] "Expected MW." (fourth from the left): represents the theoretical molecular weight (Da) of each sequence.

[0210] "Observed MW." (far right): Indicates the detected molecular weight (Da) identified for each sequence. Note that "-" indicates that it was not detected.

[0211]

[0212]

[0213] The conversion rates of the abasic reaction and cleavage reaction were calculated from the area ratio of the peaks corresponding to each detected sequence. For all substrates, the abasic reaction was 99% or more complete within 1 hour at 37°C (the substrate peak was less than 1%, with the remaining peaks consisting only of abasic forms and cleaved fragments). A graph showing the conversion rates of the cleavage reaction is shown in Figure 10. As shown in the graph, for all substrates except U-DEL8-HP and U-DEL9-HP, the cleavage reaction had progressed to 95% or more within 20 hours at 37°C. For U-DEL8-HP and U-DEL9-HP, the cleavage reaction was also 100% complete after an additional 1 hour of incubation at 90°C.

[0214] The above results indicate that the partial structures of hairpin-type DEL containing various deoxyuridines undergo an abasic reaction by USER (registered trademark) enzyme at the deoxyuridine site, followed by a cleavage reaction.

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

[0216] As shown in the schematic diagram of Figure 11, the compound (hairpin DEL) having the sequence shown in Table 4 was synthesized by the following procedure. In the sequence notation in Table 4, "S" represents the following formula (7): The 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 The compound names of the starting headpieces used to synthesize each hairpin DEL are as follows: Hairpin DEL: Starting 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 "Seq" of U-DEL1-HP, U-DEL2-HP, U-DEL4-HP, and H-DEL-HP are as shown in Table 5 below.

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

[0218] A PCR tube was charged with 2.0 μL of a 1 mM aqueous solution of each raw material headpiece; 2.4 μL of a 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; the sequence is 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. 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

[0219] The reaction solution was treated with 0.8 μL of 5 M aqueous sodium chloride solution and 17.6 μL of chilled (−20°C) ethanol and incubated 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 a solution.

[0220] To each of the resulting 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; the sequences are shown in Table 7); 0.8 μL of 10× 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 Scientific, 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

[0221] The reaction solution was treated with 0.8 μL of 5 M aqueous sodium chloride solution and 17.6 μL of chilled (−20°C) ethanol and allowed to stand at −78°C for 2 hours. After centrifugation, the supernatant was removed and the resulting pellet was air-dried. 10 μL of deionized water was added to the pellet to make it into a solution.

[0222] A 1.0 μL aliquot of the resulting solution was sampled and diluted with deionized water, and then subjected to mass analysis by ESI-MS under the analytical conditions 2 of Example 1 to identify the target substance (the theoretical molecular weight of each sequence and the detected molecular weight are shown in Table 4). The remaining solution was freeze-dried, and then deionized water was added to each solution to make the concentration 20 μM.

[0223] Of the eight hairpin DELs obtained above, H-DEL is a conventional hairpin DEL, and the remaining seven are cleavable hairpin DELs containing deoxyuridine. Real-time PCR analysis was performed to compare the PCR efficiency of various hairpin DELs before and after treatment with USER (registered trademark) enzyme. In addition, DS-DEL (prepared by annealing the compounds of sequence Nos. 47 and 48) shown in Table 7 was used as the double-stranded DEL for comparison. In addition, in the sequence notation in Table 8, "(amino-C6-L)" is the following formula (8) The other symbols are the same as in Table 1.

[0224] <Treatment Step with USER (registered trademark) enzyme> Treatment of the eight types of hairpin DEL and double-stranded DEL (DS-DEL) with USER (registered trademark) enzyme was carried out in the following procedure.

[0225] To a PCR tube, 1 μL of a 20 μM aqueous solution of each DEL, 1 μL of CutSmart® Buffer (New England BioLabs, catalog number B7204S), and 7 μL of deionized water were added. 1 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 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 value by real-time PCR> The Ct values ​​of the various DEL samples obtained above were measured by real-time PCR to compare PCR efficiencies. The conditions are as follows, and the results are shown in Figure 12. The Ct value refers to the cycle number at which the fluorescent signal generated by DNA amplification reaches a given 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 (Applied Biosystems) Plate: MicroAmp 96-Well Plate (Applied Biosystems, Catalog No. N8010560) PCR reaction solution: TB Green Premix Ex taqII (Takara Bio, Catalog No. 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 DyeII (Takara Bio, Catalog No. 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 molar numbers of the DEL samples were 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°C, 5 seconds, 52°C, 30 seconds, and 72°C, 30 seconds. The sequence 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 (registered trademark) enzyme treatment, but the Ct values ​​of the cleavable hairpin DELs containing deoxyuridine (U-DEL1, U-DEL2, U-DEL4, U-DEL7, U-DEL8, U-DEL9, and U-DEL10) decreased to the same level as that of DS-DEL, a double-stranded DEL, after USER (registered trademark) enzyme treatment.

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

[0231] Example 3 [Verification of cleavage reaction of deoxyuridine-containing hairpin DEL by USER (registered trademark) enzyme] <Synthesis of four types of hairpin DEL (U-DEL5, U-DEL11, U-DEL12, and U-DEL13)> Compounds (hairpin DEL) having the sequences shown in Table 10 were synthesized by the following procedure. In the sequence notation in Table 10, "[mdC(TEG-amino)]" represents the following formula (9): The 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 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 "Seq" of U-DEL11-HP, U-DEL12-HP, and U-DEL13-HP are as shown in Table 11 below. The notations in Table 11 are the same as those in Table 10.

[0232]

[0233] Of the raw material headpieces shown in Table 11, U-DEL12-HP and U-DEL13-HP were prepared using an automatic nucleic acid 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] As in Example 2, two-step double-stranded ligation was carried out using various starting headpieces with the double-stranded oligonucleotide Pr_TAG and CP.

[0235] A portion of the resulting solution was sampled and diluted with deionized water, and then subjected to mass analysis by ESI-MS under the following analysis condition 3 to identify the target substance (the theoretical molecular weight and detected molecular weight of each sequence are shown in Table 10). The remaining solution was freeze-dried, and then deionized water was added to each solution to make a 20 μM solution.

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

[0237] <Cleavage reaction by USER (registered trademark) enzyme> The cleavage reaction of six types of deoxyuridine-containing hairpin DEL (U-DEL5, U-DEL7, U-DEL9, U-DEL11, U-DEL12, and U-DEL13) by USER (registered trademark) enzyme was investigated by the following procedure.

[0238] To a PCR tube, 2 μL of a 20 μM aqueous solution of each hairpin DEL, 2 μL of CutSmart® Buffer (New England BioLabs, catalog number B7204S), and 14 μL of deionized water were added. 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 an additional incubation at 90°C for 1 hour.

[0239] <Confirmation of the product after cleavage by LC-MS measurement> 5.0 μL of the resulting reaction solution was sampled and diluted with deionized water, and then subjected to mass spectrometry by ESI-MS under analysis condition 3. The sequence and theoretical molecular weight of the product after cleavage expected in each reaction solution, as well as the molecular weight detected in each reaction solution, are shown in Table 12. The substrates corresponding to each experiment 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]

[0241] In all samples, MS of the substrate was not detected, and MS of the product after cleavage was observed as the main peak.

[0242] <Confirmation of cleavage reaction by gel electrophoresis> A portion of the obtained reaction solution was sampled and analyzed by denaturing polyacrylamide gel electrophoresis under the conditions shown below. The results shown in Figure 13 confirmed that the cleavage reaction proceeded with high yield for all substrates. The samples in each lane in Figure 13 are as follows: Lane 1: 20 bp DNA ladder (Lonza 20 bp DNA Ladder, 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 Denaturing polyacrylamide gel electrophoresis: Gel: Novex (trademark) 10% TBE-urea gel (Invitrogen by ThermoFisher SCIENTIFIC, catalog number EC68755BOX) Loading buffer: Novex (trademark) 10% TBE-Urea Sample Buffer (2x) (Invitrogen by ThermoFisher SCIENTIFIC, catalog number LC6876) Temperature: 60°C Voltage: 180V Electrophoresis time: 30 minutes Staining reagent: SYBER (trademark) Green II Nucleic Acid Gel Stain (Takara Bio, catalog number 5770A)

[0243] The above results indicate that hairpin-type DEL containing various deoxyuridines undergoes cleavage reaction by USER (registered trademark) enzyme at the deoxyuridine site.

[0244] Example 4 [Verification of cleavage reaction of hairpin DEL containing deoxyinosine by endonuclease V] <Synthesis of hairpin DEL containing four types of deoxyinosine (I-DEL1, I-DEL2, I-DEL3, and I-DEL4)> Compounds (hairpin DEL) having the sequences shown in Table 13 were synthesized by the following procedure. In the sequence notation in Table 13, "I" means deoxyinosine, 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. 73: I-DEL1 No. 74: I-DEL2 No. 75: I-DEL3 No. 76: I-DEL4 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 "Seq" of I-DEL1-HP, I-DEL2-HP, I-DEL3-HP, and I-DEL4-HP are as shown in Table 14 below. The notations in Table 14 are the same as those in Table 13.

[0245]

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

[0247] As in Example 2, two-step double-stranded ligation was carried out using various starting headpieces with the double-stranded oligonucleotide Pr_TAG and CP.

[0248] A portion of the resulting solution was sampled and diluted with deionized water, and then subjected to mass spectrometry by ESI-MS under analysis condition 3 to identify the target substance (the theoretical molecular weight and detected molecular weight of each sequence are shown in Table 13). The remaining solution was freeze-dried, and then deionized water was added to each solution to make the concentration 20 μM.

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

[0250] To a PCR tube, 1 μL of a 20 μM aqueous solution of each hairpin DEL, 2 μL of NEBuffer® 4 (New England BioLabs, catalog number B7004), and 15 μL of deionized water were added. 2 μL of Endnuclease V (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 resulting reaction solution was sampled and diluted with deionized water, and then subjected to mass spectrometry by ESI-MS under analysis condition 3. The sequence and theoretical molecular weight of the product after cleavage expected in each reaction solution, as well as the molecular weight detected in each reaction solution, are shown in Table 15. The substrates corresponding to each experiment 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]

[0253] In all samples, MS of the substrate was not detected, and MS of the product after cleavage 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 denaturing polyacrylamide gel electrophoresis under the same conditions as in Example 3. The results shown in Figure 14 confirmed that the cleavage reaction proceeded with high yield for all substrates. The samples in each lane in Figure 14 are as follows: Lane 1: 20 bp DNA ladder (manufactured by Lonza, Lonza 20 bp DNA Ladder, catalog number 50330) Lane 2: I-DEL1 Lane 3: Sample after the cleavage reaction of I-DEL1 Lane 4: I-DEL2 Lane 5: Sample after the cleavage reaction of I-DEL2 Lane 6: I-DEL3 Lane 7: Sample after the cleavage reaction of I-DEL3 Lane 8: I-DEL4 Lane 9: Sample after the cleavage reaction of I-DEL4

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

[0256] Example 5 [Verification of cleavage reaction of ribonucleoside-containing hairpin DEL by RNase HII] <Synthesis of ribonucleoside-containing hairpin DEL (R-DEL1)> A compound (hairpin DEL) having the sequence shown in Table 16 was synthesized by the following procedure. In the sequence notation in Table 16, "u" means uridine, and other notations are the same as in Table 2. The names of the compounds corresponding to the sequence numbers (No.) are as follows. No. 87: R-DEL1 The compound names of the raw headpieces used to synthesize each hairpin DEL are as follows: Hairpin DEL: Raw 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. The notations in Table 17 are the same as those in Table 16.

