WO2023095841A1 - Ｄｎａコード化ライブラリの評価方法 - Google Patents

Ｄｎａコード化ライブラリの評価方法 Download PDF

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WO2023095841A1
WO2023095841A1 PCT/JP2022/043405 JP2022043405W WO2023095841A1 WO 2023095841 A1 WO2023095841 A1 WO 2023095841A1 JP 2022043405 W JP2022043405 W JP 2022043405W WO 2023095841 A1 WO2023095841 A1 WO 2023095841A1
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del
group
stranded
crosslinker
double
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雅俊 丹羽
宗史 ▲徳▼川
潤 林田
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Nissan Chemical Corp
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    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B30/00—Methods of screening libraries
    • C40B30/04—Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes

Definitions

  • the present invention relates to methods for evaluating DNA-encoded libraries.
  • a compound library is a group of compound derivatives systematically collecting compounds that may have specific activities, such as drug candidate compounds. This compound library is often synthesized based on combinatorial chemistry synthetic techniques and methodologies. Combinatorial chemistry is a research field related to experimental techniques for efficiently synthesizing a series of compound libraries, which are enumerated and designed based on combinatorial theory, through systematic synthetic routes.
  • One class of compound libraries based on combinatorial chemistry are DNA-encoded libraries.
  • the DNA-encoded library is appropriately abbreviated as DEL.
  • DEL a DNA tag is added to each compound in the library. The DNA tag has a sequence designed so that each structure of each compound can be identified, and functions as a compound label (Patent Documents 1 to 3).
  • Screening using DEL has so far identified a plurality of compounds that are effective in drug development. Screening using DEL is performed, for example, as follows (Non-Patent Documents 1 to 3). 1) immobilizing the target protein on an immobilizing carrier 2) contacting the target with DEL 3) washing away the DEL with low affinity for the target 4) denaturing the target protein to achieve high affinity Elute the DEL 5) Amplify the DNA Sequence Contained in the Eluted DEL and Identify the Sequence
  • the screening method as described above has several problems. First, it is necessary to immobilize the target protein, so depending on the protein, the three-dimensional structure may change after the immobilization operation.
  • Non-Patent Documents 4 and 5 the compounds obtained by screening do not bind to the desired non-immobilized target protein, which limits the target application range of DEL.
  • high affinity binders can be recovered, but intermediate affinity binders (eg, those with a Kd value of ⁇ M order) are kinetically stable complexes with the target protein. It is difficult to recover after cleaning and removal.
  • intermediate affinity binders are also useful as hit compounds that serve as a starting point for drug discovery, and are also useful as structure-activity relationship information (Non-Patent Document 6 ).
  • a relatively short double-stranded DNA tag of about 9 to 13 mers with a 2-mer sticky end is often used, and the double-stranded DNA tag is introduced by a ligation reaction using DNA ligase.
  • the use of such a short DNA tag is made possible by the strong intramolecular formation of a double strand by the hairpin strand DNA and the fact that the DNA site other than the sticky end does 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 synthesizing DNA tags. Another advantage is that the use of shorter DNA tags reduces the total length of DEL when encoding the same number of reaction cycles.
  • Non-Patent Document 3 construction of a DEL using hairpin-strand DNA that encodes 6 cycles of reactions is achieved by using hairpin-strand DNA.
  • hairpin strands form double strands stronger than double strands (higher Tm value) if the strand length is the same. Therefore, under various chemical conditions during the introduction of building blocks, each chemical structure of the hairpin strand DNA, especially the structure of the base portion, should be more resistant to structural transformation than the double strand.
  • a hairpin strand DNA forms a double strand within the molecule, and it is difficult to newly form a double strand with another oligonucleotide strand. Therefore, it is difficult to convert to crosslinker-modified double-stranded DEL by providing newly crosslinker-modified oligonucleotides.
  • double-stranded DEL DEL using double-stranded DNA is synthesized using single-stranded DNA (single-stranded DNA that is not a hairpin strand) or double-stranded DNA having functional groups for introducing various building blocks as a starting material (headpiece).
  • DEL single-stranded DNA
  • Double-stranded DNA can be denatured into single-stranded DNA or subjected to strand exchange reaction, which is advantageous in that it can be converted into a DNA structure suitable for various uses. Therefore, it is also possible to convert to a crosslinker-modified double-stranded DEL by imparting a new crosslinker-modified single-stranded oligonucleotide (Non-Patent Documents 7, 8 and 11).
  • hairpin strand DNA and double-stranded DNA have advantages when synthesizing and evaluating DEL, respectively, but there is no known technique for achieving both advantages.
  • Non-Patent Documents 7 and 8 a single-stranded DEL having a library molecule at the 5' end is synthesized, a double strand is formed with a DNA having a photoreactive crosslinker at the 3' end, and screening is performed. A binding agent with a degree of affinity has been obtained.
  • this method since the DNA ligated with the photoreactive crosslinker does not contain the coding sequence, if it is exposed to strong separation or elution conditions for the removal of non-specific binding agents, etc., two It is possible that the strands will separate and the sequence encoding the desired structure will not be obtained.
  • the single-stranded DEL since the single-stranded DEL is used, it is a method in which the merits of the hairpin-stranded DEL cannot be utilized in the synthesis.
  • Non-Patent Document 5 Non-Patent Documents 4, 5 and 12
  • linker-modified DEL By synthesizing a single-stranded DEL having a library molecule at the 3' end and forming a double strand with a short-stranded DNA having a photoreactive crosslinker at the 5' end, a photoreactive cross It is referred to as linker-modified DEL.
  • linker-modified DEL An advantage of this approach is the presence of a photoreactive crosslinker at the 5' end, which covalently joins the coding sequence to the target through an extension reaction with a DNA polymerase.
  • Patent Document 4 describes a hairpin chain DEL having a linking site with a crosslinker. Although this method can solve the problems described in Non-Patent Document 5 above, it is necessary to perform library synthesis without damaging the functional groups for crosslinker ligation, and usable reactions and / or Another problem is that the library molecular structure can be restricted.
  • Patent Document 6 synthesis of a double-stranded DEL crosslinked by a reversible covalent bond (considered to have properties equivalent to a hairpin strand DEL in terms of double-strand formation ability and the like), and crosslinker-modified is described for conversion to DEL.
  • a reversible covalent bond a covalent bond by [2 + 2] photocyclization between a special base such as cyanovinylcarbazole and a pyrimidine base is disclosed.
  • photocyclized pyrimidine bases have lost their aromaticity, and such pyrimidine bases that have lost their aromaticity are known to be chemically unstable and decompose under basic conditions.
  • Non-Patent Document 13 Therefore, in this method, usable reactions are limited during DEL synthesis, and library molecular structures that can be constructed are also limited.
  • the present invention provides a method for inducing and evaluating a DEL containing a cleavable site in its DNA strand into a crosslinker-modified double-stranded DEL.
  • Nucleic acid cleaving technology is one of the nucleic acid chemistries such as DNA.
  • introduction of deoxyuridine into the DNA strand allows for selective cleavage by the USER® Enzyme.
  • the present inventors have found that by introducing a cleavable site such as deoxyuridine into the DNA chain, it is possible to achieve both the advantages of hairpin strand DNA and double-stranded DNA. The above problem was solved by simply inducing to DEL. Accordingly, the present invention is as follows.
  • a method for evaluating crosslinker-modified double-stranded DELs derived from hairpin DNA-encoding libraries (DELs) with "selectively cleavable sites” comprising the steps of: (1) contacting the DEL with a biological target under conditions suitable for binding of at least one library molecule of the DEL to the biological target; (2) cross-linking the cross-linker of the library molecule bound to the biological target with the biological target; (3) allowing complexes of cross-linked library molecules and biological targets to separate from non-cross-linked library molecules; (4) identifying the sequence of the oligonucleotide possessed by the library molecule in the recovered complex; (5) using the sequences determined in (4) to identify the structure of one or more compounds that bind to the biological target; A method consisting of: [2] The method of [1], wherein the crosslinker of the crosslinker-modified double-stranded DEL is linked to an oligonucleotide having a coding sequence via a covalent bond.
  • a hairpin DEL having a "selectively cleavable site” is cleaved at least one "selectively cleavable site” to convert it into a double-stranded DEL.
  • oligonucleotides to which library molecules are not bound are removed and converted to single-stranded DEL.
  • a crosslinker-modified double-stranded DEL is derived by reacting a reactive group for crosslinker modification with a crosslinker unit.
  • a crosslinker-modified primer is added to the single-stranded DEL obtained in (ii), and the attached primer is extended to induce a crosslinker-modified double-stranded DEL.
  • the method according to any one of [1] to [3], wherein the induction to crosslinker-modified double-stranded DEL comprises the following steps.
  • a hairpin DEL having a "selectively cleavable site” is cleaved at least one "selectively cleavable site” to convert it into a double-stranded DEL.
  • a crosslinker-modified double-stranded DEL is derived by reacting a reactive group for crosslinker modification with a crosslinker unit.
  • a hairpin DEL having a "selectively cleavable site” is cleaved at least one "selectively cleavable site” to convert it into a double-stranded DEL.
  • a crosslinker-modified primer is added to the double-stranded DEL obtained in (i), and the added primer is extended to induce a crosslinker-modified double-stranded DEL.
  • a hairpin DEL having a "selectively cleavable site” is cleaved at least one "selectively cleavable site” to convert it into a double-stranded DEL.
  • a modified primer having a reactive group for crosslinker modification is applied to the double-stranded DEL obtained in (i), the applied primer is extended, and a reactive group for crosslinker modification and a cross A crosslinker-modified double-stranded DEL is induced by reacting with the linker unit.
  • step (II) a modified primer having a reactive group for crosslinker modification, wherein the reactive group for crosslinker modification is directly attached to the 5′ end of the oligonucleotide or is attached via a bifunctional spacer; use.
  • the oligonucleotide to which the library molecule is not bound has a functional molecule and is removed by treatment according to the function of the functional molecule, [4] to [7] ] The method according to any one of [13] The method of [12], wherein the functional molecule is biotin.
  • step (ii) the removal of oligonucleotides to which library molecules are not bound is degradation by exonuclease.
  • step (ii) the removal of oligonucleotides to which library molecules are not bound is degradation by exonuclease.
  • the exonuclease is lambda exonuclease.
  • the crosslinker contains at least one azide group, diazirine group, sulfonyl fluoride group, diazo group, cinnamoyl group, or acrylate group.
  • the crosslinker contains at least one azide group, diazirine group, or sulfonyl fluoride group.
  • the crosslinker is represented by formulas (AA) to (AE): (Wherein, * means the 5' end of the double-stranded DEL, or the binding position to the bifunctional spacer side that is bound to the 5' end)
  • the crosslinker is represented by formulas (AA) to (AE): (Wherein, * means the 5' end of the double-stranded DEL, or the binding position to the bifunctional spacer side that is bound to the 5' end)
  • the crosslinker has the formula (BA) or (BB): (Wherein, * means the 5' end of the double-stranded DEL, or the binding position to the bifunctional spacer
  • the method according to any one of [1] to [19], which is a step of cross-linking with a biological target by [26] The method according to [25], wherein the light irradiation condition is light irradiation with a wavelength of 250 to 500 nm.