[0257]

[0258] The raw material headpieces shown in Table 17 were prepared according to a conventional method.

[0259] As in Example 2, two-step double-stranded ligation was carried out using the starting headpiece with the double-stranded oligonucleotide Pr_TAG and CP.

[0260] A portion of the resulting solution was sampled and diluted with deionized water, and then subjected to mass spectrometry by ESI-MS under analysis condition 3 to identify the target substance (the theoretical molecular weight and detected molecular weight of each sequence are shown in Table 16). The remaining solution was freeze-dried, and then deionized water was added to each solution to make the concentration 200 μM.

[0261] <Cleavage Reaction with RNase HII> The cleavage reaction of ribonucleoside-containing hairpin DEL (R-DEL1) with RNase HII was investigated by the following procedure.

[0262] To a PCR tube was added 0.5 μL of a 200 μM solution of Hairpin DEL in water, 4.9 μL of ThermoPol® Reaction Buffer Pack (New England BioLabs, catalog number B9004), and 43.6 μL of deionized water. 1 μL of RNase HII (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 cleavage products by LC-MS measurement> 10 μL of the resulting reaction solution was sampled and subjected to mass analysis by ESI-MS under analysis condition 3. The sequence and theoretical molecular weight of the expected cleavage product, as well as the detected molecular weight, are shown in Table 18. The substrates corresponding to the experiment numbers (Entry) are as follows, and other notations are the same as in Table 16. Entry 1: R-DEL1

[0264]

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

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

[0267] These results demonstrate that the phosphodiester bond at the 5' end of the ribonucleotide in hairpin-type DEL containing a ribonucleoside is cleaved by RNase HII. Example 6 [Creation of a model library using U-DEL9-HP as a starting material] As shown in the schematic diagram in Figure 16, a model library containing 3 x 3 x 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 building blocks (BB1, BB2, and BB3): 10 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." (leftmost) 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] As shown in Table 19, each double-stranded oligonucleotide tag was prepared by annealing two oligonucleotides of SEQ ID NO:2 corresponding to each tag number.

[0270]

[0271] <Synthesis of Compound "AOP-U-DEL9-HP"> The compound "AOP-U-DEL9-HP" having the sequence shown in Table 20 was synthesized by the following procedure. In the sequence notation in Table 20, "(AOP-AminoC7)" is represented by the following formula (10): The other symbols are the same as in Table 2.

[0272]

[0273] To four Violamo centrifuge tubes was added a solution of U-DEL9-HP (2.5 mL, 1 mM) in sodium borate buffer (150 mM, pH 9.4) cooled to 10°C. To each tube was added 40 equivalents of N-Fmoc-15-amino-4,7,10,13-tetraoxaoctadecanoic acid (250 μL, 0.4 M in N,N-dimethylacetamide), followed by 40 equivalents of 4-(4,6-dimethoxy[1.3.5]triazin-2-yl)-4-methylmorpholinium chloride hydrate (DMTMM) (200 μL, 0.5 M in water), and the resulting solution was shaken at 10°C for 5 hours.

[0274] The above solutions were each treated with 295 μL of 5 M aqueous sodium chloride solution and 9.7 mL of chilled (-20°C) ethanol and allowed to stand overnight at -78°C. After centrifugation, the supernatant was removed and the resulting pellets were air-dried. Each pellet was dissolved in 2.75 mL of deionized water, and 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 washed twice with 1.47 mL of deionized water. The resulting filtrate was each treated with 600 μL of 5 M aqueous sodium chloride solution and 19.8 mL of chilled (-20°C) ethanol and allowed to stand overnight at -78°C. After centrifugation, the supernatant was removed and the resulting pellets were air-dried.

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

[0276] <Introduction of double-stranded oligonucleotide tag "Pr"> The compound "AOP-U-DEL9-HP" and the double-stranded oligonucleotide tag "Pr" were ligated to synthesize the compound "AOP-U-DEL9-HP-Pr" having the sequence shown in Table 21 by the following procedure. The sequence notation in Table 21 is the same as in Table 20.

[0277]

[0278] A Violamo centrifuge tube was charged with 40 μL of a 5 mM aqueous solution of the compound "AOP-U-DEL9-HP," 160 μL of a 100 mM aqueous solution of sodium bicarbonate, 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. To the solution was added 8.0 μL of T4 DNA ligase (Thermo Fisher Scientific, catalog number EL0013), 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 chilled (-20°C) ethanol and incubated 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 subjected to ESI-MS mass spectrometry under analytical condition 2 to identify the target compound (the theoretical molecular weight and detected molecular weight of the compound are shown in Table 21). Through these steps, 133 nmol of the compound "AOP-U-DEL9-HP-Pr" with a purity of 84.5% was obtained. A 100 mM aqueous sodium bicarbonate solution was added to the resulting compound "AOP-U-DEL9-HP-Pr" to adjust the concentration to 1 mM.

[0280] Cycle A: To each of 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 to A3, 8.0 μL of 10× 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 Scientific, 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 each 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 to BB3 (4.0 μL, 200 mM N,N-dimethylacetamide solution) was added, followed by 40 equivalents of 4-(4,6-dimethoxy[1.3.5]triazin-2-yl)-4-methylmorpholinium chloride hydrate (DMTMM) (4.0 μL, 200 mM aqueous solution), and the mixture was shaken for 2 hours at 10° C. Furthermore, 20 equivalents of a building block (2.0 μL, 200 mM N,N-dimethylacetamide solution) was added to each tube, followed by 20 equivalents of DMTMM (2.0 μL, 200 mM aqueous solution), 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, after which the three solutions were mixed in one PCR tube.

[0284] To the mixed solution, 6.0 μL of piperidine was added at 0°C, and the mixture was 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. This solution was used as the starting material for the next step.

[0285] Cycle B: To each of three PCR tubes was added 13.7 μL of a 1 mM solution of the starting material obtained in Cycle A, 20.6 μL of a 1 mM aqueous solution of one of the double-stranded oligonucleotide tags B1 to B3, 5.5 μL of 10× 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 Scientific, catalog number EL0013) was added, 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 each dissolved in 13.7 μL of 150 mM sodium borate buffer (pH 9.4).

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

[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, and the three solutions were then mixed in one PCR tube.

[0289] To the mixed solution, 4.1 μL of piperidine was added at 0°C, and the mixture was 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. This was used as the starting material for the next step.

[0290] Cycle C: To each of three PCR tubes was added 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 to C3, and 2.8 μL of 10× 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 Scientific, catalog number EL0013) was added, 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 each dissolved in 7.0 μL of 150 mM sodium borate buffer (pH 9.4).

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

[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, and the three solutions were then mixed in one PCR tube.

[0294] To the mixed solution, 2.1 μL of piperidine was added at 0°C, and the mixture was 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. This was used as the starting material for the next step.

[0295] <CP Ligation> A PCR tube was charged with 12.2 μL of a 0.41 mM solution of the starting material obtained in cycle C, 6.0 μL of a 1 mM aqueous solution of CP (the same as that used in Example 2), 2.1 μL of 10× 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. 0.1 μL of T4 DNA ligase (Thermo Fisher Scientific, 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 adjust the concentration to 20 μM.

[0297] <Results> Samples obtained after ligation of the double-stranded oligonucleotide tags in each cycle were analyzed by electrophoresis using a 2.2% agarose gel (Lonza, FlashGel® Cassette, Catalog No. 57031). The results shown in Figure 17 confirmed that encoding by the double-stranded oligonucleotide tags was achieved with high efficiency in each cycle. The samples in each lane in Figure 17 are as follows: Lane 1: AOP-U-DEL9-HP-Pr; Lane 2: Sample after ligation of double-stranded oligonucleotide tag A1 in cycle A; Lane 3: Sample after ligation of double-stranded oligonucleotide tag A2 in cycle A; Lane 4: Sample after ligation of double-stranded oligonucleotide tag A3 in cycle A; Lane 5: Sample after ligation of double-stranded oligonucleotide tag B1 in cycle B; Lane 6: Sample after ligation of double-stranded oligonucleotide tag B2 in cycle B; Lane 7: Sample after ligation of double-stranded oligonucleotide tag B3 in cycle B; Lane 8: Sample after ligation of double-stranded oligonucleotide tag C1 in cycle C; Lane 9: Sample after ligation of double-stranded oligonucleotide tag C2 in cycle C; Lane 10: Sample after ligation of double-stranded oligonucleotide tag C3 in cycle C; Lane 11: Sample after ligation of CP; Lane 12: 20 bp DNA ladder (Lonza 20 bp DNA) Ladder, Catalog No. 50330)

[0298] The sample after cycle C was analyzed under analysis condition 3. Figure 18 shows the chromatograph and mass spectrum results. Deconvolution of the obtained mass spectrum revealed an average molecular weight of 35,532.4. This result is consistent with the average molecular weight expected after cycle C (35,514.2), indicating that the library synthesis reactions (ligation of double-stranded oligonucleotide tags and introduction of building blocks) were achieved with high efficiency.

[0299] As a result, a model library containing 3×3×3 (27) compound species was synthesized using U-DEL9-HP as a raw material according to the above synthesis procedure.

[0300] <Cleavage of Obtained Model Library with USER (registered trademark) enzyme> The cleavage reaction of the above-obtained model library with USER (registered trademark) enzyme was carried out according to the following procedure.

[0301] To a PCR tube, 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. 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 an additional incubation at 90°C for 1 hour.

[0302] A portion of the resulting reaction solution was sampled and analyzed by denaturing polyacrylamide gel electrophoresis under the same conditions as in Example 3. The results shown in Figure 19 confirmed that the model library, which was made from U-DEL9-HP as the starting material, underwent a highly efficient cleavage reaction with USER (registered trademark) enzyme. The samples in each lane in Figure 19 are as follows: Lane 1: 20 bp DNA ladder (manufactured by Lonza, Lonza 20 bp DNA Ladder, catalog number 50330) Lane 2: Model library Lane 3: Sample of the model library after cleavage reaction with USER (registered trademark) enzyme

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

[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), and subsequently 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (72 μL, 0.1 M dimethyl sulfoxide solution) were added to each of eight PCR tubes, and the resulting solutions were shaken at 30°C for 30 minutes. To each solution, 150 μL of triethylamine hydrochloride buffer (500 mM, pH 10) was added, followed by an aqueous solution of AOP-U-DEL9-HP (synthesized in Example 6) (75 μL, 0.67 mM), and the mixture was shaken at 37° C. for 6 hours.

[0305] The reaction solution was combined into one Violamo centrifuge tube, treated with 800 μL of 5 M aqueous sodium chloride 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 binary mobile phase gradient profile with 50 mM triethylammonium acetate buffer (pH 7.5) and acetonitrile / water (100:1, v / v). Fractions containing the target product were collected, mixed, and concentrated. The resulting solution was desalted using an Amicon® Ultra Centrifugal filter (3 kD cutoff), precipitated with ethanol, and then the pellet was diluted with deionized water to a 1 mM aqueous solution.

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

[0307] <Synthesis of raw material head pieces of three types of DEL compounds (SABA-DEL-HP, ClSABA-DEL-HP, mSABA-DEL-HP)> The compounds of the sequences shown in Table 23 were synthesized by the following procedure. In the sequence notation in Table 23, "(SABA-AOP-AminoC7)" is represented by the following formula (12): "(ClSABA-AOP-AminoC7)" means a group represented by the following formula (13): "(mSABA-AOP-AminoC7)" means a group represented by the following formula (14): 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. 114: SABA-DEL-HP No. 115: ClSABA-DEL-HP No. 116: mSABA-DEL-HP The raw carboxylic acids used to synthesize each compound are as follows: Compound: Raw carboxylic acid SABA-DEL-HP: 4-sulfamoylbenzoic acid ClSABA-DEL-HP: 4-chloro-3-sulfamoylbenzoic acid mSABA-DEL-HP: 3-sulfamoylbenzoic acid

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

[0309] Each of the above solutions was treated with 20 μL of 5 M aqueous sodium chloride solution and 660 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 pellet was 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. The target product was eluted using a binary mobile phase gradient profile with 50 mM triethylammonium acetate buffer (pH 7.5) and acetonitrile / water (100:1, v / v). Fractions containing the target product were collected, mixed, and concentrated. Each resulting solution was desalted using an Amicon® Ultra Centrifugal filter (3 kD cutoff), precipitated with ethanol, and then the pellet was diluted with deionized water to a 1 mM aqueous solution.

[0310] A portion of the obtained solution was sampled and diluted with deionized water, and then subjected to mass analysis by ESI-MS under the analytical conditions 2 of 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 having biotin at the 3' end ("AAZ-BIO-DEL", "SABA-BIO-DEL", "ClSABA-BIO-DEL", "mSABA-BIO-DEL", and "Amino-BIO-DEL")> The DEL compounds having the sequences shown in Table 24 were synthesized by the following procedure. In the sequence notation in Table 24, "(BIO)" represents the following formula (15): The 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 The 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] A PCR tube was charged with 10 μL of a 1 mM aqueous solution of each raw material headpiece; 12 μL of a 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 sequence is 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. 4 μL of a 10-fold diluted aqueous solution of T4 DNA ligase (Thermo Fisher Scientific, 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 each sequence number (No.) are as follows: No. 122: Pr_TAG2_CP_a No. 123: Pr_TAG2_CP-BIO_b

[0313] The reaction solution was treated with 4 μL of 5 M aqueous sodium chloride solution and 132 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 pellet was air-dried and 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 and diluted with deionized water, and then subjected to mass spectrometry by ESI-MS under the analytical conditions 3 of Example 3 to identify each target compound (the theoretical molecular weight and detected molecular weight of each compound are shown in Table 24).

[0315] <Cleavage of five 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> The five DEL compounds obtained above, "AAZ-BIO-DEL", "SABA-BIO-DEL", "ClSABA-BIO-DEL", "mSABA-BIO-DEL", and "Amino-BIO-DEL", were subjected to cleavage reaction with USER (registered trademark) enzyme according to the following procedure, and converted into the DEL compounds "DS-AAZ-BIO-DEL", "DS-SABA-BIO-DEL", "DS-ClSABA-BIO-DEL", "DS-mSABA-BIO-DEL", and "DS-Amino-BIO-DEL", respectively, having double-stranded nucleic acids having the sequences shown in Table 26. The sequence notations in Table 26 are the same as those in Table 24, and each of the five compounds is formed by a double strand of oligonucleotide chains of SEQ ID NO: 124 and SEQ ID NO: 125, SEQ ID NO: 126 and SEQ ID NO: 127, SEQ ID NO: 128 and SEQ ID NO: 129, SEQ ID NO: 130 and SEQ ID NO: 131, and SEQ ID NO: 132 and SEQ ID NO: 133.

[0316]

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

[0318] The resulting reaction solutions were each desalted and concentrated using an Amicon (registered trademark) Ultra Centrifugal filter (3 kD cutoff), and then precipitated with ethanol. Deionized water was added to each of the resulting pellets to prepare an aqueous solution.

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

[0320] In addition, a portion of the obtained reaction solution was sampled and analyzed by denatured polyacrylamide gel electrophoresis under the same conditions as in Example 3. From the results shown in Figure 20, "AAZ-BIO-DEL", "SABA-BIO-DEL", "ClSABA-BIO-DEL", "mSABA-BIO-DEL", and "Amino-BIO-DEL" were cleaved with a high yield and respectively converted to "DS-AAZ-BIO-DEL", "DS-SABA-BIO-DEL", "DS-ClSABA-BIO-DEL", "DS-mSABA-BIO-DEL", and "DS-Amino-BIO-DEL". The samples in each lane of Figure 20 are as follows. Furthermore, concentration 1 and concentration 2 mean 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 20 bp DNA Ladder, catalog number 50330) Lane 2: AAZ-BIO-DEL (concentration 1) Lane 3: AAZ-BIO-DEL (concentration 2) Lane 4: AAZ-BIO-DEL sample after cleavage reaction with USER (registered trademark) enzyme (concentration 1) Lane 5: AAZ-BIO-DEL sample after cleavage reaction with USER (registered trademark) enzyme (concentration 2) Lane 6: SABA-BIO-DEL (concentration 1) Lane 7: SABA-BIO-DEL (concentration 2) Lane 8: SABA-BIO-DEL sample after cleavage reaction with USER (registered trademark) enzyme (concentration 1) Lane 9: Sample after cleavage reaction with SABA-BIO-DEL USER (registered trademark) enzyme (concentration 2) Lane 10: ClSABA-BIO-DEL (concentration 1) Lane 11: ClSABA-BIO-DEL (concentration 2) Lane 12: Sample after cleavage reaction with ClSABA-BIO-DEL USER (registered trademark) enzyme (concentration 1) Lane 13: Sample after cleavage reaction with ClSABA-BIO-DEL USER (registered trademark) 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 USER (registered trademark) enzyme (concentration 1) Lane 17: mSABA-BIO-DEL sample after cleavage reaction with USER (registered trademark) enzyme (concentration 2) Lane 18: Amino-BIO-DEL (concentration 1) Lane 19: Amino-BIO-DEL (concentration 2) Lane 20: Amino-BIO-DEL sample after cleavage reaction with USER (registered trademark) enzyme (concentration 1) Lane 21: Amino-BIO-DEL sample after cleavage reaction with USER (registered trademark) enzyme (concentration 2) Lane 22: 20 bp DNA ladder (Lonza, Lonza 20 bp DNA Ladder, catalog number 50330).

[0321] <Preparation of DEL having single-stranded DNA using streptavidin beads> The DEL compounds having double-stranded nucleic acids obtained above, "DS-AAZ-BIO-DEL", "DS-SABA-BIO-DEL", "DS-ClSABA-BIO-DEL", "DS-mSABA-BIO-DEL", and "DS-Amino-BIO-DEL", were each treated with streptavidin beads, and the DEL compounds having single-stranded DNA, "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] To each of five PCR tubes, 450 μL of Magnosphere (trademark) MS160 / Streptavidin (JSR Life Sciences, catalog number J-MS-S160S) was added, and the supernatant was removed by magnetic separation. 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 Tween 20) was added, and the supernatant was removed by magnetic separation. To the obtained particles, 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 Tween 20) were added, mixed, and shaken at room temperature for 20 minutes.

[0323] The supernatant was removed from each mixture by magnetic separation, and the particles were washed twice 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 Tween 20) and the supernatant was removed by magnetic separation. Then, 900 μL of denaturing solution (0.1 M sodium hydroxide; 0.1 M sodium chloride) was added to each mixture, and the supernatant was recovered by magnetic separation.

[0324] To each supernatant, 900 μL of 3-(N-morpholino)propanesulfonic acid buffer (1.0 M, pH 7.0) was added, and the mixture was desalted using an Amicon® Ultra Centrifugal filter (3 kD cutoff). Each supernatant was subjected to ethanol precipitation, and the pellet was dissolved in deionized water.

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

[0326] <Synthesis of photoreactive crosslinker-modified primer> Photoreactive crosslinker-modified primer "PXL-Pr" with the sequence shown in Table 27 was synthesized by the following procedure. In the sequence notation in Table 27, "X" represents the following formula (16): The other symbols are the same as in Table 2.

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

[0328] The reaction mixture was treated with 23.6 μL of 5 M aqueous sodium chloride solution and 778.8 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. 180 μL of deionized water was added to the pellet to form a solution, after which 20 μL of piperidine was added and the mixture was shaken at 10°C for 3 hours.

[0329] The resulting 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 for 30 minutes at −78° C. 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) and subsequently 50 equivalents of sodium 1-((3-(3-methyl-3H-diazirin-3-yl)propanoyl)oxy)-2,5-dioxopyrrolidine-3-sulfonate (Sulfo-SDA) (25 μL, 200 mM aqueous solution) were added, and the mixture was shaken at 37° C. for 2 hours.

[0331] The resulting 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 at −78°C for 30 minutes. After centrifugation, the supernatant was removed, and 100 μL of deionized water was added to the resulting pellet. Subsequently, 75 μL of triethylamine hydrochloride buffer (500 mM, pH 10) and 50 equivalents of Sulfo-SDA (25 μL, 200 mM aqueous solution) were added, and the mixture was shaken at 37°C for 1 hour and 20 minutes. An additional 50 equivalents of Sulfo-SDA (25 μL, 200 mM aqueous solution) was added, and the mixture was shaken at 37°C for 40 minutes.

[0332] The resulting solution was treated with 22.5 μL of 5 M aqueous sodium chloride solution and 743 μL of chilled (−20° C.) ethanol and allowed 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 50 equivalents of Sulfo-SDA (25 μL, 200 mM aqueous solution), and the mixture was shaken at 37° C. for 3 hours.

[0333] The resulting 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 product was eluted using a binary mobile phase gradient profile with 50 mM triethylammonium acetate buffer (pH 7.5) and acetonitrile / water (100:1, v / v). Fractions containing the target product were collected, mixed, and concentrated. The resulting solution was desalted using an Amicon® Ultra Centrifugal filter (3 kD cutoff), precipitated with ethanol, and then the pellet was dissolved in 100 μL of deionized water.

[0334] A portion of the obtained solution was sampled and diluted with deionized water, and then subjected to mass spectrometry by ESI-MS under the analytical conditions 3 of Example 3, to identify the target 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 DEL> The DEL compounds having the single-stranded DNA obtained above ("SS-AAZ-DEL", "SS-SABA-DEL", "SS-ClSABA-DEL", "SS-mSABA-DEL", and "SS-Amino-DEL") were used as template DNA, and a primer extension reaction using each "PXL-Pr" was carried out 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") having the sequences shown in Table 29. The sequence notations in Table 29 are the same as those in Tables 26 and 27, and mean that the five compounds are formed by double strands of 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.

[0336] To a PCR tube were added 30 μL of a 10 μM aqueous solution of DEL compound with each type of single-stranded DNA; 0.505 μL of a 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. 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 resulting solution was desalted using an Amicon® Ultra Centrifugal filter (3 kD cutoff). Deionized water was added to the resulting supernatant to make a 60 μL solution, which was then treated with 6 μL of 5 M aqueous sodium chloride solution and 198 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 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 and diluted with deionized water, and then subjected to mass spectrometry by ESI-MS under the analytical conditions 3 of Example 3. The target photoreactive crosslinker-modified double-stranded DEL "PXL-DS-AAZ-DEL", "PXL-DS-SABA-DEL", "PXL-DS-ClSABA-DEL", "PXL-DS-mSABA-DEL", and "PXL-DS-Amino-DEL" were identified (the theoretical molecular weight of the compound and the detected molecular weight are shown in Table 29).