  • the step (2) of "crosslinking the crosslinker of the library molecule bound to the biological target with the biological target” is replaced by “crosslinking the crosslinker of the library molecule bound to the biological target by incubation.
  • the step (3) of "separating the complexes of the crosslinked library molecules and the biological targets from the non-crosslinked library molecules” includes “separating the complexes of the crosslinked library molecules and the biological targets.” , separating from non-crosslinked library molecules by electrophoresis”.
  • the method of [31], wherein the electrophoresis is gel electrophoresis.
  • a hairpin-type DEL having a "selectively cleavable site” has the formula (I) (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 form a double-stranded oligonucleotide, LP is the loop site; L is a linker; D is a divalent group derived from a reactive functional group, Sp is a bond or a bifunctional spacer; An is a substructure composed of at least one building block.
  • ) is a compound represented by 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 attached to F at the 3' end, It has at least one selectively cleavable site in at least one of the E, F, or LP sites.
  • DEL represented by The method according to any one of [1] to [34].
  • a hairpin DEL with a "selectively cleavable site" is represented by formula (III) An-Sp-C-Bn (III) (In the formula, An and Sp have the same meaning as in [35], Bn represents a double-stranded oligonucleotide tag formed by oligonucleotide chain X and oligonucleotide chain Y; C is of formula (I) (wherein E, LP, L, D and F have the same meaning as in [35], except that D is directly attached to An or via a bifunctional spacer, and E and F binds to the respective ends of the double-stranded oligonucleotide tag Bn.) is DEL represented by The method of [35].
  • An is the same as [35] and is a partial structure constructed from n building blocks ⁇ 1 to ⁇ n (n is an integer from 1 to 10); Bn is a double-stranded oligonucleotide tag formed by oligonucleotide chain X and oligonucleotide chain Y, and is a partial structure containing an oligonucleotide containing a base sequence capable of identifying the structure of An. The method of [35] or [36]. [38] L.P.
  • LP1 is a loop site represented by p-LS-(LP2) q
  • LS is a partial structure selected from the group of compounds described in (A) to (C) below
  • Nucleotide B
  • Nucleic acid analogue C1-14 trivalent group
  • LP1 optionally having a substituent may be alone or different from the group of compounds described in (1) and (2) below is each partial structure selected by p pieces
  • Nucleotide (2) Nucleic acid analog LP2 is each partial structure selected singly or differently from the group of compounds described in (1) and (2) below
  • (1) the total number of nucleotides (2) nucleic acid analogs p and q is from 0 to 40; [35] The method according to any one of [37].
  • LP1, LP2 and LS each have the following structure: (A) a nucleotide or (B) a nucleic acid analogue requiring (B11) to (B15) below (B11) a phosphate (or equivalent site) and a hydroxyl group (or equivalent site thereof), (B12) composed of carbon, hydrogen, oxygen, nitrogen, phosphorus or sulfur, (B13) has a molecular weight of 142 to 1,500; (B14) the number of atoms between residues is 3 to 30, (B15) the bonding patterns of atoms between residues are all single bonds, or contain 1 to 2 double bonds and the rest are single bonds; The method of any one of [38]-[42], wherein the structure is selected singly or differently from [44] LP1, LP2 and LS each have the following structure: (A) a nucleotide or (B) a nucleic acid analogue requiring (B21) to (B25) below (B21) having a phosphat
  • LS is represented by formulas (a) to (g): (Wherein, * means the bonding position with the linker, ** means the bonding position with LP1 or LP2, and R is a hydrogen atom or a methyl group.) The method according to any one of [38] to [50].
  • LS is of formula (h): (Wherein, * means the binding position with the linker, ** means the binding position with LP1 or LP2) The method according to any one of [38] to [50].
  • [53] The method according to any one of [38] to [50], wherein LS is polyalkylene glycol phosphate.
  • LS is represented by formulas (i) to (k): (Wherein, n1, m1, p1, and q1 are each independently an integer of 1 to 20, * means the binding position with the linker, ** means the binding position with LP1 or LP2 .) The method according to any one of [38] to [50].
  • LS is of formula (l): (Wherein, * means the binding position with the linker, ** means the binding position with LP1 or LP2) The method according to any one of [38] to [50].
  • LS is (B42), (B43) or (B44): (B42) Amino C6 dT (B43) mdC (TEG-Amino) (B44) Uni-Link (registered trademark) Amino Modifier The method according to any one of [38] to [50]. [57] The method of any one of [38]-[50], wherein LS is a nucleotide.
  • LS is (C) a C1-14 trivalent group optionally having a substituent, and (C) has the following structure: (1) optionally substituted C1-10 aliphatic hydrocarbon optionally substituted with 1 to 3 heteroatoms; (2) optionally substituted C6-14 aromatic hydrocarbons, (3) an optionally substituted C2-9 aromatic heterocyclic ring, or (4) an optionally substituted C2-9 non-aromatic heterocyclic ring, [38]-[ 42] and the method according to any one of [46] to [50].
  • LS is (C) a C1-14 trivalent group optionally having a substituent, and (C) has the following structure: (1) optionally substituted C1-6 aliphatic hydrocarbons, (2) optionally substituted C6-10 aromatic hydrocarbon, or (3) optionally substituted C2-5 aromatic heterocycle [38]-[42] and the method according to any one of [46] to [50].
  • LS is (C) a C1-14 trivalent group optionally having a substituent
  • (C) has the following structure: (1) C1-6 aliphatic hydrocarbons, (2) benzene, or (3) a C2-5 nitrogen-containing aromatic heterocyclic ring wherein the above (1) to (3) are unsubstituted, or 1 to which are independently or differently selected from the substituent group ST1 It may be substituted with three substituents, and the substituent group ST1 is a group composed of a C1-6 alkyl group, a C1-6 alkoxy group, a fluorine atom and a chlorine atom, provided that the substituent group ST1 is When substituting with an aliphatic hydrocarbon, an alkyl group is not selected from the substituent group ST1,
  • LS is (C) a C1-14 trivalent group optionally having a substituent, and (C) has the following structure: (1) a
  • LS is (C) a C1-14 trivalent group optionally having a substituent, and (C) has the following structure: (1) The method according to any one of [38] to [42] and [46] to [50], which is a C1-6 alkyl group.
  • E and F are each independently an oligomer composed of nucleotides or nucleic acid analogs; the chain length of E and F is 3 to 40, respectively; [35] The method according to any one of [62].
  • E and F are each independently an oligomer composed of nucleotides or nucleic acid analogs; The method according to any one of [35] to [63], wherein E and F have chain lengths of 4 to 30, respectively.
  • E and F are each independently an oligomer composed of nucleotides or nucleic acid analogs; The method according to any one of [35] to [64], wherein E and F have chain lengths of 6 to 25, respectively.
  • E and F are each independently an oligomer composed of nucleotides or nucleic acid analogs; E and F comprise complementary base sequences to form a double-stranded oligonucleotide; The E and F double-stranded oligonucleotides are overhanging ends, [35] The method according to any one of [65]. [67] The method of [66], wherein the overhang of the protruding end has a length of 2 or more bases.
  • E and F are each independently an oligomer composed of nucleotides or nucleic acid analogs; E and F comprise complementary base sequences to form a double-stranded oligonucleotide; the E and F double-stranded oligonucleotides are blunt-ended; [35] The method according to any one of [65]. [69] The method according to any one of [35] to [68], wherein each of complementary base sequences contained in E and F has a length of 3 bases or more. [70] The method according to any one of [35] to [69], wherein each of complementary nucleotide sequences contained in E and F has a chain length of 4 or more bases.
  • nucleotide is deoxyadenosine, deoxyguanosine, thymidine, or deoxycytidine.
  • E and F are each independently oligomers composed of nucleic acid analogues.
  • L is (1) optionally substituted C1-20 aliphatic hydrocarbons optionally substituted with 1 to 3 heteroatoms; Or (2) the method according to any one of [35] to [76], which is a C6-14 aromatic hydrocarbon optionally having a substituent.
  • 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 having a substituent is a C6-10 aromatic hydrocarbon.
  • L is a C1-6 aliphatic hydrocarbon that can be substituted with a substituent group ST1, or a benzene that can be substituted with a substituent group ST1, wherein the substituent group ST1 is a C1-6 alkyl group, A group consisting of a C1-6 alkoxy group, a fluorine atom and a chlorine atom (however, when the substituent group ST1 is substituted with an aliphatic hydrocarbon, no alkyl group is selected from the substituent group ST1.), [35 ] to [78].
  • the reactive functional group of D is Any one of [35] to [81], which is a reactive functional group capable of forming a C—C, amino, ether, carbonyl, amide, ester, urea, sulfide, disulfide, sulfoxide, sulfonamide, or sulfonyl bond described method.
  • the reactive functional group of D is a C hydrocarbon having a halogen atom, a C 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
  • 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, [35 ] to [84].
  • the reactive functional group of D is a primary amino group.
  • the selectively cleavable site is a deoxyribonucleoside that is neither deoxyadenosine, deoxyguanosine, thymidine, nor deoxycytidine; [35] The method according to any one of [86].
  • the selectively cleavable site is deoxyuridine, bromodeoxyuridine, deoxyinosine, 8-hydroxydeoxyguanosine, 3-methyl-2'-deoxyadenosine, N6-etheno-2'-deoxyadenosine, 7-
  • the method of any one of [35] to [88], wherein the selectively cleavable site is deoxyuridine or deoxyinosine.
  • An is a low-molecular-weight organic compound having substituents selected singly or differently from the substituent group consisting of an aryl group, a non-aromatic cyclyl group, a heteroaryl group and a non-aromatic heterocyclyl group, [35] The method according to any one of -[102]. [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.
  • the bifunctional spacers are each 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; The method according to any one of [1] to [107].
  • oligonucleotide chain X and oligonucleotide chain Y contain complementary nucleotide sequences.
  • the present invention provides a method for inducing and evaluating a DEL containing a cleavable site in its DNA strand into a crosslinker-modified double-stranded DEL. That is, the present invention provides a compound screening technique that combines "simple DEL synthesis technique” and “expansion/improvement of DEL evaluation technique” than conventionally. Therefore, according to the present invention, opportunities for obtaining useful hit compounds in the development of pharmaceuticals, agricultural chemicals, and medical materials are expanded.
  • FIG. 1 shows an exemplary DEL manufacturing method of Modality 1.
  • FIG. Starting with a first oligonucleotide strand containing a cleavable site in the DNA strand, a headpiece containing a loop site and a second oligonucleotide strand, binding building blocks, and oligonucleotide tags corresponding to the building blocks. (3 times in FIG. 1) and, if desired, double-stranded ligation of the oligonucleotide tag containing the primer region, the production of DEL is achieved.
  • FIG. 11 shows an exemplary DEL usage of Modality 1.
  • a cleaving means such as an enzyme is used to cleave the cleavable site into a double-stranded oligonucleotide that is not bound at the loop site.
  • PCR can be performed with high efficiency.
  • FIG. 11 shows an exemplary DEL usage of mod 2.
  • a cleaving means such as an enzyme is used to cleave the cleavable site into a double-stranded oligonucleotide that is not bound at the loop site.
  • PCR can be performed with high efficiency.
  • 3 illustrates an exemplary DEL usage of mod 3.
  • a cleaving means such as an enzyme is used to cleave both the cleavable sites so that the loop site is
  • PCR can be performed with high efficiency.
  • 4 shows an exemplary DEL usage of form 4.
  • the cleavage conditions are selected to One of the second oligonucleotide strands can be selectively cleaved.
  • 4 shows an exemplary DEL usage of Form 5.
  • FIG. New overhanging ends can be generated by providing a cleavable site near the end of the DNA tag and cleaving the site if desired. The overhanging ends can be used as sticky ends to ligate desired nucleic acid sequences such as UMIs (specific molecular identification sequences) to impart new functions.
  • Figure 6 shows an exemplary DEL usage of form 6;
  • a cleavable site, a modifying group, or a functional molecule can be used in combination.
  • DEL can be prepared by converting hairpin strand DNA to single-stranded DNA.
  • a functional molecule e.g., biotin
  • the synthesized DEL compound ligate a double-stranded oligonucleotide chain having a functional molecule (e.g., biotin) at the 3' end (A), cleave the cleavable site (B), and (C).
  • the functional molecule is biotin
  • streptavidin beads or the like having biotin affinity are used to selectively remove biotin-bound oligonucleotide chains from the system.
  • FIG. 6 shows an exemplary usage of DEL obtained in Form 6.
  • FIG. DEL having a single-stranded DNA obtained in mode 6 forms a double strand with a modified oligonucleotide having a desired functional site (for example, a crosslinker-modified DNA such as a photoreactive crosslinker) to form a new function.
  • a desired functional site for example, a crosslinker-modified DNA such as a photoreactive crosslinker
  • Figure 7 shows an exemplary DEL usage of form 7;
  • a cleavable site can be used to introduce a crosslinker.
  • a cleavable site is cleaved from the synthesized DEL compound (A), a modified primer is provided (B), and a crosslinker-modified double-stranded DEL compound can be synthesized based on the provided primer. (C).
  • a crosslinker-modified double-stranded DEL compound can markedly improve detection sensitivity in DEL library screening (see Non-Patent Documents 7 and 11, etc.).
  • Example 1 the partial structures of hairpin-type DEL containing deoxyuridine (U-DEL1-sh, U-DEL2-sh, U-DEL3-sh, U-DEL4-sh, U-DEL5-HP, U-DEL6- 10 types of HP, U-DEL7-HP, U-DEL8-HP, U-DEL9-HP, and U-DEL10-HP) each incubation when verifying the cleavage reaction by USER (registered trademark) enzyme 1 is a graph representing the conversion rate of a cleavage reaction over time.
  • hairpin DELs U-DEL1, U-DEL2, U-DEL4, U-DEL7, U-DEL8, U-DEL9, U-DEL10, H-DEL, 1 is a schematic diagram showing the synthesis procedure of U-DEL5, U-DEL11, U-DEL12, U-DEL13, I-DEL1, I-DEL2, I-DEL3, R-DEL1, and BIO-DEL).
  • FIG. Hairpin DEL synthesis is achieved by two-step double-stranded ligation with double-stranded oligonucleotides Pr_TAG and CP using the corresponding headpieces as starting materials.
  • Example 2 the eight hairpin DELs (U-DEL1, U-DEL2, U-DEL4, U-DEL7, U-DEL8, U-DEL9, U-DEL10, and H-DEL) and the double-stranded DEL Fig. 3 is a graph showing Ct values measured by real-time PCR of (DS-DEL) for each sample amount. Samples treated with USER (registered trademark) enzyme for various DELs are indicated as "USER (+)", and untreated samples are indicated as "USER (-)”.
  • Deoxyuridine-containing cleavable hairpins DEL (U-DEL1, U-DEL2, U-DEL4, U-DEL7, U-DEL8, U-DEL9, and U-DEL10) are 2-fold after USER® enzyme treatment. It shows Ct values comparable to main-strand DEL (DS-DEL).
  • a hairpin DEL (U-DEL5, U-DEL7, U-DEL9, U-DEL11, U-DEL12 and U-DEL13) containing six deoxyuridines was cleaved using USER (registered trademark) enzyme.
  • FIG. 2 is an image of a gel obtained by denaturing polyacrylamide gel electrophoresis showing progress.
  • Example 4 denaturing polyacrylamide gel electrophoresis showing the progress of cleavage reaction by endonuclease V of hairpins DEL containing four deoxyinosines (I-DEL1, I-DEL2, I-DEL3 and I-DEL4) It is an image of the resulting gel. Note that the numbers in the figure indicate the number of each lane.
  • FIG. 10 is a gel image obtained by denaturing polyacrylamide gel electrophoresis, showing the progress of cleavage reaction of ribonucleoside-containing hairpin DEL (R-DEL1) by RNase HII in Example 5.
  • R-DEL1 ribonucleoside-containing hairpin DEL
  • FIG. 2 is a schematic showing the synthesis procedure of a model library containing 3 ⁇ 3 ⁇ 3 (27) compound species starting from U-DEL9-HP.
  • the synthesis of the model library is accomplished by three (cycles A, B, C) split-and-pool steps. Each cycle also includes a double-stranded oligonucleotide tag ligation reaction and a chemical reaction for introducing building blocks.
  • FIG. 10 is an image of a gel obtained by agarose gel electrophoresis, showing the progress of each cycle of ligation reaction in model library synthesis of Example 6.
  • FIG. 18A is a chromatograph obtained from a sample after completion of cycle C in the model library synthesis of Example 6.
  • FIG. 18B is the deconvolution result of the MS spectrum obtained from the sample after completion of Cycle C in the model library synthesis of Example 6.
  • FIG. 10 is a gel image obtained by denaturing polyacrylamide gel electrophoresis, showing the progress of the cleavage reaction of the model library with USER (registered trademark) enzyme in Example 6.
  • USER registered trademark
  • Example 7 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”) are images of gels obtained by denaturing polyacrylamide gel electrophoresis, showing the progress of the cleavage reaction by USER (registered trademark) enzyme. Note that the numbers in the figure indicate the number of each lane.
  • DEL compounds 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” are images of gels obtained by polyacrylamide gel electrophoresis, showing the results of primer extension reactions. Note that the numbers in the figure indicate the number of each lane.
  • Example 8 in order to compare the binding agent recovery efficiency of various photoreactive crosslinker-modified double-stranded DELs with or without photocrosslinking reaction, the recovered amount was measured by real-time PCR, and the Ct value and ⁇ Ct value (negative control It is a graph showing the difference from the Ct value). Samples without UV irradiation are indicated as "UV(-)", and samples with UV irradiation are indicated as "UV(+)”. Also, “solution S” is indicated as “S”, and “solution E” is indicated as "E”. Each notation in the graph corresponds to each sample as follows.
  • DEL compounds with 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” are images of gels obtained by polyacrylamide gel electrophoresis, showing the results of primer extension reactions. Note that the numbers in the figure indicate the number of each lane.
  • Example 11 the ⁇ Ct value calculated using the Ct value measured by real-time PCR was used to compare the binding agent recovery efficiency of various photoreactive crosslinker-modified double-stranded DELs with different linker structures with or without photocrosslinking reaction.
  • Fig. 3 is a graph showing (difference from negative control). A sample without UV irradiation is indicated as "UV(-)", and a sample with UV irradiation is indicated as "UV(+)”. Each notation in the graph corresponds to each sample as follows.
  • Fig. 10 is a graph showing the ⁇ Ct value (difference from negative control) calculated using the Ct value measured by real-time PCR, in order to compare the binding agent recovery efficiency with "modified double-stranded DEL".
  • Each bar in the graph corresponds to each sample in order from the left as follows.
  • Leftmost bar “PXL-DS-SABA-DEL3” without UV irradiation Second bar from left: “PXL-DS-ClSABA-DEL3” without UV irradiation
  • Third bar from left: Without UV irradiation "PXL-DS-mSABA-DEL3” 4th bar from the left: “PXL-DS-SABA-DEL3” with UV irradiation 5th bar from the left: “PXL-DS-ClSABA-DEL3” with UV irradiation 6th bar from left: “PXL-DS-mSABA-DEL3” with UV irradiation 7th bar from left: "PXL-DS-SABA-DEL4" without UV irradiation 8th bar from left: UV "PXL-DS-ClSABA-DEL4" without irradiation 9th bar from the left: "PXL-DS
  • FIG. 10 is a graph showing the ⁇ Ct value (difference from negative control) calculated using the Ct value measured by real-time PCR, in order to compare the binding agent recovery efficiency with "modified double-stranded DEL".
  • ⁇ Ct value difference from negative control
  • FIG. 10 is a gel image obtained by denaturing polyacrylamide gel electrophoresis, showing the progress of the cleavage reaction of the hairpin DEL compound (“mSABA-DEL5”) with USER® enzyme in Example 14.
  • mSABA-DEL5 hairpin DEL compound
  • a DEL compound (“SS-mSABA-DEL5") having a single-stranded DNA and a photoreactive crosslinker-modified primer "PXL-Pr5" were used to perform a primer extension reaction.
  • 1 is an image of a gel obtained by polyacrylamide gel electrophoresis.
  • FIG. 2 is an image of a gel obtained by polyacrylamide gel electrophoresis showing the results of performing an extension reaction.
  • FIG. 16 a DEL compound (“SS-mSABA-DEL") having a single-stranded DNA and a reactive group-modified primer "BCN-Pr" for crosslinker modification were used to perform a primer extension reaction.
  • Example 17 is an image of a gel obtained by polyacrylamide gel electrophoresis showing . Note that the numbers in the figure indicate the number of each lane.
  • a primer extension reaction was performed using a single-stranded DEL model library, a photoreactive crosslinker-modified primer "PXL-Pr” and a reactive group-modified primer for crosslinker modification "BCN-Pr”. is an image of a gel obtained by polyacrylamide gel electrophoresis, showing the results of performing . Note that the numbers in the figure indicate the number of each lane.
  • the compound library is a group of compound derivatives systematically collecting compounds that may have specific activities, such as drug candidate compounds.
  • This compound library is often synthesized based on combinatorial chemistry synthetic techniques and methodologies.
  • Combinatorial chemistry is a field of research related to experimental techniques for efficiently synthesizing a series of compound libraries, which are enumerated and designed based on combinatorial theory, through systematic synthetic routes.
  • DNA-encoded library As mentioned above and well known to those skilled in the art, one type of compound library based on combinatorial chemistry is the DNA-encoded library.
  • DNA-encoding libraries are abbreviated DEL where appropriate. DEL is also essentially synonymous with DNA-encoded compound library.
  • a DNA-encoded library means a library in which DNA tags are added to each compound in the library. The DNA tag has a sequence designed so that each structure of each compound can be identified, and functions as a compound label.
  • Nucleotides are generally understood as substances in which a phosphate group is attached to a nucleoside.
  • Nucleotide and nucleoside are terms well known to those skilled in the art, and one general aspect of nucleoside is that a nucleobase such as a purine base or a pyrimidine base is glycoside-bonded to the 1-position of a sugar such as a pentasaccharide. understood.
  • Nucleosides and nucleotides are also units constituting nucleic acids such as DNA and RNA.
  • Nucleic acid is also a concept well known to those skilled in the art, but is generally understood as a polymer of nucleotides.
  • the nucleic acid of the present invention is a polymer composed of nucleotides and nucleic acid analogues described below.