[0339] In addition, a portion of the obtained reaction solution was sampled and analyzed by polyacrylamide gel electrophoresis under the conditions shown below. From the results shown in Figure 21, it was confirmed that the primer extension reaction resulted in high yields of conversion to "PXL-DS-AAZ-DEL", "PXL-DS-SABA-DEL", "PXL-DS-ClSABA-DEL", "PXL-DS-mSABA-DEL", and "PXL-DS-Amino-DEL". The samples in each lane of Figure 21 are as follows. Furthermore, concentration 1 and concentration 2 mean 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, Lonza 20 bp DNA Ladder, 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, Lonza 20 bp DNA Ladder, 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 (Lonza) 20 bp DNA Ladder, Catalog No. 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 (Fujifilm Wako Pure Chemical Industries, Ltd., catalog number 190-15481) Loading buffer: 6x Loading Buffer (Takara Bio Inc., catalog number 9156) Temperature: room temperature Voltage: 200 V Electrophoresis time: 50 minutes Staining reagent: SYBER (trademark) Green II Nucleic Acid Gel Stain (Takara Bio Inc., catalog number 5770A)

[0341] Example 8 [Comparison of binder recovery efficiency of photoreactive crosslinker-modified double-stranded DEL with and without photocrosslinking reaction] <Preparation of DEL samples> Each of the five types of photoreactive crosslinker-modified double-stranded DEL obtained above was diluted with deionized water to prepare 50 nM DEL samples.

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

[0343] <Recovery of DEL cross-linked to protein> Dynabeads Histag isolation & pulldown (Invitrogen, catalog number 10104D): 10.0 μL Tween 20 (Sigma, catalog number P7949-100ML) 10% SDS (NIPPON GENE, catalog number 311-90271) Wash buffer (D-PBS(-) diluted with Tween 20 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 fixed to a magnetic stand and left to stand for 2 minutes, after which the supernatant was removed and 200 μL of wash buffer was added to suspend the Dynabeads. This washing procedure was repeated five times. 80 μL of D-PBS(-) was added to the washed Dynabeads and reacted at 95°C for 10 minutes. After the reaction, the Dynabeads were placed on a magnetic stand, and the supernatant was collected after 2 minutes (Solution E).

[0345] <Preparation of Samples Not Subjected to Photocrosslinking Reaction> The above series of operations was carried out without UV irradiation to prepare solutions S and E as samples.

[0346] <Measurement of Ct Values ​​by Real-Time PCR> The Ct values ​​of the various DEL samples obtained above were measured by real-time PCR to compare PCR efficiencies. The conditions were as follows, and the results are shown in Figure 22.

[0347] Apparatus: 7500 Real-Time PCR System (Applied Biosystems) Plate: MicroAmp 96-Well Plate (Applied Biosystems, Catalog No. N8010560) PCR reaction solution: TaqMan Gene Expression Master Mix (Applied Biosystems, Catalog No. 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 TaqMan (registered trademark) probe labeled with FAM (registered trademark) fluorescent dye at the 5' end and NFQ and MGB at the 3' end of the base sequence of SEQ ID NO: 139 in Table 30 (manufactured by Fisher, product number 4316034): 0.50 μL Aqueous solutions of various DEL samples (S and E samples): 2.0 μL Deionized water: 5.5 μL Temperature conditions: After holding at 50°C for 2 minutes, the following cycle was repeated 40 times: 95°C, 15 seconds 59°C, 1 minute

[0348] The strength of the affinity between CA9 protein and each compound as a binding agent 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 of Figure 22, in solution E without UV irradiation, "PXL-DS-AAZ-DEL" and "PXL-DS-SABA-DEL" having high affinity binders showed a significant decrease in Ct value compared to the negative control. However, "PXL-DS-ClSABA-DEL" and "PXL-DS-mSABA-DEL" having medium affinity binders showed no significant change in Ct value compared to the negative control. This result suggests that in DEL screening, if a photocrosslinking reaction is not performed, it is expected that a high affinity binder can be obtained, but it is difficult to obtain a medium affinity binder.

[0350] On the other hand, in Solution E, which was irradiated with UV light, the Ct values ​​of all compounds were significantly lower than those of the negative control. This suggests that photocrosslinking in DEL screening can be used to obtain binders with moderate affinity.

[0351] These results indicate that photoreactive crosslinker-modified double-stranded DEL derived from hairpin-type DEL having a "selectively cleavable site" is useful in DEL screening using photocrosslinking reactions.

[0352] Example 9 [Preparation of DEL Having Single-Stranded DNA Using Lambda Exonuclease] The DEL compound "DS-Amino-BIO-DEL" having double-stranded nucleic acid was converted to the DEL compound "SS-Amino-DEL" having single-stranded DNA by treatment with Lambda Exonuclease according to the following procedure.

[0353] To a PCR tube, an aqueous solution of DS-Amino-BIO-DEL (500 pmol), 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, and then deionized water was added to bring the total volume to 50 μL. The resulting solution was incubated at 37°C for 30 minutes.

[0354] A 10 μL aliquot of the resulting reaction solution was sampled and subjected to mass spectrometry by ESI-MS under the analytical conditions 3 of Example 3. 24024.9 was observed, identifying the DEL compound "SS-Amino-DEL" containing the desired single-stranded DNA.

[0355] MS of one of the oligonucleotide strands (SEQ ID NO: 132) contained 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 reaction to form single strands proceeded with high yield.

[0356] Example 10 [Synthesis of photo-crosslinker-modified double-stranded DEL having a different linker structure from the photoreactive crosslinker-modified double-stranded DEL used in Example 8] <Preparation of DEL having five types of single-stranded DNA>

[0357] Similarly to Example 7, five types of DEL compounds having single-stranded DNA ("SS-AAZ-DEL", "SS-SABA-DEL", "SS-ClSABA-DEL", "SS-mSABA-DEL", and "SS-Amino-DEL" were prepared. However, in the step of <Synthesis of five types of DEL compounds having biotin at the 3' end> ​​described in Example 7, Pr_TAG2_CP (similar to Example 1) was used instead of Pr_TAG2_CP-BIO. Similarly, Pr_TAG2_CP_a and Pr_TAG2_CP_b were synthesized and annealed to prepare DEL having single-stranded DNA (sequences shown in Table 31). Instead of the step <Preparation of DEL having single-stranded DNA using streptavidin beads>, DEL having single-stranded DNA was prepared in the same manner as in Example 9. 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

[0358] <Synthesis of photoreactive crosslinker-modified primer "PXL-Pr2"> The photoreactive crosslinker-modified primer "PXL-Pr2" having the sequence shown in Table 32 was synthesized by the following procedure. In the sequence notation in Table 32, "(X2)" represents the following formula (17): The other symbols are the same as in Table 2.

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

[0360] The solution was treated with 11 μL of 5 M aqueous sodium chloride solution and 363 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 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 and diluted with deionized water, and then subjected to mass spectrometry by ESI-MS under the analytical conditions 3 of Example 3, to identify the target photoreactive crosslinker-modified primer "PXL-Pr2" (the theoretical molecular weight and detected molecular weight of each compound are shown in Table 32).

[0362] <Synthesis of photoreactive crosslinker-modified double-stranded DEL> The DEL compounds having the single-stranded DNA obtained above ("SS-AAZ-DEL", "SS-SABA-DEL", "SS-ClSABA-DEL", "SS-mSABA-DEL", and "SS-Amino-DEL") were used as template DNA, and the primer extension reaction using "PXL-Pr2" was carried out in the same manner as in Example 7, to synthesize the photoreactive crosslinker-modified double-stranded DEL compounds ("PXL-DS-AAZ-DEL2", "PXL-DS-SABA-DEL2", "PXL-DS-ClSABA-DEL2", "PXL-DS-mSABA-DEL2", and "PXL-DS-Amino-DEL2") of the sequences shown in Table 33. The sequence notations in Table 33 are the same as those in Tables 26 and 32, and mean that the five compounds are formed by double strands of 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.

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

[0364] In addition, a portion of the obtained reaction solution was sampled and analyzed by polyacrylamide gel electrophoresis under the conditions shown in Example 7. From the results shown in Figure 23, it was confirmed that the primer extension reaction resulted in high yields of conversion to "PXL-DS-AAZ-DEL2", "PXL-DS-SABA-DEL2", "PXL-DS-ClSABA-DEL2", "PXL-DS-mSABA-DEL2", and "PXL-DS-Amino-DEL2". The samples in each lane of Figure 23 are as follows. Samples were prepared so that the loading amount of each DEL compound was approximately 40 ng. Lane 1: 20 bp DNA ladder (Lonza 20 bp DNA Ladder, 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 20 bp DNA Ladder, catalog number 50330)

[0365] <Preparation of DEL compound "SS-Amino-DEL3" having single-stranded DNA> DEL compound "SS-Amino-DEL3" having single-stranded DNA (sequence shown in Table 34) was prepared using an automated nucleic acid synthesizer nS-8II (manufactured by Gene Design) in the same manner as in Example 1. Note that "SS-Amino-DEL3" has the same structure as the oligonucleotide derived by the same procedure as in <Preparation of DEL having five types of single-stranded DNA> above, using "U-DEL12-HP" (sequence shown in Table 11) as the raw material headpiece. The sequence notation in Table 34 is the same as in Table 10.

[0366] <Synthesis of DEL compound "SS-AAZ-DEL3" having single-stranded DNA> DEL compound "SS-AAZ-DEL3" having single-stranded DNA of the sequence shown in Table 35 was synthesized by the following procedure. In the sequence notation in Table 35, "[AAZ-mdC(TEG-amino)]" is the following formula (18): The other symbols are the same as in Table 10.

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

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

[0369] <Synthesis of three types of DEL compounds having single-stranded DNA ("SS-SABA-DEL3", "SS-ClSABA-DEL3", and "SS-mSABA-DEL3")> Three types of DEL compounds having single-stranded DNA ("SS-SABA-DEL3", "SS-ClSABA-DEL3", and "SS-mSABA-DEL3") having the sequences shown in Table 36 were synthesized by the following procedure. In addition, in the sequence notation in Table 36, "[SABA-mdC (TEG-amino)]" is the following formula (19) "[ClSABA-mdC(TEG-amino)]" means a group represented by the following formula (20): "mSABA-mdC(TEG-amino)]" means a group represented by the following formula (21): The 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 The raw carboxylic acids used to synthesize each compound are as follows: Compound: Raw carboxylic acid SABA-DEL-HP: 4-sulfamoylbenzoic acid ClSABA-DEL-HP: 4-chloro-3-sulfamoylbenzoic acid mSABA-DEL-HP: 3-sulfamoylbenzoic acid

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

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

[0372] The resulting 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 binary mobile phase gradient profile, 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). Fractions containing the target product were collected, mixed, and concentrated. The resulting solution was desalted using an Amicon® Ultra Centrifugal filter (3 kD cutoff), precipitated with ethanol, and then the pellet was dissolved in deionized water.

[0373] A portion of the obtained solution was sampled and diluted with deionized water, and then subjected to mass spectrometry by ESI-MS under the analytical conditions 3 of Example 3. The target "SS-SABA-DEL3", "SS-ClSABA-DEL3", and "SS-mSABA-DEL3" were identified (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"> Photoreactive crosslinker-modified primer "PXL-Pr3" having the sequence shown in Table 37 was synthesized using the same procedure as in <Synthesis of photoreactive crosslinker-modified primer "PXL-Pr2"> above. However, instead of "L-Pr", "L-Pr3" (synthesized in the same manner as in Example 1, sequence shown in Table 38) was used as the starting material. The sequence notation in Table 37 is the same as in Table 32. The sequence notation in Table 38 is the same as in Table 8.