  • nucleic acid polymers composed of nucleotides and nucleic acid analogues
  • nucleic acid monomers such as nucleotides and nucleic acid analogues
  • nucleic acids are sometimes simply referred to as nucleic acids.
  • the latter usage is also a usage in accordance with common general technical knowledge, and can be understood by a person skilled in the art according to the appropriate context.
  • Nucleotide in a broad sense includes not only natural nucleotides (original nucleotides) but also artificial nucleotides (various nucleic acid analogues).
  • the broad definition of nucleotides in the present invention includes the following aspects.
  • Natural nucleoside nucleotides include adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxyuridine, deoxyguanosine, deoxycytidine, inosine or diaminopurine deoxyriboside).
  • nucleotides of nucleosides having 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 or 2-thiocytidine.
  • C a nucleotide having an intercalated nucleobase
  • D a non-natural nucleotide having ribose or 2'-deoxyribose
  • E a nucleot
  • nucleic acid analogs examples 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 acids (SNA), acyclic threoninol nucleic acids (aTNA), or nucleic acids in which the oxygen in ribose is replaced.
  • PMO locked nucleic acid
  • LNA locked nucleic acid
  • GAA glycol nucleic acid
  • TAA threose nucleic acid
  • SNA serinol Nucleic acids
  • aTNA acyclic threoninol nucleic acids
  • (F1) PMO PMO is a nucleic acid analogue having a morpholine ring at the sugar moiety and an uncharged phosphorodiamidate structure at the phosphodiester moiety.
  • (F2) LNA LNA is a nucleic acid analogue having a cross-linked structure in the sugar moiety. is bridged by Examples of bridge structures include methylene, propylene, ether or amino bridge structures. Typical LNAs include 2',4'-BNA (2'-O,4'-C-methano bridged nucleic acids).
  • F3 GNA Glycol nucleic acid is also called GNA. Examples include R-GNA or S-GNA.
  • TNA Threose nucleic acid is also called TNA. In this case ribose is replaced by ⁇ -L-threofuranosyl-(3′ ⁇ 2′).
  • SNA Serinol nucleic acids are also called SNA. In this case ribose is replaced by a serinol unit attached to a phosphodiester bond.
  • F6 aTNA Acyclic threoninol nucleic acids are also referred to as aTNA. Examples include D-aTNA or L-aTNA.
  • ribose is replaced by a threoninol unit attached to a phosphodiester bond.
  • F7 Sugar in which the oxygen in ribose is replaced Specific examples include those in which oxygen is replaced with S, Se, or alkylene (eg, methylene or ethylene).
  • G backbone-modified nucleotides (examples of the backbone-modified nucleotides include peptide nucleic acids (the peptide nucleic acids are also called PNAs.
  • the 2-aminoethyl-glycine linkage is attached to the ribose and phosphodiester backbones supersedes.
  • a nucleotide with a modified phosphate group examples include phosphorothioate, 5′-N-phosphoramidite, phosphoroselenate, boranophosphoric acid, boranophosphate, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, phosphotriesters, bridged phosphoramidates, bridged phosphorothioates, bridged methylene-phosphonates, and the like.
  • nucleotide when a nucleotide is described without particular limitation, it means a natural nucleotide.
  • Natural nucleotide is a term well known to those skilled in the art and is not particularly limited as long as it is essentially a naturally occurring nucleotide.
  • the natural nucleotide in the present invention is the nucleotide described in (A) above.
  • Nucleic acid analog Nucleic acid analog is a term well known to those skilled in the art, and the structure of the nucleic acid analog in the present invention is not limited as long as it has the effect of the present invention.
  • the nucleic acid analogue is a compound according to aspects (B) to (H) above.
  • the nucleic acid analogue in the present invention is a compound having a phosphate-corresponding site and a hydroxyl group-corresponding site in a nucleic acid monomer. Nucleic acid analogues are more preferably compounds having a phosphate moiety and a hydroxyl group. As one aspect, the nucleic acid analogue in the present invention is a compound that can be used as a monomer in a nucleic acid synthesizer. As is well known to those skilled in the art, in a nucleic acid synthesizer, the phosphoric acid (or corresponding site) of a nucleic acid analogue is phosphoramidized, and the hydroxyl group (or its corresponding site) is protected with a protecting group.
  • Nucleic acid oligomers can be synthesized.
  • a partial structure other than the phosphate site (or corresponding site) and hydroxyl group (or corresponding site) in the nucleic acid analog 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 effect of the present invention, but when the structural features of natural nucleic acids (deoxyadenosine, thymidine, deoxycytidine, deoxyguanosine) are confirmed as a reference here, the molecular weight is 322 (thymidine monophosphate) to 347 (deoxyguanosine monophosphate), and the number of atoms (oxygen atoms and phosphorus including atoms (hereinafter also referred to as the number of inter-residue atoms) is six.
  • the following are known as nucleic acid analogs that can be used in nucleic acid synthesizers.
  • the nucleic acid analogue is a compound (B1) characterized by: (B11) has a phosphoric acid (or corresponding site) and a hydroxyl group (or its corresponding site). (B12) composed of carbon, hydrogen, oxygen, nitrogen, phosphorus or sulfur; (B13) It has a molecular weight of 142 to 1,500. (B14) The number of inter-residue atoms is 5-30. (B15) The bond mode of atoms between residues are all single bonds or contain 1 to 2 double bonds and the rest are single bonds.
  • the nucleic acid analogue is compound (B2) characterized by: (B21) having a phosphoric acid and a hydroxyl group; (B22) composed of carbon, hydrogen, oxygen, nitrogen or phosphorus; (B23) It has a molecular weight of 142 to 1,000. (B24) The number of inter-residue atoms is 5-20. (B25) The bonding mode of atoms between residues are all single bonds.
  • the nucleic acid analogue is a compound (B3) characterized by: (B31) having a phosphoric acid and a hydroxyl group; (B32) composed of carbon, hydrogen, oxygen, nitrogen or phosphorus; (B33) has a molecular weight of 142 to 700; (B34) The number of inter-residue atoms is 5-12. (B35) The bonding modes of atoms between residues are all single bonds.
  • the nucleic acid analog is the following compounds (B41), (B42), (B43), (B44), (B5), (B51), or (B52).
  • B41 d-Spacer
  • B42 Amino C6 dT
  • B43 mdC
  • TAG-Amino B44
  • Uni-Link registered trademark
  • Amino Modifier B5
  • B51 Diethylene glycol phosphate or triethylene glycol phosphate
  • Oligonucleotides and oligonucleotide chains in the present invention mean polymers of nucleotides having one or more nucleotides at internal positions between the 5' and 3' ends and between the 5' and 3' ends.
  • a base sequence complementary to each other means that two oligonucleotides of a nucleic acid form a fixed pair of adenine and thymine (or uracil) or guanine and cytosine to form a so-called complementary base pair connected by hydrogen bonding.
  • a sequence of nucleotides that can be Formation of complementary base pairs is also called hybridization.
  • Complementary base pairing is a concept generally called "Watson-Crick base pairing" or "natural base pairing".
  • base pairs are Watson-Crick type, Hoogsteen base pairs, or other hydrogen bonding motifs (for example, diaminopurine and T, 5-methyl C and G, 2-thiothymine and A, 6- Hydroxypurine and C, pseudoisocytosine and G) base pairs formed by formation may also be used.
  • base pairs are Watson-Crick type, Hoogsteen base pairs, or other hydrogen bonding motifs (for example, diaminopurine and T, 5-methyl C and G, 2-thiothymine and A, 6- Hydroxypurine and C, pseudoisocytosine and G) base pairs formed by formation may also be used.
  • 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 the order of preference.
  • to hybridize in the present invention means the act of forming a double strand between oligonucleotides or oligonucleotide strands containing complementary base sequences, and the act of forming a double strand between oligonucleotides or oligonucleotide strands containing complementary sequences. It means a phenomenon in which they form a double strand.
  • a double strand means a state in which two nucleic acid strands form complementary base pairs (hybridize).
  • the two nucleic acid strands may be derived from two nucleic acid strands or from two nucleic acid sequences within one nucleic acid strand molecule.
  • a double-stranded oligonucleotide and a double-stranded oligonucleotide chain in the present invention mean a secondary structure formed by hybridizing two or more different oligonucleotide chains.
  • the two oligonucleotides may differ in length and may have non-hybridized regions.
  • the region where the double strands hybridize is the double strand.
  • double-stranded DNA means a secondary structure formed by hybridizing two different DNA strands.
  • Each DNA strand may have a different length and may have a non-hybridized region.
  • the DNA strand is not limited to naturally occurring deoxyribonucleotides, but means all oligonucleotide strands that can be amplified by DNA polymerase.
  • forming a double strand means that a double strand may be formed 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. .
  • a temperature of 4 to 40°C such as a temperature of 4 to 40°C, an aqueous solvent, and a pH of 4 to 10.
  • the nucleic acid is a nucleic acid that forms a double strand. .
  • the Tm value refers to the temperature at which half of the DNA molecules are annealed with the complementary strand.
  • a blunt end in the present invention means that the ends of a double-stranded oligonucleotide are paired without protruding from either end.
  • a protruding end in the present invention means that one of the ends of a double-stranded oligonucleotide has a protruding portion.
  • the overhang of the protruding end can be of any length, but is preferably 1 to 50 bases, more preferably 1 to 30 bases, even more preferably 1 to 15 bases, most preferably 2 to 6 bases. is the length of In certain embodiments, the overhangs can be used as hybridizing regions when performing sticky-end ligations.
  • PCR means polymerase chain reaction.
  • PCR is a means of amplifying oligonucleotide strands and is a technique well known to those skilled in the art.
  • (1) a double-stranded oligonucleotide chain to be amplified is dissociated into two single strands by heat treatment or the like, and (2) the temperature is adjusted to be suitable for the enzymatic reaction.
  • an enzyme such as DNA polymerase
  • an enzyme such as DNA polymerase
  • a primer means an oligonucleotide that can be annealed to a template oligonucleotide chain and extended by a polymerase in a template-dependent manner.
  • 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. It is preferably present at the end of the nucleotide chain.
  • a nick means a portion of a double-stranded oligonucleotide chain that lacks an internucleotide bond and breaks the oligonucleotide chain.
  • the 5' side of this missing portion may or may not have a phosphate group.
  • a gap means a portion where one or more consecutive nucleotides are deleted in a double-stranded oligonucleotide chain and the oligonucleotide chain is dissociated.
  • the 5' side of the deleted portion may or may not have a phosphate group.
  • a hairpin strand is a single-stranded structure in which two complementary nucleic acid strands are linked, and the characteristics of the hairpin strand and the hairpin strand DEL are as described above.
  • the terms "hairpin site”, “hairpin structure”, and “hairpin type” used in the present invention are understood as terms derived from the same concept as the above-mentioned "hairpin chain”.
  • nucleic acid ligation reaction and "ligation” means a reaction that connects the ends of nucleic acids.
  • Enzymatic nucleic acid ligation reaction and enzymatic ligation refer to a reaction that uses an enzyme to join the ends of nucleic acids.
  • Enzymes that can be used for nucleic acid ligation reactions are, for example, DNA ligase, RNA ligase, DNA polymerase, RNA polymerase, or topoisomerase.
  • DNA ligase is an enzyme that joins the ends of DNA strands with phosphodiester bonds.
  • a DNA ligase is understood as 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 ligases include DNA ligase I, II, III, IV and T4 DNA ligase.
  • RNA ligase is an enzyme that joins the ends of RNA strands with phosphodiester bonds.
  • RNA ligase is understood as a ligase belonging to EC number: 6.5.1.3.
  • the RNA ligase belongs to the poly(ribonucleotide):poly(ribonucleotide) ligase family.
  • RNA ligase is also called polyribonucleotide synthase or polyribonucleotide ligase.
  • chemical ligation means a reaction that joins the ends of nucleic acids together without using an enzyme.
  • Paired functional groups for chemical reactions include, for example, optionally substituted alkynyl groups and optionally substituted azide groups, optionally substituted dienes having a 4 ⁇ -electron system (e.g., substituted optionally substituted 1,3-unsaturated compounds such as optionally substituted 1,3-butadiene, 1-methoxy-3-trimethylsilyloxy-1,3-butadiene, cyclopentadiene, cyclohexadiene or furan.
  • optionally substituted alkynyl groups and optionally substituted azide groups optionally substituted dienes having a 4 ⁇ -electron system
  • substituted optionally substituted 1,3-unsaturated compounds such as optionally substituted 1,3-butadiene, 1-methoxy-3-trimethylsilyloxy-1,3-butadiene, cyclopentadiene, cyclohexadiene or furan.
  • an optionally substituted dienophile or optionally substituted heterodienophile having a 2 ⁇ -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 e.g., a pair of a phosphorothioate group and an iodo group (e.g., a 3′-terminal phosphorothioate group and a 5′-terminal iodo group), or a phosphoric acid and a hydroxy group pair (eg, a 5'-terminal phosphate group and a 3'-terminal hydroxy group pair or a 5'-terminal hydroxy group and a 3'-terminal phosphate group pair).
  • 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 common technical knowledge.
  • Artificial DNA PNA & XNA, 2014, Vol. 5, e27896, Current Opinion in Chemical Biology, 2015, Vol. 26, pp. 80-88.
  • selectively cleavable means that in a certain compound, only a specific site can be selectively cleaved under predetermined conditions without changing other molecular structures of the compound.
  • selectively cleavable site means a site that can be selectively cleaved under predetermined conditions in a certain compound.
  • the 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 a plurality of nucleic acids and cleaved by a specific sequence, or may be a site composed of a single nucleic acid.
  • the cleavable site is a nucleic acid
  • an established production method such as a nucleic acid synthesizer can be used and production efficiency is high
  • the reaction conditions for constructing the building block of DEL are the DNA tag Since it is essential that the part of the nucleic acid is not degraded, it is preferable from the viewpoint that if the cleavable site is a nucleic acid, it should not be degraded.
  • a more preferred structure of the "selectively cleavable nucleic acid” is a nucleic acid containing nucleotides not included in the DNA tag sequence of DEL. If the cleavable site is a nucleotide that is not included 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.
  • Deoxyadenosine, deoxyguanosine, thymidine, and deoxycytidine are preferable as nucleic acids used for DNA tag sequences. Accordingly, preferred structures for selectively cleavable sites are nucleic acids that are neither deoxyadenosine, deoxyguanosine, thymidine, nor deoxycytidine.
  • Examples of “selectively cleavable sites” include “nucleotides having cleavable bases”.
  • a “nucleotide with a cleavable base” in DEL has the N-glycosidic bond between the base and sugar moieties cleaved by the action of DNA glycosylase, leaving an abasic site.
  • Phosphodiester bonds flanking the abasic site can be altered by chemical conditioning (e.g., temperature elevation, basic hydrolysis, etc.) or by enzymes with apurinic/apyrimidinic (AP) endonuclease or AP lyase activity (e.g., endonuclease activity).
  • chemical conditioning e.g., temperature elevation, basic hydrolysis, etc.
  • AP apurinic/apyrimidinic
  • nuclease III endonuclease IV, endonuclease V, endonuclease VI, endonuclease VII, endonuclease VIII, APE1 (human-derived AP endonuclease), Fpg (formamidopyridine-DNA glycosylase), etc.) and cut into 1 base Form a gap or nick.
  • 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, 5,6-dihydroxydeoxythymidine and the like. Nucleotides with other cleavable bases will be apparent to those skilled in the art. DEL is selectively abasic by incorporating these "nucleotides with cleavable bases" into DEL and using a DNA glycosylase that specifically recognizes the structure.
  • DNA glycosylase refers to any enzyme having glycosylase activity, which recognizes any nucleobase portion in an oligonucleotide, cleaves the N-glycosidic bond between the base portion and the sugar portion, and decomposes.
  • An enzyme that creates a base site is an enzyme having glycosylase activity, which recognizes any nucleobase portion in an oligonucleotide, cleaves the N-glycosidic bond between the base portion and the sugar portion, and decomposes.
  • uracil DNA glycosylase (recognizes deoxyuridine), alkyladenine DNA glycosylase (recognizes 3-methyl-2'-deoxyadenosine, 7-methyl-2'-deoxyguanosine, and deoxyinosine), Fpg (8-hydroxydeoxy guanosine), endonuclease VIII (recognizes degraded pyrimidine bases such as 5,6-dihydroxydeoxythymidine and uracil glycol), SUMG1 (abbreviation for single-strand selective uracil-DNA glycosylase, recognizes deoxyuridine), etc. mentioned.
  • More preferred examples of the "selectively cleavable site" in the present invention include deoxyinosine and deoxyuridine.
  • a particularly preferred example of the "selectively cleavable site" in the present invention is deoxyuridine.
  • the "selectively cleavable site" in 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 and the compound portion constructed from multiple building blocks as substrates, and act by recognizing only “selectively cleavable sites”. It is preferable because Cleavage using the enzyme may also be achieved by changing the chemical conditions after structurally changing the "selectively cleavable site” with the enzyme. Examples of such enzymes include glycosylases and nucleases.
  • a glycosylase is an enzyme that has the function of hydrolyzing a glycosidic bond (a covalent bond formed by dehydration condensation between a sugar molecule and another organic compound).
  • DNA glycosylase is an enzyme that recognizes a nucleobase portion in an oligonucleotide and hydrolyzes its glycosidic bond, as described above.
  • a nuclease in the present invention is an enzyme that has the function of hydrolyzing the phosphodiester bond between the sugar and phosphate of nucleic acids.
  • Nucleases include, for example, AP endonucleases, nicking endonucleases, ribonucleases.
  • DNA glycosylase cleaves the phosphodiester bond adjacent to the abasic site generated by the action of any DNA glycosylase. Therefore, in the present invention, it is preferable to use DNA glycosylase and AP endonuclease in combination.
  • a nicking endonuclease (eg, Nb.BbvCI, Nb.BsmI, Nb.BsrDI, etc.) recognizes a specific DNA sequence and produces a nick in which the phosphodiester bond is cleaved on only one of the double strands.
  • Endonuclease V is also capable of generating nicks with the second phosphodiester bond cleaved 3' from the deoxyinosine and is useful in the practice of the present invention.
  • a ribonuclease is an enzyme that degrades RNA.
  • a ribonucleoside is used as a "selectively cleavable site" and can be used by allowing ribonuclease to act.
  • RNase HII a type of ribonuclease, is capable of generating nicks in which the 5' phosphodiester bonds of ribonucleotides incorporated into DNA sequences are cleaved and is useful in the practice of the present invention.
  • USER (registered trademark) means "Uracil-Specific Excision Reagent” Enzyme.
  • USER is an endonuclease cocktail that removes uracil containing uracil DNA glycosylase (UDG) and endonuclease VIII. USER removes uracil in double-stranded DNA to create a 1-base gap and breaks the DNA strand.
  • UDG uracil DNA glycosylase
  • UDG uracil DNA glycosylase
  • USER removes uracil in double-stranded DNA to create a 1-base gap and breaks the DNA strand.
  • UDG first removes the uracil base to create an abasic site.
  • An endonuclease then cleaves the phosphodiester bond to release baseless deoxyribose, creating a one-base gap.
  • USER® Enzyme and USER® Enzyme are USER® as defined above.
  • an exonuclease is an enzyme that has the function of sequentially hydrolyzing phosphodiester bonds from the 5' or 3' end of nucleic acids.
  • Exonucleases include, for example, lambda exonuclease, exonuclease III, T7 exonuclease.
  • Lambda exonuclease is an enzyme that degrades DNA phosphorylated at the 5' end of double-stranded DNA.
  • the present invention is useful in converting double-stranded DEL to single-stranded DEL.
  • a building block is a portion that has a functional group and can constitute a part of a compound, and may be in the form of a compound.
  • the base sequence that can identify each building block means a specific base sequence designed to correspond to the structure of each building block.
  • Designing a sequence means assigning a nucleobase sequence to each structure, for example, nucleobase sequence AAA for building block structure A, nucleobase sequence TTT for structure B, and nucleobase sequence CGC for structure C. means that The sequence can be freely designed as long as the object of the present invention can be achieved. For example, any number of base sequences can be assigned to one building block.
  • an oligonucleotide tag is a partial structure containing an oligonucleotide containing a base sequence that allows identification of the structure of the partial structure constructed by building blocks.
  • Oligonucleotide tags in the present invention may be oligonucleotides corresponding to each building block, or longer oligonucleotides containing oligonucleotides corresponding to multiple building blocks.
  • Nucleotides constituting the oligonucleotide tag of the present invention are not limited as long as the effect of the present invention is achieved, but from the viewpoint of easiness of amplification by PCR and analysis by a sequencer, nucleotides suitable for these operations are desirable. .
  • nucleotides examples include nucleotides having the above-described natural nucleobase as the base portion and having the above-described ribose or 2′-deoxyribose as the sugar portion. More preferred examples are deoxyadenosine, thymidine, deoxycytidine or deoxyguanosine.
  • a head piece in the present invention means a starting compound for preparing a compound library such as DEL.
  • the structure of the headpiece of the present invention is not limited as long as the object of the present invention is achieved, but in a most typical embodiment, 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 at least one selectively cleavable site in the structure.
  • the DNA tag is preferably a double-stranded oligonucleotide strand and there are preferably two sites to which the oligonucleotide tag can be ligated.
  • the headpiece is the compound shown in the schematic below.
  • the headpiece is desirably chemically stable.
  • the headpiece preferably has a structure that allows the DNA tag and the building block to be arranged in appropriate spaces.
  • the headpiece preferably has moderate flexibility.
  • more appropriate spatial arrangement and flexibility will be explained.
  • the structural characteristics of the headpiece described here may be achieved by the headpiece alone, or by combining the headpiece and a bifunctional spacer.
  • preferable structural characteristics of the headpiece are such that the headpiece and the DNA tag do not inhibit the formation reaction of the building block, and conversely, the headpiece and the building block do not inhibit the elongation reaction of the DNA tag. be.
  • preferred structural properties of the headpiece are such that the headpiece and the DNA tag portion do not affect the interaction between the building block compound (library compound) and the target (target protein, etc.). be.
  • preferred headpiece structural features are those that orient DNA tags and building block moieties in opposite directions (eg, 90 degrees or more opposite).