[0375]

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

[0377] <Synthesis of five types of photoreactive crosslinker-modified double-stranded DEL "PXL-DS-DEL3"> The DEL compounds having the single-stranded DNA obtained above ("SS-AAZ-DEL3", "SS-SABA-DEL3", "SS-ClSABA-DEL3", "SS-mSABA-DEL3", and "SS-Amino-DEL3") were used as template DNA, and the primer extension reaction using "PXL-Pr3" was carried out in the same manner as in Example 7, to synthesize five types of photoreactive crosslinker-modified double-stranded DEL compounds ("PXL-DS-AAZ-DEL3", "PXL-DS-SABA-DEL3", "PXL-DS-ClSABA-DEL3", "PXL-DS-mSABA-DEL3", and "PXL-DS-Amino-DEL3") having the sequences shown in Table 39. The sequence notations in Table 39 are the same as those in Tables 34 to 37, and mean that the five compounds are formed by double strands of 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.

[0378] A portion of the obtained solution was sampled and diluted with deionized water, and then subjected to mass spectrometry by ESI-MS under the analytical conditions 3 of Example 3. The target photoreactive crosslinker-modified double-stranded 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 (the theoretical molecular weights of the compounds and the detected molecular weights are shown in Table 39).

[0379] In addition, a portion of the obtained reaction solution was sampled and analyzed by polyacrylamide gel electrophoresis under the conditions shown in Example 7. From the results shown in Figure 24, it was confirmed that the primer extension reaction resulted in high yields of conversion to "PXL-DS-AAZ-DEL3", "PXL-DS-SABA-DEL3", "PXL-DS-ClSABA-DEL3", "PXL-DS-mSABA-DEL3", and "PXL-DS-Amino-DEL3". The samples in each lane of Figure 24 are as follows. Samples were prepared so that the loading amount of each DEL compound was approximately 40 ng. Lane 1: 20 bp DNA ladder (Lonza 20 bp DNA Ladder, 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 20 bp DNA Ladder, catalog number 50330)

[0380] Example 11 [Comparison of binder recovery efficiency between the presence or absence of photocrosslinking reaction of photoreactive crosslinker modified double-stranded DEL synthesized in Example 10] <Preparation of DEL sample> Four kinds of photoreactive crosslinker modified double-stranded DEL (PXL-DS-mSABA-DEL2, PXL-DS-Amino-DEL2, PXL-DS-mSABA-DEL3 and PXL-DS-Amino-DEL3) obtained in Example 10 were diluted with deionized water to prepare 50 nM DEL sample.

[0381] <Photocrosslinking reaction> Apparatus: CL-1000 Ultraviolet Crosslinker (manufactured by UVP, INC.) Microtube: 1.5 mL siliconized microtube, round bottom (manufactured by Watson Co., Ltd., catalog number 131-615CH) Reaction tube: 96-well bottom vial (manufactured by Techno Labs. 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 (Sino Biological, Catalog No. 10107-H08H): 10.0 μL Aqueous solutions of various DEL samples (50 nM): 1.0 μL Reaction conditions: The CA9 protein and DEL solution of the above composition were mixed in a microtube and reacted on ice for 1 hour. The entire volume was dispensed into reaction tubes, kept on ice, and irradiated with 365 nm UV for 20 minutes.

[0382] <Recovery of DEL cross-linked to protein> Dynabeads Histag isolation & pulldown (Invitrogen, catalog number 10104D): 10.0 μL Tween 20 (Sigma, catalog number P7949-100ML) 10% SDS (NIPPON GENE, catalog number 311-90271) Wash buffer (D-PBS(-) diluted with Tween 20 and 10% SDS to prepare 0.2%) Imidazole (FUJIFILM, catalog number 097-05391) Elution Buffer (Imidazole was dissolved in D-PBS(-) to a concentration of 200 mM, and the pH was adjusted to 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 mixture was fixed to a magnetic stand and left to stand for 2 minutes, after which the supernatant was removed and 200 μL of wash buffer was added to suspend the Dynabeads. This procedure was repeated five times. 100 μL of elution buffer was added to the washed Dynabeads and left to stand at room temperature for 10 minutes. After the reaction, the Dynabeads were placed on a magnetic stand, and after 2 minutes the supernatant was collected as a sample.

[0384] <Preparation of Samples Not Subjected to Photocrosslinking Reaction> The above series of operations was carried out without UV irradiation, and each sample was collected.

[0385] <Measurement of Ct Values ​​by Real-Time PCR> The Ct values ​​of the various DEL samples obtained above were measured by real-time PCR in the same manner as in Example 8. The results of comparing 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 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 of Example 8, it is difficult to obtain a binder with moderate affinity in DEL screening without performing a photocrosslinking reaction.

[0388] On the other hand, the ΔCt values ​​of the UV-irradiated samples were all elevated compared to the UV-irradiated samples, suggesting 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 used to obtain a binder with moderate affinity.

[0389] These results suggest that photoreactive crosslinker-modified double-stranded DELs with various linker structures derived from hairpin-type DELs with "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 having 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 with no covalent bond between the crosslinker and the 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 using "PXL-Pr3" was carried out according to the following procedure, and four types of photoreactive crosslinker-modified double-stranded DEL compounds ("PXL-DS-SABA-DEL4", "PXL-DS-ClSABA-DEL4", "PXL-DS-mSABA-DEL4", and "PXL-DS-Amino-DEL4") with the sequences shown in Table 40 were synthesized. The sequence notations in Table 40 are the same as those in Tables 34, 36, and 37, and mean that the four compounds are formed by double strands of oligonucleotide chains 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, respectively.

[0392] To a PCR tube, 30 μL of a 10 μM aqueous solution of DEL compounds containing various single-stranded DNAs and 3.77 μL of a 159 μM aqueous solution of "PXL-Pr3" were added. Dehydrated water was added to the resulting solution to bring the total volume to 60 μL. The solution was then incubated at 90°C for 2 minutes and then cooled to room temperature over 30 minutes.

[0393] <Preparation of DEL sample> The four types of "photoreactive crosslinker-modified double-stranded DEL without a covalent bond between the crosslinker and the coding sequence" obtained above ("PXL-DS-SABA-DEL4", "PXL-DS-ClSABA-DEL4", "PXL-DS-mSABA-DEL4", and "PXL-DS-Amino-DEL4") and the four types of "photoreactive crosslinker-modified double-stranded DEL with a covalent bond between the crosslinker and the 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> Apparatus: CL-1000 Ultraviolet Crosslinker (manufactured by UVP, INC.) Microtube: 1.5 mL siliconized microtube, round bottom (manufactured by Watson, catalog number 131-615CH) Reaction tube: 96-well bottom vial (manufactured by Techno Labs. 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 (Sino Biological Co., Ltd., catalog number 10107-H08H): 10.0 μL Aqueous solutions of various DEL samples (50 nM): 1.0 μL Reaction conditions: The CA9 protein and DEL solution in the above composition were mixed and incubated on ice for 2 hours. Then, the mixture was kept on ice and exposed to UV light at 365 nm for 20 minutes.

[0395] <Recovery of DEL cross-linked to protein> Dynabeads Histag isolation & pulldown (Invitrogen, catalog number 10104D): 10.0 μL Tween 20 (Sigma, catalog number P7949-100ML) 10% SDS (NIPPON GENE, catalog number 311-90271) Wash buffer (D-PBS(-) diluted with Tween 20 and 10% SDS to prepare 0.2%) Imidazole (FUJIFILM, catalog number 097-05391) Elution Buffer (Imidazole was dissolved in D-PBS(-) to a concentration of 200 mM, and the pH was adjusted to 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 fixed to a magnetic stand and left to stand for 2 minutes, after which the supernatant was removed and 200 μL of wash buffer was added to suspend the Dynabeads. This procedure was repeated five times. 100 μL of elution buffer was added to the washed Dynabeads and allowed to react at room temperature for 10 minutes. After the reaction, the Dynabeads were placed on a magnetic stand, and after 2 minutes the supernatant was collected as a sample.

[0397] <Preparation of Samples Not Subjected to Photocrosslinking Reaction> The above series of operations was carried out without UV irradiation, and each sample was collected.

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

[0399] As in Example 8, the strength of the affinity between CA9 protein and each compound as a binder is assumed to be 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 in Figure 26, in the samples subjected to UV irradiation, for both binders, the "photoreactive crosslinker-modified double-stranded DEL (PXL-DS-DEL3) having a covalent bond between the crosslinker and the coding sequence" had a significantly higher ΔCt value than the "photoreactive crosslinker-modified double-stranded DEL (PXL-DS-DEL4) without a covalent bond between the crosslinker and the coding sequence." Even though the type of binder is the same, the ΔCt values ​​differ due to differences in the structure of the photoreactive crosslinker-modified double-stranded DEL, 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] These results indicate that a "photoreactive crosslinker-modified double-stranded DEL having a covalent bond between the crosslinker and the coding sequence," derived from a hairpin-type DEL having a "selectively cleavable site," is more useful in DEL screening using photocrosslinking reactions than a "photoreactive crosslinker-modified double-stranded DEL without a covalent bond between the crosslinker and the coding sequence."

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

[0402] <Preparation of DEL sample> In the same manner as in Example 12, four types of "photoreactive crosslinker-modified double-stranded DEL without a covalent bond between the crosslinker and the 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 a covalent bond between the crosslinker and the 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> Apparatus: CL-1000 Ultraviolet Crosslinker (manufactured by UVP, INC.) Reaction tube: 96-well bottom vial (manufactured by Techno Labssy 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): 39.4 μL Carbonic Anhydrase IX / CA9 (Sino Biologica, Catalog No. 10107-H08H): 3.0 μL Aqueous solution of various DEL samples (50 nM): 1.0 μL Reaction conditions: The CA9 protein and DEL solution in the above composition were mixed and incubated on ice for 2 hours. After that, the mixture was kept on ice and irradiated with 365 nm UV for 20 minutes.

[0404] <Recovery of DEL cross-linked to protein> Dynabeads Histag isolation & pulldown (Invitrogen, catalog number 10104D): 10.0 μL Tween 20 (Sigma, catalog number P7949-100ML) Wash buffer (D-PBS(-) diluted with Tween 20 to prepare 0.2%) 200 mM imidazole solution (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 fixed to a magnetic stand and left to stand for 2 minutes, after which the supernatant was removed. 200 μL of wash buffer was added to suspend the Dynabeads and reacted at 90 °C for 10 minutes. This procedure was repeated three times. 30 μL of 200 mM imidazole solution was added to the washed Dynabeads and reacted at room temperature for 10 minutes. After the reaction, the Dynabeads were placed on a magnetic stand, and after 2 minutes the supernatant was collected as a sample.

[0406] <Preparation of Samples Not Subjected to Photocrosslinking Reaction> The above series of operations was carried out without UV irradiation, and each sample was collected.

[0407] As described above in <Recovery of DEL cross-linked with protein>, during recovery of DEL cross-linked with protein, it is subjected to heating conditions, which are strong separation and elution conditions.

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

[0409] As shown in the graph in Figure 27, in the samples subjected to UV irradiation, for both binders, the "photoreactive crosslinker-modified double-stranded DEL (PXL-DS-DEL3) having a covalent bond between the crosslinker and the coding sequence" had a significantly higher ΔCt value than the "photoreactive crosslinker-modified double-stranded DEL (PXL-DS-DEL4) without a covalent bond between the crosslinker and the coding sequence." Even though the type of binder is the same, the ΔCt values ​​differ due to differences in the structure of the photoreactive crosslinker-modified double-stranded DEL, 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).