  • the preferred structural characteristics of the headpiece are such that the loop portion of the headpiece and the building block are separated by several atoms to ten and several atoms in terms of the skeleton of the organic compound.
  • the headpiece preferably has a moderate affinity for the DNA tag portion and building block portion.
  • Moderate affinity means chemical reactivity and stability such that a bond can be formed, maintained, and cleaved under desired conditions, eg, to practice the present invention.
  • a bifunctional spacer means a spacer moiety that has at least two reactive groups that allow bonding between the building block moiety and the headpiece.
  • headpiece In the description of the present invention, the terms “headpiece”, “headpiece compound”, and “compound for headpiece” are terms indicating compounds of the same concept.
  • a compound for use as a headpiece can be understood in essentially the same way as “the use of a compound as a headpiece” from the point of use, and from the point of view of the method, "a compound used as a headpiece”. can be understood essentially the same as “method of using as a piece”. The same is true for compound libraries.
  • a preferred structure of the headpiece will be described below, but the structure of the headpiece is not limited as long as the effects of the present invention are achieved.
  • the headpiece includes: (D) a reactive functional group that has at least one site that can be directly linked to a building block or indirectly via a bifunctional spacer; (L) a linker extending from a 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 with one binding site that can be linked to the other strand of the oligonucleotide tag, and (LP) a loop site that connects the linker and the two oligonucleotide strands; is composed of It has at least one selectively cleavable site in at least one of the E, F, or LP sites.
  • the headpiece is a compound represented by formula (I) below.
  • E and F are independently oligomers composed of nucleotides or nucleic acid analogues, provided that E and F contain base sequences complementary to each other to form a double-stranded oligonucleotide
  • LP is the loop site
  • L is a linker
  • D is a reactive functional group.
  • the partial structure of the site that binds to the linker may be referred to as a linking site or (LS).
  • E-LP-F may be collectively referred to as a hairpin site.
  • first and second oligonucleotide strands Preferred embodiments of the first oligonucleotide strand (E) and the second oligonucleotide strand (F) are described below.
  • the first oligonucleotide strand (E) and the second oligonucleotide strand (F) form a double strand in the molecule via the loop site (LP), and the headpiece forms a hairpin structure.
  • the preferred chain length for intramolecular double chain formation is 3 bases or more, more preferably 4 bases or more, and still more preferably 6 bases or more.
  • the chain length of E and F is 3 to 40 each in one embodiment.
  • the chain length of E and F is 4 to 40 each in one embodiment.
  • the chain length of E and F is 6 to 25 each in one embodiment.
  • the site where the oligonucleotide tag is ligated preferably has a structure suitable for enzymatic ligation or chemical ligation.
  • the ligation of the headpiece and the oligonucleotide tag is performed by enzymatic double-stranded ligation.
  • the first and second oligonucleotide strands preferably form overhanging ends for ligation.
  • the chain length of the protruding end is preferably 2 bases or more, more preferably 2 to 10 bases, still more preferably 2 to 5 bases. Therefore, one of the first and second oligonucleotide strands is preferably longer than the other strand by the length of the protruding end.
  • the 5' end of the strand having the 5' end of the headpiece among the first and second oligonucleotide strands is preferably phosphorylated.
  • first and second oligonucleotide strands may contain part or all of the primer binding sequences for PCR.
  • a suitable chain length for the primer binding sequence is 17 to 25 bases.
  • linker Preferred embodiments of the linker (L) are described below.
  • a linker as described above, is a moiety that extends from a reactive functional group and binds to a linking moiety.
  • the linker is a divalent group (-L-) derived from the following aspects.
  • linker is the following embodiment (L1).
  • (L1) optionally substituted C1-20 aliphatic hydrocarbon optionally substituted by 1 to 3 heteroatoms, or (2) optionally substituted C6-14 aromatic hydrocarbon.
  • L is the following aspect (L2), (L3), (L4) or (L5).
  • (L2) optionally substituted C1-6 aliphatic hydrocarbons, optionally substituted C1-6 aliphatic hydrocarbons optionally substituted by 1 or 2 oxygen atoms, or optionally substituted C6-10 aromatic family hydrocarbons.
  • (L3) C1-6 aliphatic hydrocarbons substitutable with the substituent group ST1, or benzene substitutable with the substituent group ST1.
  • the substituent group ST1 is a group composed of a C1-6 alkyl group, a C1-6 alkoxy group, a fluorine atom and a chlorine atom.
  • a reactive functional group as described above, has at least one site that can be directly linked to a building block or indirectly via a bifunctional spacer, and is the site of attachment to the linker group.
  • the reactive functional group becomes a monovalent group (D-) in the headpiece, and in DEL a "reactive functional group-derived divalent group" (-D- ).
  • D is an amino group
  • R-HN- a specific structure for (D-)
  • R is a substituent as described below.
  • R is not limited as long as the effects of the present invention are achieved, but in the following aspects (D1) to (D5), R is preferably (1) a hydrogen atom, or (2) unsubstituted or A C1-6 alkoxy group, a C1-6 alkyl group substituted with 1 to 3 substituents selected singly or differently from the substituent group consisting of a fluorine atom and a chlorine atom.
  • R is more preferably a hydrogen atom or a C1-3 alkyl group, still more preferably a hydrogen atom.
  • (D-) is a methylene group having a leaving group (X-)
  • the specific structure of (D-) is (X-CH 2 -), for example, amino group, hydroxy or with nucleophiles such as thiol groups to form carbon-nitrogen, carbon-oxygen, or carbon-sulfur bonds.
  • the specific structure of (-D-) is (-CH 2 -).
  • the specific structure of (D-) is (HOC-).
  • Aldehyde groups for example, by reductive amination reactions with amino groups, form carbon-nitrogen bonds, in which (-D-) become -CH 2 -, and, for example, by reaction with phosphorus ylide groups, carbon-carbon double bonds are formed.
  • the site (D-) is the following aspect (D1).
  • D1 A functional group capable of forming a C—C, amino, ether, carbonyl, amide, ester, urea, sulfide, disulfide, sulfoxide, sulfonamide, or sulfonyl bond.
  • (-D-) becomes a C-C, amino, ether, carbonyl, amide, ester, urea, sulfide, disulfide, sulfoxide, sulfonamide, or sulfonyl bond.
  • (D-) is the following aspect (D2), (D3), (D4) or (D5).
  • (D2) A C1 hydrocarbon with a leaving group, an amino group, a hydroxyl group, a precursor of a carbonyl group, a thiol group, or an aldehyde group.
  • (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;
  • (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 carboxy group.
  • (D5) Primary amino group. In this case, (-D-) becomes -NH-.
  • the loop site (LP) is designed so that the first oligonucleotide strand (E) and the second oligonucleotide strand (F) form an intramolecular duplex and the headpiece can form a hairpin structure. preferably. That is, the loop site (LP) preferably has a chain length and binding flexibility that make the loop structure thermodynamically stable.
  • the loop site (LP) is: LP is (LP1) is a loop site represented by p-LS-(LP2) q, LS is a partial structure selected from the group of compounds described in (A) to (C) below, (A) Nucleotide (B) Nucleic acid analogue (C) C1-14 trivalent group LP1 optionally having a substituent may be alone or different from the group of compounds described in the following (1) and (2) is each partial structure selected by p pieces, (1) Nucleotide (2) Nucleic acid analog LP2 is each partial structure selected individually or differently from the group of compounds described in (1) and (2) below, (1) The total number of nucleotides (2) nucleic acid analogues p and q is 0-40.
  • loop site Further preferred aspects of the loop site are as described above.
  • the structure of the loop site will be supplemented below.
  • nucleotides are the natural nucleotides described above, and the nucleic acid analogs are as described above.
  • LP1 is each partial structure selected singly or differently from the group of compounds described in (1) and (2) below, and LP2 is described in (1) and (2) below. It is each partial structure selected singly or differently from the group of compounds represented by q.
  • Nucleotides (2) Nucleic acid analogues Individually or p different selected means that, for example, when p is 4, LP1 is AATG, ATCG, TC (d-Spacer) G or A (d-Spacer) ( d-Spacer) C, may be selected singly or differently from the group of compounds described in (1) and (2). The same is true for LP2.
  • the loop site may contain part or all of the primer binding sequence for PCR.
  • LS is (A) a nucleotide or (B) a nucleic acid analogue.
  • 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 analogues are linked as monomers. Oligomers can also be referred to as chain compounds.
  • nucleic acid oligomers of the invention are either oligonucleotide strands, nucleic acid analog strands, or mixed strands of nucleotides and nucleic acid analogs.
  • the loop site becomes a nucleic acid oligomer.
  • the headpiece can be manufactured by a nucleic acid synthesizer, which is significantly preferable in practice.
  • LS is (A) a nucleotide or (B) a nucleic acid analogue
  • a linker site (L) and a reactive functional group site (D) are combined with LS for nucleic acid synthesis.
  • Monomers can be prepared and then nucleic acid oligomers synthesized. Examples of such nucleic acid synthesis monomers include the aforementioned Amino C6 dT, mdC (TEG-Amino), Uni-Link (registered trademark) Amino Modifier, and the like.
  • the nucleotide portion corresponds to the linking site (LS), and the side chain portion extending from the base corresponds to the linker site (L) and the reactive functional It corresponds to the base site (D).
  • the reactive functional group (D) may be protected with a protecting group.
  • the nucleic acid analogue is the following compound (B6).
  • (B6) A compound in which the (-LD) is bound to the base portion of a nucleotide.
  • the nucleic acid analog is the following compounds (B61), (B62), (B63), (B64) or (B65).
  • (B61) (-L-D) is (-L1-D1) (B6) (B62) (-LD) is (-L2-D2) (B6).
  • (B63) (-LD) is (-L3-D3) (B6).
  • (B64) (-LD) is (-L4-D4) (B6).
  • B65 The compound according to any one of (B61) to (B64), wherein (-D) is (-D5).
  • LS is (A) a nucleotide or (B) a nucleic acid analogue
  • a nucleic acid oligomer is first synthesized, and then the linker site (L) and the reactive functional group site ( D) can be combined.
  • the linker site (L) and the reactive functional group site ( D) can be combined.
  • the "specific nucleic acid analogue” include the aforementioned Amino C6 dT, mdC (TEG-Amino), and Uni-Link (registered trademark) Amino Modifier.
  • mdC (TEG-Amino) itself corresponds to the linking site (LS), and from the base side chain, the additional site that further binds to the linker site (L) and the reactive functional group site (D). Equivalent to.
  • the chain length of the loop site is such that the first oligonucleotide chain (E) and the second oligonucleotide chain (F) form a double chain in the molecule, and the headpiece forms a hairpin structure.
  • Chain length is preferred.
  • the sum of p and q is 1-40.
  • the sum of p and q is 2-20.
  • the sum of p and q is 2-10.
  • the sum of p and q is 2-7.
  • the loop site of the present invention is (A) is composed of nucleotides and the following nucleic acid analogues (B41), (B42), (B43), (B44) or (B52).
  • B41 d-Spacer
  • B42 Amino C6 dT
  • B43 mdC
  • TAG-Amino B44
  • Uni-Link registered trademark
  • Amino Modifier B52
  • LS is preferably B42, B43 or B44.
  • LP1 and LP2 are preferably A, B41 or B52.