[0410] These results indicate that a "photoreactive crosslinker-modified double-stranded DEL having a covalent bond between the crosslinker and the coding sequence," derived from a hairpin-type DEL having a "selectively cleavable site," is more useful in DEL screening using photocrosslinking reactions than a "photoreactive crosslinker-modified double-stranded DEL without a covalent bond between the crosslinker and the coding sequence."

[0411] Furthermore, these results suggest 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," can also be adapted for DEL screening under strong separation and elution conditions for the purpose of removing nonspecific binders, etc.

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

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

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

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

[0416] <Cleavage of hairpin DEL compound ("mSABA-DEL5") by USER (registered trademark) enzyme> The cleavage reaction of the hairpin DEL compound "mSABA-DEL5" obtained above by USER (registered trademark) enzyme was carried out in the same manner as in Example 7 <Five types of DEL compounds having biotin at the 3' end (AAZ-BIO-DEL, SABA-BIO-DEL, ClSABA-BIO-DEL, mSABA-BIO-DEL, Amino-BIO-DEL) by USER (registered trademark) enzyme>, and converted to a DEL compound having a double-stranded nucleic acid of the sequence shown in Table 43 "DS-mSABA-DEL5". Note that the sequence notation in Table 43 is the same as Table 36, which means that it is formed by a double strand of oligonucleotide chains of SEQ ID NO: 153 and SEQ ID NO: 154.

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

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

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

[0420] A portion of the obtained supernatant is sampled, diluted with deionized water, and then subjected to ESI-MS mass spectrometry under the analytical conditions 3 of Example 3. As a result, a molecular weight of 23825.8 is observed, and the DEL compound "SS-mSABA-DEL5" having the target single-stranded DNA is identified.

[0421] <Synthesis of photoreactive crosslinker-modified primer "PXL-Pr5"> The photoreactive crosslinker-modified primer "PXL-Pr5" having 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", "L-Pr5" (synthesized in the same manner as in Example 1, the sequence is shown in Table 45) was used as the raw material. The sequence notation in Table 44 is the same as in Table 32. The sequence notation in Table 45 is the same as in Table 8.

[0422]

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

[0424] <Synthesis of photoreactive crosslinker-modified double-stranded DEL compound ("PXL-DS-mSABA-DEL5")> Using the DEL compound "SS-mSABA-DEL5" having the single-stranded DNA obtained above as template DNA, the primer extension reaction using "PXL-Pr5" was carried out in the same manner as in Example 7, and the photoreactive crosslinker-modified double-stranded DEL compound "PXL-DS-mSABA-DEL5" of the sequence shown in Table 46 was synthesized. Note that the sequence notation in Table 46 is the same as in Table 36 and Table 44, which means that it is formed by a double strand of the oligonucleotide chain of SEQ ID NO: 157 and SEQ ID NO: 154.

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

[0426] In addition, a portion of the obtained reaction solution was sampled and analyzed by polyacrylamide gel electrophoresis under the conditions shown in Example 7. From the results shown in Figure 29, it was confirmed that the primer extension reaction resulted in high conversion to "PXL-DS-mSABA-DEL5". The samples in each lane of Figure 29 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, Lonza 20 bp DNA Ladder, 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, Lonza 20 bp DNA Ladder, catalog number 50330)

[0427] Example 15 [Synthesis of crosslinker-modified double-stranded DEL] <Synthesis of three types of crosslinker-modified primers (PA-Pr, TPD-Pr, ACA-Pr)> Various crosslinker-modified primers having the sequences shown in Table 47 were synthesized by the following procedure. In the sequence notation in Table 47, "(PA)" represents the following formula (22): "(TPD)" means a group represented by the following formula (23): "(ACA)" means a group represented by the following formula (24): The other symbols are the same as in Table 2. The raw material activated esters used to synthesize each compound are as follows: Compound: Raw material activated ester PA-Pr: N-succinimidyl 4-azidobenzoate TPD-Pr: N-succinimidyl 4-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzoate ACA-Pr: N-succinimidyl acrylic acid

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

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

[0430] The resulting 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 binary mobile phase gradient profile, 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). Fractions containing the target product were collected, mixed, and concentrated. The resulting solution was desalted using an Amicon® Ultra Centrifugal filter (3 kD cutoff), precipitated with ethanol, and then the pellet was dissolved in deionized water.

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

[0432] <Synthesis of Compound "AOP-L-Pr"> Compound "AOP-L-Pr" having the sequence shown in Table 48 was synthesized by the following procedure. In the sequence notation in Table 48, "(AOP-aminoC6-L)" represents the following formula (25): The other symbols are the same as in Table 2.

[0433] To a PCR tube was added 200 μL of a 1 mM solution of L-Pr (synthesized as in Example 1, sequence shown in Table 28) in sodium borate buffer (150 mM, pH 9.4) cooled to 10° C. To the tube was added 40 equivalents of N-Fmoc-15-amino-4,7,10,13-tetraoxaoctadecanoic acid (20 μL, 0.4 M solution in N,N-dimethylacetamide), followed by 40 equivalents of 4-(4,6-dimethoxy[1.3.5]triazin-2-yl)-4-methylmorpholinium chloride hydrate (DMTMM) (16 μL, 0.5 M aqueous solution), and 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 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. 180 μL of deionized water was added to the pellet to form a solution, after which 20 μL of piperidine was added and the mixture was shaken at 10°C for 3 hours.

[0435] The resulting 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 for 30 minutes at −78° C. 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 and diluted with deionized water, and then subjected to mass analysis by ESI-MS under the analytical conditions 2 of Example 1 to identify the target substance (the theoretical molecular weight and detected molecular weight of each sequence are shown in Table 48).

[0437] <Synthesis of Crosslinker-Modified Primer (BMP-Pr)> The crosslinker-modified primer (BMP-Pr) having the sequence shown in Table 49 was synthesized by the following procedure. In the sequence notation in Table 49, "(BMP)" represents the following formula (26): The other symbols are the same as in Table 2.

[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-maleimidopropionate (5 μL, 0.2 M dimethyl sulfoxide solution) was 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 an additional 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 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 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 resulting supernatant was sampled and diluted with deionized water, and then subjected to mass spectrometry by ESI-MS under the analytical conditions 3 of Example 3 to identify the target crosslinker-modified primer "BMP-Pr" (the theoretical molecular weight and detected molecular weight of each compound are shown in Table 49).

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

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

[0443] <Synthesis of crosslinker-modified double-stranded DEL ("ACA-DS-ClSABA-DEL")> Using the DEL compound "SS-ClSABA-DEL" having the single-stranded DNA obtained in Example 10 as template DNA, a primer extension reaction using the crosslinker-modified primer "ACA-Pr) obtained above was carried out in the same manner as in Example 7, and the crosslinker-modified double-stranded DEL compound "ACA-DS-ClSABA-DEL" having the sequence shown in Table 51 was synthesized. Note that the sequence notation in Table 51 is the same as in Table 26 and Table 47, and indicates that "ACA-DS-ClSABA-DEL" is formed by a double strand of the oligonucleotide chain of SEQ ID NO: 164 and SEQ ID NO: 129.

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

[0445] <Confirmation of primer extension reaction by gel electrophoresis> A portion of the solution obtained in the above <Synthesis of crosslinker-modified double-stranded DEL ("TPD-DS-mSABA-DEL")> and <Synthesis of crosslinker-modified double-stranded DEL ("ACA-DS-ClSABA-DEL")> was sampled and analyzed by polyacrylamide gel electrophoresis under the conditions shown in Example 7. From the results shown in Figure 30, it was confirmed that the primer extension reaction was converted to "TPD-DS-mSABA-DEL" and "ACA-DS-ClSABA-DEL" in high yield. The samples in each lane of 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, Lonza 20 bp DNA Ladder, 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, Lonza 20 bp DNA Ladder, catalog number 50330)

[0446] Example 16 [Synthesis of crosslinker-modified double-stranded DEL using double-stranded DEL having a reactive group for crosslinker modification] <Synthesis of primer ("BCN-Pr") having a reactive group for crosslinker modification> A primer "BCN-Pr" having a reactive group for crosslinker modification, having the sequence shown in Table 52, was synthesized by the following procedure. In the sequence notation in Table 52, "(BCN)" represents the following formula (27): The other symbols are the same as in Table 2.

[0447]

[0448] In the same manner as in Example 15, "BCN-Pr" was synthesized using "AOP-L-Pr" as the raw material and succinimidyl (1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethyl carbonate as the raw material activated ester.

[0449] A portion of the obtained solution was sampled and diluted with deionized water, and then subjected to mass analysis by ESI-MS under the analytical conditions 3 of Example 3, to identify the target "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 having a reactive group for crosslinker modification> Using "SS-mSABA-DEL" obtained in Example 10 as template DNA, the primer extension reaction using "BCN-Pr" was carried out in the same manner as in Example 7, and a double-stranded DEL compound "BCN-DS-mSABA-DEL" having a reactive group for crosslinker modification of the sequence shown in Table 53 was synthesized. The sequence notation in Table 53 is the same as that in Table 26 and Table 52, and indicates that "BCN-DS-mSABA-DEL" is formed by a double strand of oligonucleotide chains of SEQ ID NO: 166 and SEQ ID NO: 131.

[0451] A portion of the obtained solution was sampled and diluted with deionized water, and then subjected to ESI-MS mass spectrometry under the analytical conditions 3 of Example 3. The double-stranded DEL "BCN-DS-mSABA-DEL" having a 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] In addition, a portion of the obtained reaction solution was sampled and analyzed by polyacrylamide gel electrophoresis under the conditions shown in Example 7. From the results shown in Figure 31, it was confirmed that the primer extension reaction resulted in high conversion to "BCN-DS-mSABA-DEL". The samples in each lane of 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, Lonza 20 bp DNA Ladder, 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, Lonza 20 bp DNA Ladder, catalog number 50330)

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

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

[0455] Dimethyl sulfoxide was added to the resulting mixture to dilute it 10 times (diluted solution).

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

[0457] 1 μL of the obtained solution was sampled and diluted with deionized water, and then subjected to ESI-MS mass spectrometry under the analytical conditions 3 of Example 3. The desired click-modified double-stranded DEL "PSF-DS-mSABA-DEL" was identified (the theoretical molecular weight of the compound and the detected molecular weight are shown in Table 54).

[0458] <Synthesis of crosslinker-modified double-chain DEL ("BMP-DS-mSABA-DEL") by click reaction with double-chain DEL having a reactive group for crosslinker modification> A crosslinker was introduced into the "BCN-DS-mSABA-DEL" obtained above by click reaction to synthesize the crosslinker-modified double-chain DEL compound "BMP-DS-mSABA-DEL" having the sequence shown in Table 55. The synthesis procedure is shown below. In the sequence notation in Table 55, "(BMP-t)" is the following formula (29) The other notations are the same as in Table 26, and "BMP-DS-mSABA-DEL" is formed by a double strand of oligonucleotide chains of SEQ ID NO: 131 and SEQ ID NO: 168.

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

[0460] Dimethyl sulfoxide was added to the resulting mixture to dilute it 10 times (diluted solution).

[0461] A BCN-DS-mSABA-DEL solution (2 μL, 0.1 mM) in sodium phosphate buffer (250 mM, pH 7.0) was added to a PCR tube, followed by 1.8 μL of dimethyl sulfoxide. Then, 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 obtained solution was sampled and diluted with deionized water, and then subjected to ESI-MS mass spectrometry under the analytical conditions 3 of Example 3 to identify the desired click-modified double-stranded DEL "BMP-DS-mSABA-DEL" (the theoretical molecular weight of the compound and the detected molecular weight are shown in Table 55).