  • the loop site is a nucleic acid oligomer according to the sequences (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
  • the number of cuttable parts is preferably 5 or less, more preferably 1 to 2.
  • At least one cleavable site is in the first oligonucleotide strand or between the first oligonucleotide strand and the linker attachment site. and the at least one cleavable site is preferably in the second oligonucleotide strand or between the second oligonucleotide strand and the linker attachment site.
  • the position of the cleavable site is preferably within 20 bases starting from the binding portion between the loop site and the first oligonucleotide strand or the second oligonucleotide strand. Yes, more preferably within 10 bases, still more preferably within 3 bases.
  • the present invention provides suitable conditions in a method of deriving and evaluating a DEL containing a cleavable site in its DNA strand into a cross-linker-modified double-stranded DEL.
  • the position of the cleavable site is in the 3′ direction from the binding portion between the loop site and the first oligonucleotide strand or the second oligonucleotide strand. , preferably within 20 bases, more preferably within 10 bases, still more preferably within 3 bases, and most preferably within 1 base.
  • the compound constituting the DEL of the present invention is a compound represented by the following formula (II).
  • 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 form a double-stranded oligonucleotide, LP is the loop site; L is a linker; D is a divalent group derived from a reactive functional group, Sp is a bond or a bifunctional spacer; An is a substructure composed of at least one building block.
  • X is attached to E at the 5' end
  • Y is attached to F at the 3' end
  • preferred embodiments of E, F, LP, L and D in the compounds of formula (II) above are the preferred embodiments of E, F, LP, L and D described with respect to formula (I) above. It is the same as the aspect. Preferred aspects of X, Y, Sp, and An are described separately.
  • a bifunctional spacer is a spacer moiety that has at least two reactive groups that allow bonding of the substructure An of the compound library to the headpiece.
  • 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.
  • the reactive group (SpX) becomes a monovalent group (-SpX) in the bifunctional spacer alone (reagent state before bonding to the headpiece), and DEL ( In the state where it is combined with the headpiece), it becomes "a divalent group derived from a reactive group" (-SpX-) based on the above (-SpX).
  • the reactive group (SpD) becomes a monovalent group (SpD-) in the state before bonding to An, and in DEL (the state bonded to An), the "reactive group-derived is a divalent group” (-SpD-).
  • SpX are reactive groups that form amino, carbonyl, amide, ester, urea, or sulfonamide bonds.
  • SpX is 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, carboxyl halide group, aldehyde group, or sulfonyl halide group
  • SpX2 carboxy group
  • SpX3 carboxy group
  • SpD is (D1), (D2), (D3), (D4) or (D5) as described above.
  • SpL is (L1), (L2), (L3), (L4) or (L5) described above.
  • SpL is the following (SpL1), (SpL2) or (SpL3).
  • SpL1 Polyalkylene glycol, polyethylene, C1-20 aliphatic hydrocarbon optionally substituted with heteroatom, peptide, oligonucleotide, or combinations thereof.
  • SpL2 polyalkylene glycol, polyethylene, C1-10 aliphatic hydrocarbon, or peptide
  • SpL3 polyethylene glycol, or polyethylene
  • 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)
  • the (Sp-DL) portion of the compound constituting DEL is the following (SpDL1), (SpDL2), (SpDL3) (SpDL4), (SpDL5), (SpDL6), (SpDL7), (SpDL8), (SpDL9), or (SpDL10).
  • the headpiece can be synthesized on a nucleic acid synthesizer.
  • a linker site (L) and a reactive functional group site (D) are bound to LS to prepare a monomer for nucleic acid synthesis, and then synthesize a nucleic acid oligomer.
  • Examples of such nucleic acid synthesis monomers include the aforementioned Amino C6 dT, mdC (TEG-Amino), Uni-Link (registered trademark) Amino Modifier, and the like.
  • the length of the linker site may be limited.
  • introduction of a suitable bifunctional spacer makes it possible to adjust the distance between the headpiece and An, which is advantageous in carrying out the invention.
  • C1-C6 and “C1-6” in terms such as “C1-C6 alkyl group” and “C1-6 alkyl group” have 1 to 6 carbon atoms. means that Similarly, when m and n are integers, the description “Cm to Cn” or “Cm to n” means that the number of carbon atoms is m to n. Therefore, “C1-C6 alkyl group” and “C1-6 alkyl group” refer to alkyl groups having 1 to 6 carbon atoms, and “C1-C6 alkylene” and “C1-6 alkylene” refer to is 1 to 6 alkylene.
  • C1-6 alkyl in the present invention means a linear or branched alkyl group having 1 to 6 carbon atoms. Specific examples include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl and the like.
  • C1-3 alkyl in the present invention means a linear or branched alkyl group having 1 to 3 carbon atoms. Specific examples are methyl, ethyl, propyl, isopropyl.
  • C1-6 alkoxy means linear or branched alkoxy having 1 to 6 carbon atoms. Specific examples include methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentyloxy, hexyloxy and the like.
  • C1-3 alkoxy means linear or branched alkoxy having 1 to 3 carbon atoms. Specific examples are methoxy, ethoxy, propoxy, isopropoxy.
  • hydrocarbon means a chain, branched or cyclic saturated or unsaturated compound composed only of carbon atoms and hydrogen atoms.
  • aliphatic hydrocarbons mean non-aromatic hydrocarbons.
  • An “aliphatic hydrocarbon” may be linear, branched or cyclic, and saturated or unsaturated. Exemplary structures include structures with alkyl, alkenyl, alkynyl, cycloalkyl or cycloalkenyl, or combinations thereof.
  • C1-20 aliphatic hydrocarbon means an aliphatic hydrocarbon having 1 to 20 carbon atoms.
  • C1-10 aliphatic hydrocarbon means an aliphatic hydrocarbon having 1 to 10 carbon atoms.
  • C1-6 aliphatic hydrocarbon means an aliphatic hydrocarbon having 1 to 6 carbon atoms.
  • aromatic hydrocarbon as used in the present invention means an aromatic hydrocarbon among hydrocarbons.
  • C6-14 aromatic hydrocarbon means aromatic hydrocarbon having 6 to 14 carbon atoms. Specific examples include benzene, naphthalene and anthracene.
  • C6-10 aromatic hydrocarbon means aromatic hydrocarbon having 6 to 10 carbon atoms. Specific examples are benzene or naphthalene.
  • the heteroaromatic ring of the present invention is an aromatic heterocyclic ring having as a heteroatom in the ring structure an element selected singly or differently from the group consisting of nitrogen, oxygen and sulfur.
  • the aromatic heterocycle is a "C1-9 aromatic heterocycle” having 1 to 9 carbon atoms, and in one embodiment, the "C1-9 aromatic heterocycle” has 5 to 10 membered aromatic heterocycle”.
  • the aromatic heterocyclic ring is a "C1-5 aromatic heterocyclic ring” having 1 to 5 carbon atoms, and in one aspect, the "C1-5 aromatic heterocyclic ring” membered aromatic heterocycle”.
  • the aromatic heterocycle is a "C2-9 aromatic heterocycle” having 2 to 9 carbon atoms, and in one embodiment, the "C2-9 aromatic heterocycle” membered aromatic heterocycle”. In one embodiment, the aromatic heterocycle is a "C2-5 heteroaromatic ring” having 2 to 5 carbon atoms, and in one embodiment, the "C2-5 heteroaromatic ring” has 5 to 6 membered aromatic heterocycle”.
  • the nitrogen-containing aromatic heterocycle of the present invention is an aromatic heterocycle having nitrogen as a heteroatom in the ring structure.
  • the nitrogen-containing aromatic heterocycle is a "C1-5 nitrogen-containing aromatic heterocycle” having 1 to 5 carbon atoms, and in one embodiment, "C1-5 nitrogen-containing aromatic heterocycle ”is a 5- to 6-membered aromatic heterocycle”.
  • the nitrogen-containing aromatic heterocycle is a "C2-5 nitrogen-containing aromatic heterocycle” having 2 to 5 carbon atoms, and in one embodiment, a "C2-5 nitrogen-containing aromatic heterocycle ”is a 5- to 6-membered aromatic heterocycle”.
  • the non-aromatic heterocycles of the present invention are non-aromatic heterocycles having as heteroatoms in the ring structure an element selected singly or differently from the group consisting of nitrogen, oxygen and sulfur.
  • Non-aromatic heterocycles may contain partially unsaturated bonds.
  • the non-aromatic heterocycle is a "C2-9 non-aromatic heterocycle” having 2 to 9 carbon atoms, and in one embodiment, the "C2-9 non-aromatic heterocycle” is 5- to 10-membered non-aromatic heterocyclic ring”.
  • the "C1-14 trivalent group” means a trivalent group derived from a compound having 1 to 14 carbon atoms.
  • the structure is not limited as long as the effects of the present invention are achieved.
  • 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, more preferably an oxygen atom, a nitrogen atom or a sulfur atom.
  • propyl (—CH 2 —CH 2 —CH 3 ) as an example of a hydrocarbon
  • “propyl optionally substituted with heteroatoms” means that methylene (—CH 2 —) in alkyl is Ether ((--CH 2 --O--CH 3 ) or (--O--CH 2 --CH 3 )) substituted with oxygen, or amine ((--CH 2 --NH--CH 3 ) or (--CH 2 --NH---CH 3 ) substituted with nitrogen —NH—CH 2 —CH 3 )) and other structures.
  • 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.
  • polypeptides and peptides mean compounds or partial structures formed by linking amino acids.
  • Amino acid is a general term for organic compounds having both amino and carboxyl functional groups.
  • Amino acids constituting the polypeptides and peptides of the present invention are not particularly limited, and include modified amino acids and the like.
  • proline which is classified as an imino acid
  • Amino acids constituting the polypeptides and peptides of the present invention are preferably ⁇ -amino acids, more preferably "protein-constituting amino acids”.
  • halogen atoms include fluorine atoms, chlorine atoms, bromine atoms and iodine atoms.
  • the substituent on the nitrogen atom is preferably a C1-6 alkyl group or a hydrogen atom, more preferably a hydrogen atom.
  • a CC bond means a carbon-carbon bond.
  • CC bonds include single, double and triple bonds.
  • steps a and/or c of the production method of the present invention a bond appropriately selected from the above 11 types is constructed. These eleven types of bonds are among the most fundamental bonding modes in organic chemistry, and the reactions to build them are also well known to those skilled in the art. Therefore, in designing and constructing the partial structure An of the compound library of the present invention, those skilled in the art can appropriately combine these 11 types of bonds and use them.
  • the organic compound composed of elements selected singly or differently from the element group consisting of H, B, C, N, O, Si, P, S, F, Cl, Br and I is It is an organic compound that is built up by bonding.
  • the partial structure An of the compound library of the present invention is constructed from the above 12 elements. These twelve elements are particularly basic elements in organic compounds, and the reactions to build them are also well known to those skilled in the art. Therefore, in designing and constructing the partial structure An of the compound library of the present invention, those skilled in the art can appropriately combine these 12 elements and use them.
  • a low-molecular-weight organic compound having substituents selected singly or differently from the substituent group consisting of an aryl group, a non-aromatic cyclyl group, a heteroaryl group and a non-aromatic heterocyclyl group means a chemical structure understood by each name. It is a low-molecular-weight organic compound having A low-molecular-weight compound is a concept well known to those skilled in the art, and examples of preferred molecular weights of low-molecular-weight compounds in the present invention are mentioned separately.