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

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

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

[0466] The present invention provides a method for utilizing a nucleic acid compound containing a selectively cleavable site. Furthermore, the present invention provides a method for converting a DEL containing a cleavable site in a DNA strand into a crosslinker-modified double-stranded DEL and evaluating the resulting DEL. This enables compound screening that combines a "simple DEL synthesis method" with "expanded and improved DEL evaluation methods" compared to conventional methods.

Claims

1. (i) A step of cleaving at least one "selectively cleavable site" of a hairpin-type DNA-encoded library (DEL) having a "selectively cleavable site" to convert it into a double-stranded DEL. A method for producing a crosslinker-modified double-stranded DEL, comprising any one of the following (A) to (D): (a) (ii) removing oligonucleotides to which no library molecules are bound from the double-stranded DEL obtained in (i) and converting them into single-stranded DEL; and (iii) a step of attaching a crosslinker-modified primer to the single-stranded DEL obtained in (ii), and extending the attached primer to derive a crosslinker-modified double-stranded DEL; or (stomach) (ii) removing oligonucleotides to which no library molecules are bound from the double-stranded DEL obtained in (i) and converting them into single-stranded DEL; (iii) A step of attaching a modified primer having a reactive group for crosslinker modification to the single-stranded DEL obtained in (ii), and extending the attached primer to derive a double-stranded DEL having a reactive group for crosslinker modification; (iv) reacting a reactive group for crosslinker modification with a crosslinker unit to derive a crosslinker-modified double-stranded DEL; or (cormorant) (ii) adding a crosslinker-modified primer to the double-stranded DEL obtained in (i), and extending the added primer to derive a crosslinker-modified double-stranded DEL; or (workman) (ii) A step of adding a modified primer having a reactive group for crosslinker modification to the double-stranded DEL obtained in (i), extending the added primer, and reacting the reactive group for crosslinker modification with the crosslinker unit to derive a crosslinker-modified double-stranded DEL. The method further comprises:

2. A method for evaluating a crosslinker-modified double-stranded DEL obtained by the method of claim 1, comprising: (1) contacting the DEL 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 the library molecule bound to the biological target with the biological target; (3) separating the cross-linked library molecule-biological target complexes from non-cross-linked library molecules; (4) identifying the sequences of the oligonucleotides contained in the library molecules in the recovered complexes; and (5) using the sequences determined in (4) to identify the structure of one or more compounds that bind to the biological target. A method comprising:

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

4. 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 to the 5' end of the oligonucleotide via a bifunctional spacer.

5. In (a) or (c), (I) A hairpin-type DEL is used in which at least one "selectively cleavable site" is located in the 3' direction from the site where the library molecule is bound; and (II) Using a crosslinker-modified primer in which the crosslinker is directly attached to the 5' end of the oligonucleotide or attached via a bifunctional spacer; 3. The method according to claim 1 or 2.

6. In (a) or (d), (I) A hairpin-type DEL is used in which at least one "selectively cleavable site" is located in the 3' direction from the site where the library molecule is bound; and (II) A modified primer having a reactive group for crosslinker modification, which is directly bound to the 5' end of the oligonucleotide or bound via a bifunctional spacer, is used; 3. The method according to claim 1 or 2.

7. In the above (a) or (b) (ii), Oligonucleotides to which library molecules are not bound have functional molecules, and are removed by a treatment according to the function of the functional molecules.

3. The method according to claim 1 or 2.

8. The method of claim 7, wherein the functional molecule is biotin.

9. In the above (a) or (b) (ii), Removal of oligonucleotides not bound to library molecules is by exonuclease degradation.

3. The method according to claim 1 or 2.

10. 10. The method of claim 9, wherein the exonuclease is a lambda exonuclease.

11. 3. The method of claim 1, wherein the crosslinker comprises at least one of an azide group, a diazirine group, a sulfonyl fluoride group, a diazo group, a cinnamoyl group, or an acrylate group.

12. 3. The method of claim 1, wherein the crosslinker comprises at least one azide group, a diazirine group, or a sulfonyl fluoride group.

13. The crosslinker may be represented by the formula (AA) to (AE): 【Chemical 1】 (In the formula, * means the 5' end of the double-stranded DEL or the binding position of the bifunctional spacer bound to the 5' end.) The method according to claim 1 or 2, comprising any one of the following structures:

14. The crosslinker may be represented by the formula (AA) to (AE): 【Chemistry 2】 (In the formula, * means the 5' end of the double-stranded DEL or the binding position of the bifunctional spacer bound to the 5' end.) The method according to claim 1 or 2, wherein the structure is any one of the following:

15. The crosslinker has the formula (BA) or (BB): 【Chemistry 3】 (In the formula, * means the 5' end of the double-stranded DEL or the binding position of the bifunctional spacer bound to the 5' end.) The method according to claim 1 or 2, comprising any one of the following structures:

16. The crosslinker has the formula (BA) or (BB): 【Chemistry 4】 (In the formula, * means the 5' end of the double-stranded DEL or the binding position of the bifunctional spacer bound to the 5' end.) The method according to claim 1 or 2, wherein the structure is any one of the following:

17. In (a) or (d), The reactive group for crosslinker modification is a reactive group for Click reaction; 3. The method according to claim 1 or 2.

18. In the above (a) or (d), The reactive group for crosslinker modification is an alkynyl group, an alkenyl group, an azide group, or a tetrazinyl group; The method according to claim 1 or 2.

19. In the above (a) or (d), The reactive group for crosslinker modification is represented by the formulae (CA) to (CL): 【Chemistry 5】 (In the formula, * means the 5' end of the double-stranded DEL or the binding position to the bifunctional spacer side bound to the 5' end.) The structure is one of 3. The method according to claim 1 or 2.

20. The method according to claim 2, wherein the step (2) of "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."

21. The method according to claim 20, wherein the light irradiation condition is light irradiation with a wavelength of 250 to 500 nm.

22. The method according to claim 21, wherein the light irradiation conditions are light irradiation with a wavelength of 365 nm.

23. The method according to any one of claims 20 to 22, wherein the light irradiation conditions are light irradiation for 10 seconds to 180 minutes.

24. The method according to claim 23, wherein the light irradiation conditions are light irradiation for 30 seconds to 30 minutes.

25. The method according to claim 2, wherein the step (2) of "crosslinking the crosslinker of the library molecule bound to the biological target to the biological target" is the step of "crosslinking the crosslinker of the library molecule bound to the biological target to the biological target by incubation."

26. The method according to claim 2, wherein the step (3) of "separating the complexes of the cross-linked library molecules and the biological target from the non-cross-linked library molecules" is the step of "separating the complexes of the cross-linked library molecules and the biological target from the non-cross-linked library molecules by electrophoresis."

27. 27. The method of claim 26, wherein the electrophoresis is gel electrophoresis.

28. 27. The method of claim 26, wherein the electrophoresis is capillary electrophoresis.

29. The method according to claim 2, wherein the step (3) of "separating the complexes of the cross-linked library molecules and the biological target from the non-cross-linked library molecules" is "separating the complexes of the cross-linked library molecules and the biological target by immobilizing the complexes on a carrier for immobilizing the biological target and removing the non-cross-linked library molecules by washing."

30. The hairpin-type DEL having a "selectively cleavable site" is represented by the formula (I): 【Chemistry 6】 (In the formula, X and Y are oligonucleotide chains; E and F are each independently An oligomer composed of nucleotides or nucleic acid analogs, provided that E and F contain complementary base sequences to each other and form a double-stranded oligonucleotide; LP is the loop region, 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 partial structure composed of at least one building block, X and Y have a sequence capable of forming a double strand at least in part, X is attached to E at the 5' end, Y is bound to F at the 3' end, At least one selectively cleavable site is present in at least one of E, F, or LP. DEL represented by 3. The method according to claim 1 or 2.

31. The hairpin-type DEL having a "selectively cleavable site" is represented by the formula (III): An-Sp-C-Bn (III) (In the formula, An and Sp have the same meanings as in claim 30; Bn represents a double-stranded oligonucleotide tag formed by the oligonucleotide strand X and the oligonucleotide strand Y; C is a compound of formula (I) 【Chemistry 7】 (wherein E, LP, L, D, and F have the same meanings as in claim 30, with the proviso that D is bound to An directly or via a bifunctional spacer, and E and F are bound to the corresponding ends of the double-stranded oligonucleotide tag Bn.) DEL represented by 31. The method of claim 30.

32. An is the same as in claim 30 and is a partial structure constructed of n building blocks α1 to αn (n is an integer from 1 to 10), Bn is a double-stranded oligonucleotide tag formed of an oligonucleotide strand X and an oligonucleotide strand Y, and is a partial structure containing an oligonucleotide containing a base sequence that can identify the structure of An.

31. The method of claim 30.

33. LP, a loop region represented by (LP1)p-LS-(LP2)q, LS is a partial structure selected from the group of compounds described in the following (A) to (C), (A) Nucleotide (B) Nucleic acid analog (C) a C1-14 trivalent group which may have a substituent LP1 is a partial structure selected from the group of compounds described in the following (1) and (2), and p of the partial structures are selected independently or differently: (1) Nucleotide (2) Nucleic acid analogs LP2 is a partial structure selected from the group of compounds described in the following (1) and (2) in a quantity of q, either singly or differently, (1) Nucleotide (2) Nucleic acid analogs The sum of p and q is 0 to 40; 31. The method of claim 30.

34. 34. The method of claim 33, wherein the sum of p and q is 2 to 20.

35. 35. The method of claim 34, wherein the sum of p and q is 2 to 10.

36. 36. The method of claim 35, wherein the sum of p and q is 2 to 7.

37. 34. The method of claim 33, wherein the sum of p and q is 0.

38. LP1, LP2 and LS each have the following structure: (A) Nucleotide or (B) A nucleic acid analog satisfying the following requirements (B11) to (B15): (B11) having a phosphate group or a moiety equivalent thereto and a hydroxyl group or a moiety equivalent thereto; (B12) composed of carbon, hydrogen, oxygen, nitrogen, phosphorus or sulfur; (B13) The molecular weight is 142 to 1500. (B14) the number of atoms between residues is 3 to 30; (B15) The bonding modes of atoms between residues are all single bonds, or contain one or two double bonds and the rest are single bonds.

34. The method of claim 33, wherein the structure is selected, alone or differently, from

39. LP1, LP2 and LS each have the following structure: (A) Nucleotide or (B) A nucleic acid analog satisfying the following requirements (B21) to (B25): (B21) having a phosphoric acid and a hydroxyl group, (B22) composed of carbon, hydrogen, oxygen, nitrogen or phosphorus; (B23) the molecular weight is 142 to 1000; (B24) The number of atoms between residues is 3 to 15. (B25) The bonding modes of atoms between residues are all single bonds.

34. The method of claim 33, wherein the structure is selected, alone or differently, from

40. LP1, LP2 and LS each have the following structure: (A) Nucleotide or (B) A nucleic acid analog satisfying the following requirements (B31) to (B35): (B31) having a phosphoric acid and a hydroxyl group, (B32) composed of carbon, hydrogen, oxygen, nitrogen or phosphorus; (B33) The molecular weight is 142 to 700. (B34) The number of atoms between residues is 4 to 7. (B35) The bonding modes of atoms between residues are all single bonds.

34. The method of claim 33, wherein the structures are selected, alone or differently, from

41. LP1 and LP2 are each the following: (B41) d-Spacer, (B5) Polyalkylene glycol phosphate ester 34. The method of claim 33, wherein

42. 34. The method of claim 33, wherein LP1 and LP2 are diethylene glycol phosphate ester or triethylene glycol phosphate ester, respectively.

43. 34. The method of claim 33, wherein LP1 and LP2 are each triethylene glycol phosphate esters.

44. 34. The method of claim 33, wherein LP1 and LP2 are each a d-Spacer.

45. 34. The method of claim 33, wherein LP1 and LP2 are each a nucleotide.

46. LS is a compound represented by formula (a) to formula (g): 【Chemistry 8】 (In the formula, * represents the bonding position with the linker, ** represents the bonding position with LP1 or LP2, and R represents a hydrogen atom or a methyl group.) 34. The method of claim 33, wherein

47. LS is a compound of formula (h): 【Chemistry 9】 (In the formula, * means the bonding position with the linker, and ** means the bonding position with LP1 or LP2.) 34. The method of claim 33, wherein:

48. 34. The method of claim 33, wherein LS is a polyalkylene glycol phosphate ester.

49. LS is a compound represented by formula (i) to formula (k): 【Chemistry 10】 (In the formula, n1, m1, p1, and q1 each independently represent an integer of 1 to 20, * represents the bonding position to the linker, and ** represents the bonding position to LP1 or LP2.) 34. The method of claim 33, wherein:

50. LS is a compound of formula (l): 【Chemistry 11】 (In the formula, * means the bonding position with the linker, and ** means the bonding position with LP1 or LP2.) 34. The method of claim 33, wherein:

51. LS is (B42), (B43) or (B44): (B42) Amino C6 dT (B43) mdC (TEG-Amino) (B44) Uni-Link (registered trademark) Amino Modifier 34. The method of claim 33, wherein

52. 34. The method of claim 33, wherein the LS is a nucleotide.

53. L S is (C) a C 1-14 trivalent group which may have a substituent, and (C) is the following structure: (1) C1-10 aliphatic hydrocarbons which may have a substituent and may be substituted with 1 to 3 heteroatoms; (2) optionally substituted C6-14 aromatic hydrocarbons; (3) an optionally substituted C2-9 aromatic heterocycle, or (4) Optionally substituted C2-9 non-aromatic heterocycle 34. The method of claim 33, wherein

54. L S is (C) a C 1-14 trivalent group which may have a substituent, and (C) is the following structure: (1) a C1-6 aliphatic hydrocarbon which may have a substituent; (2) an optionally substituted C6-10 aromatic hydrocarbon, or (3) Optionally substituted C2-5 aromatic heterocycle 34. The method of claim 33, wherein

55. L S is (C) a C 1-14 trivalent group which may have a substituent, and (C) is the following structure: (1) C1-6 aliphatic hydrocarbons, (2) benzene, or (3) C2-5 nitrogen-containing aromatic heterocycle wherein (1) to (3) may be unsubstituted or substituted with 1 to 3 substituents selected, either singly or differently, from substituent group ST1, and substituent group ST1 is a group consisting of a C1-6 alkyl group, a C1-6 alkoxy group, a fluorine atom, and a chlorine atom, provided that when substituent group ST1 substitutes an aliphatic hydrocarbon, no alkyl group is selected from substituent group ST1; 34. The method of claim 33, wherein

56. L S is (C) a C 1-14 trivalent group which may have a substituent, and (C) is the following structure: (1) a C1-6 alkyl group, or (2) Benzene that is unsubstituted or substituted with one or two C1-3 alkyl or C1-3 alkoxy groups 34. The method of claim 33, wherein

57. L S is (C) a C 1-14 trivalent group which may have a substituent, and (C) is the following structure: (1) C1-6 alkyl group 34. The method of claim 33, wherein:

58. The method described in claim 30, wherein the chain lengths of E and F are each 3 to 40.

59. The method described in claim 58, wherein the chain lengths of E and F are each 4 to 30.

60. The method described in claim 59, wherein the chain lengths of E and F are each 6 to 25.

61. The method described in claim 30, wherein the double-stranded oligonucleotide formed by E and F has a protruding end.

62. 62. The method of claim 61, wherein the overhang of the cohesive end is 2 or more bases in length.

63. The method described in claim 30, wherein the double-stranded oligonucleotide formed by E and F has blunt ends.

64. The method according to claim 30, wherein the chain lengths of the complementary base sequences contained in E and F are each 3 or more bases.

65. The method of claim 64, wherein the chain lengths of the complementary base sequences contained in E and F are each 4 or more bases.

66. The method of claim 65, wherein the chain lengths of the complementary base sequences contained in E and F are each 6 bases or more.

67. The method of claim 30, wherein E and F are each independently an oligomer composed of nucleotides.

68. 31. The method of claim 30, wherein the nucleotide is a ribonucleotide or a deoxyribonucleotide.

69. 69. The method of claim 68, wherein the nucleotide is a deoxyribonucleotide.

70. 31. The method of claim 30, wherein the nucleotide is deoxyadenosine, deoxyguanosine, thymidine, or deoxycytidine.

71. The method of claim 30, wherein E and F are each independently an oligomer composed of a nucleic acid analog.

72. L, (1) C1-20 aliphatic hydrocarbons which may have a substituent and may be substituted with 1 to 3 heteroatoms; or (2) C6-14 aromatic hydrocarbons which may have a substituent 31. The method of claim 30, wherein:

73. 31. The method of claim 30, wherein L is a C1-6 aliphatic hydrocarbon optionally having a substituent, a C1-6 aliphatic hydrocarbon optionally substituted with 1 or 2 oxygen atoms, or a C6-10 aromatic hydrocarbon optionally having a substituent.

74. The method according to claim 30, wherein L is a C1-6 aliphatic hydrocarbon that can be substituted with substituent group ST1, or a benzene that can be substituted with substituent group ST1, wherein substituent group ST1 is a group consisting of a C1-6 alkyl group, a C1-6 alkoxy group, a fluorine atom, and a chlorine atom (however, when substituent group ST1 substitutes an aliphatic hydrocarbon, an alkyl group is not selected from substituent group ST1).

75. 31. The method of claim 30, wherein L is a C1-6 alkyl group or benzene unsubstituted or substituted with one or two C1-3 alkyl or C1-3 alkoxy groups.

76. 31. The method of claim 30, wherein L is a C1-6 alkyl group.

77. The reactive functional group of D is 31. The method of claim 30, wherein the reactive functional group is capable of forming a C-C, amino, ether, carbonyl, amide, ester, urea, sulfide, disulfide, sulfoxide, sulfonamide, or sulfonyl bond.

78. 31. The method of claim 30, 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.

79. The method of claim 30, wherein the reactive functional group of D is a C1 hydrocarbon having a halogen atom, a C1 hydrocarbon having a sulfonic acid-based leaving group, an amino group, a hydroxyl group, a carboxy group, a halogenated carboxy group, a thiol group, or an aldehyde group.

80. The reactive functional group of D is —CH ２ Cl, —CH ２ Br, —CH ２ OSO ２ CH ３ , -CH ２ OSO ２ CF ３ , an amino group, a hydroxyl group, or a carboxy group.

81. 31. The method of claim 30, wherein the reactive functional group of D is a primary amino group.

82. 31. The method of claim 30, wherein the selectively cleavable site is a deoxyribonucleoside that is other than deoxyadenosine, deoxyguanosine, thymidine, and deoxycytidine.

83. 31. The method of claim 30, wherein the selectively cleavable site is 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.

84. 84. The method of claim 83, wherein the selectively cleavable site is deoxyuridine or deoxyinosine.

85. 85. The method of claim 84, wherein the selectively cleavable site is deoxyuridine.

86. 85. The method of claim 84, wherein the selectively cleavable site is deoxyinosine.

87. 31. The method of claim 30, wherein the selectively cleavable site is the second phosphodiester bond 3' from the deoxyinosine.

88. 31. The method of claim 30, wherein the selectively cleavable site is a ribonucleoside.

89. 31. The method of claim 30, wherein the selectively cleavable site is one.

90. 31. The method of claim 30, wherein at least one cleavable site is contained in E or (LP1)p and at least one cleavable site is contained in F or (LP2)q.

91. The method of claim 90, wherein the cleavable site contained in E or (LP1)p and the cleavable site contained in F or (LP2)q are cleavable under different conditions.

92. The method according to claim 30, wherein An is a partial structure constructed by n building blocks α1 to αn (n is an integer of 1 to 10).

93. 31. The method of claim 30, wherein An is a small molecule organic compound.

94. The method of claim 30, wherein the building block of An is a compound having a molecular weight of 500 or less.

95. 95. The method of claim 94, wherein the building block of An is a compound having a molecular weight of 300 or less.

96. 96. The method of claim 95, wherein the building block of An is a compound having a molecular weight of 150 or less.

97. 31. The method according to claim 30, wherein An is an organic compound composed of elements selected, alone or differently, from the group consisting of H, B, C, N, O, Si, P, S, F, Cl, Br and I.

98. The method according to claim 30, wherein An is a low molecular weight organic compound having substituents, either alone or differently, selected from the group consisting of aryl groups, non-aromatic cyclyl groups, heteroaryl groups, and non-aromatic heterocyclyl groups.

99. 31. The method of claim 30, wherein An has a molecular weight of 5000 or less.

100. 100. The method of claim 99, wherein An has a molecular weight of 800 or less.

101. 101. The method of claim 100, wherein An has a molecular weight of 500 or less.

102. 31. The method of claim 30, wherein An is a polypeptide.

103. 31. The method of claim 30, wherein Sp is a bifunctional spacer.

104. the bifunctional spacer is SpD-SpL-SpX; SpD is a divalent group derived from a reactive group capable of forming a C-C, amino, ether, carbonyl, amide, ester, urea, sulfide, disulfide, sulfoxide, sulfonamide, or sulfonyl bond; SpL is a polyalkylene glycol, polyethylene, a C1-20 aliphatic hydrocarbon optionally substituted with a heteroatom, a peptide, an oligonucleotide, or a combination thereof; SpX is a divalent group derived from a reactive group that forms an amino, carbonyl, amide, ester, urea, or sulfonamide bond; 31. The method of claim 30.

105. the bifunctional spacer is SpD-SpL-SpX; 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 carboxy group; 31. The method of claim 30.

106. The method according to claim 30, wherein the oligonucleotide strand X and the oligonucleotide strand Y have sequences capable of forming a duplex.

107. 31. The method of claim 30, wherein oligonucleotide strand X and oligonucleotide strand Y comprise complementary base sequences.

108. The method according to claim 30, wherein the oligonucleotide strand X and the oligonucleotide strand Y each have a length of 1 to 200 bases.

109. The method of claim 108, wherein the oligonucleotide strand X and the oligonucleotide strand Y each have a length of 3 to 150 bases.

110. The method of claim 109, wherein the oligonucleotide strand X and the oligonucleotide strand Y each have a length of 30 to 150 bases.

111. 31. The method of claim 30, wherein oligonucleotide strand X and oligonucleotide strand Y have blunt ends.

112. 31. The method of claim 30, wherein oligonucleotide strand X and oligonucleotide strand Y have overhanging ends.

113. 113. The method of claim 112, wherein the overhang of the cohesive end is 1 to 30 bases in length.

114. 114. The method of claim 113, wherein the overhang of the cohesive end is 2 to 5 bases in length.

115. The method described in claim 112, wherein a specific molecular recognition sequence is further bound to the protruding end.

116. The method according to claim 30, wherein a functional molecule is bound to either X or Y.

117. The method of claim 30, wherein biotin is bound to either X or Y.

118. 31. The method of claim 30, wherein Sp is a bond.

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