  • the aryl group of the present invention is preferably a C6-10 aryl group, more preferably a phenyl group.
  • the non-aromatic cyclyl group of the present invention is preferably a 5- to 8-membered non-aromatic cyclyl group, more preferably a 5- or 6-membered non-aromatic cyclyl group.
  • the non-aromatic cyclyl group may contain a partially unsaturated bond.
  • the heteroaryl group and non-aromatic heterocyclyl group of the present invention are groups having an element selected singly or differently from the group consisting of nitrogen, oxygen and sulfur as a heteroatom within the ring structure.
  • the heteroaryl group, non-aromatic heterocyclyl group of the present invention is preferably a 5- to 8-membered group, more preferably a 5- or 6-membered group, and the non-aromatic heterocyclyl group is a partially unsaturated bond may contain
  • the partial structure An of the compound library of the present invention has the above four groups. These four groups are particularly basic substructures in organic compounds, and reactions for building them into compounds are also well known to those skilled in the art. Therefore, in designing and constructing the partial structure An of the compound library of the present invention, those skilled in the art can appropriately combine these four groups and use them.
  • the synthesis history of An means a record of all the operations performed until An was synthesized, and in particular, the structure and order of the building blocks used until An was synthesized. For example, if the reaction is carried out in two or more separate reaction vessels, each using different building blocks and/or using different reaction conditions, the reaction may be carried out with oligos of predetermined sequence before or after the reaction. By ligating a nucleotide chain to the product in each reaction vessel, the synthesis history is given as sequence information of the oligonucleotide. By repeating such operations until An is constructed, an oligonucleotide of Bn having the synthesis history of An is constructed.
  • Split-and-pool synthesis is a synthetic method developed by Geisen et al. as a combinatorial chemical construction method for peptide libraries using solid-phase synthesis during the early days of combinatorial chemistry.
  • Split-and-pool synthesis is also called a split-mix method.
  • a sample is cut out from a solid-phase carrier to which amino acids are peptide-bonded at each step of peptide extension. After the N types of carriers are mixed and homogenized, they are equally divided to extend the next N types of amino acids.
  • one type of peptide chain will be generated for each carrier, and if all 20 natural amino acids are applied at each step, a peptide library that can be combined with all peptides of a specific length will be constructed.
  • assays can be performed using peptides on a solid-phase carrier using the ELISA method.
  • the carrier particles that have reacted with the assay are picked up (for example, fluorescence-labeled carrier particles of about 0.1 mm are picked up with an optical microscope).
  • the target peptide sequence is determined by an instrumental analyzer (peptide analyzer, etc.) from the peptides of the particles, or peptide sequences that are candidates for screening are indirectly identified by other combinatorial chemical identification methods (eg, tagging method). can decide.
  • A2-Sp-C-B2 (A2(a)-Sp-C-B2(a), A2(b)-Sp-C-B2(b)...
  • w types of structures ⁇ 3 ( ⁇ 3(a ⁇ w)) and w types of ⁇ 3 ( ⁇ 2(a ⁇ w)) corresponding thereto are prepared, and w pieces of (A2(a ⁇ v)-Sp- C-B2 (a-v) mixtures) are subjected to steps (c) and (d), respectively.
  • a mixture of (v ⁇ w) types of A3-Sp-C-B3 compound library is obtained by mixing the obtained w products. For example, if this mixture is subjected to a drug receptor binding test, (v ⁇ w) kinds of compounds can be screened in one go. By washing away compounds that did not bind to the drug receptor, only bound compounds can be isolated.
  • the DNA of the isolated A3-Sp-C-B3 compound is amplified to a sequence-readable amount, and the sequence information allows the structure of A3 to be determined.
  • compound library building block, split-and-pool, and the like are terms well known to those skilled in the art in fields such as combinatorial chemistry, and can be performed as appropriate with reference to the following literature.
  • Takashi Takahashi, Takayuki Doi “Combinatorial Chemistry”, Synthetic Organic Chemistry Association Journal, 2002, Vol. 60, pp. 426-433 (2) Combinatorial Chemistry Study Group ed.
  • a DNA-encoded library is a compound library consisting of a group of compounds (DNA-encoded compounds) labeled with DNA or oligonucleotides having substantially the same function as DNA.
  • DNA-encoded compounds are screened in the form of mixtures of 10 2 -10 20 compounds, and the DNA sequences contained in the compounds obtained are identified by techniques known in the art (e.g., next-generation Identification by use of sequencers and/or use of microarrays) makes it possible to identify the structure of the compound.
  • a technique of contacting a target such as a protein with a DNA-encoded library and selecting compounds that bind to the target can be selected.
  • Bio target is a term well known to those skilled in the art, but in one aspect, in the present invention, "biological target” is a target in the development of drugs such as pharmaceuticals and agricultural chemicals.
  • a group of biological substances such as enzymes (e.g., kinases, phosphatases, methylases, demethylases, proteases, and DNA repair enzymes), proteins involved in protein:protein interactions (e.g., receptor ligands), Included are receptor targets (eg, GPCRs), ion channels, cells, bacteria, viruses, parasites, DNA, RNA, prions, or carbohydrates.
  • evaluation of biological activity is a term well known to those skilled in the art.
  • evaluation of biological activity refers to the biological activity of a compound (e.g., the ability to bind to a biological target). , enzyme activity inhibition function, enzyme activity promotion function, etc.).
  • a compound e.g., the ability to bind to a biological target.
  • enzyme activity inhibition function e.g., enzyme activity promotion function, etc.
  • the aforementioned Patent Documents 2 and 3, Non-Patent Documents 1 to 6, etc. can also be referred to.
  • “Functional evaluation” is a term well known to those skilled in the art, but in one aspect, in the present invention, “functional evaluation” refers to a specific function of a compound (e.g., binding ability, biological activity, luminescence It is to evaluate the presence or absence or strength of characteristics, etc.).
  • the present invention provides a plurality of approaches with several advantages regarding DEL and methods for producing DEL by using DNA strands with cleavable sites. Forms 1 through 7 are described in detail below.
  • Form 1 The present invention provides a DEL using the "hairpin-shaped headpiece having a cleavable site”.
  • PCR can be performed with high efficiency by deriving double-stranded oligonucleotides that are not bound at loop sites.
  • Mode 2 (Regarding Form 2)
  • the cleavable site may be present in the second oligonucleotide strand.
  • the features of Mode 2 are similar to Mode 1, except for the cleavable site.
  • the cleavable site is present in both the first and second oligonucleotide strands. good.
  • the loop site is cleaved from both oligonucleotide strands, which is expected to further improve PCR efficiency.
  • the cleavable site may be present in both the first oligonucleotide strand (E) and the second oligonucleotide strand (F), and the cleavable
  • the structure of each site may be different. In such cases, differences in the properties of the two (or more) cleavable sites can be exploited to control the cleavage site.
  • deoxyuridine may be used as the cleavable site in the first oligonucleotide strand (E) and deoxyinosine as the cleavable site in the second oligonucleotide strand (F).
  • the USER enzyme can be used to selectively cleave the deoxyuridine of the first oligonucleotide strand (E).
  • alkyladenine DNA glycosylase and endonuclease VIII can be used to selectively cleave the second oligonucleotide chain (F) at a cleavage site starting from deoxyinosine.
  • the present invention can also have a cleavable site in the DNA tag portion (eg, oligonucleotide strand (Y)).
  • New overhanging ends can be generated by providing a cleavable site near the end of the DNA tag and cleaving the site if desired.
  • the overhanging ends can be used as sticky ends to ligate desired nucleic acid sequences such as UMIs (specific molecular identification sequences).
  • UMIs specific molecular identification sequences
  • UMIs specific molecular identification sequences
  • UMIs are molecular identifiers that give individual DNA sequences to individual DNA molecules by attaching them to DNA contained in a sample (Document Nature Method, 2012, 9, pp. 72-74).
  • PCR overlap sequences derived from the same molecule
  • PCR amplification bias can be reduced. quantification becomes possible.
  • a cleavable site, a modification group, or a functional molecule can be used in combination.
  • DEL is prepared by converting hairpin strand DNA into single-stranded DNA. Is possible.
  • a DEL compound using a headpiece having a cleavable site in the E section is taken as an example.
  • Step A A double-stranded oligonucleotide chain having a solid phase-supported removable modification group (eg, biotin) at the 3′ end is ligated to the synthesized DEL compound.
  • Step B Cut the cleavable site.
  • Step C Add a treatment according to the function of the modifying group.
  • a treatment according to the function of the modifying group for example, in the case of biotin, streptavidin beads or the like having biotin affinity are used to selectively remove biotin-bound oligonucleotide chains from the system. It is thereby possible to obtain DEL with single-stranded DNA.
  • the functional molecule is a molecule having a specific chemical or biological function (e.g., solubility, photoreactivity, substrate-specific reactivity, target proteolysis-inducing properties). By assigning it, it becomes possible to evaluate and purify DEL according to its function.
  • a specific chemical or biological function e.g., solubility, photoreactivity, substrate-specific reactivity, target proteolysis-inducing properties
  • biotin means all biotins that bind to avidin, including not only vitamin B7 but also desthiobiotin, for example.
  • the present invention provides suitable conditions in a method of deriving and evaluating a DEL containing a cleavable site in its DNA strand into a cross-linker-modified double-stranded DEL.
  • Another method for preparing DEL in which hairpin-stranded DNA is converted to single-stranded DNA is the method using the following exonuclease.
  • Step A A hairpin type having a "selectively cleavable site" is cut with an enzyme such as USER (registered trademark) Enzyme that is cleaved with the 5' end of the cleavage site phosphorylated after the cleavage reaction. Use to cut.
  • Step B One oligonucleotide strand phosphorylated at the 5' end is degraded and removed, for example, by treatment with lambda exonuclease. Thereby, a DEL having single-stranded DNA (single-stranded DEL) is obtained.
  • the single-stranded DEL obtained above is preferably a single-stranded DEL having library molecules in the 3' direction of the oligonucleotide strand.
  • the single-stranded DEL is capable of performing a primer extension reaction using a crosslinker-modified primer with a crosslinker at its 5' end. This technique enables easy synthesis of a “crosslinker-modified double-stranded DEL” in which a crosslinker is covalently linked to an oligonucleotide having a coding sequence.
  • the “selectively cleavable site” of the raw hairpin-type DEL is 3′ from the site to which the library molecule is bound. Existing.
  • a DEL having a single-stranded DNA is a modified oligonucleotide having a desired functional site (for example, a crosslinker-modified DNA such as a photoreactive crosslinker, or a crosslinker such as a photoreactive crosslinker).
  • a new function can be imparted by forming a double strand with a linker-modified primer).
  • the optionally provided primer may be extended to lead to a crosslinker-modified double-stranded DEL compound.
  • Such crosslinker-modified double-stranded DEL compounds are useful in the present invention because, after screening, a covalent bond is formed between the biological target and the coding sequence.
  • the present invention utilizes cleavable sites to introduce cross-linkers.
  • a DEL compound using a headpiece having a cleavable site in the E section is taken as an example.
  • Step A Cleavage the cleavable site to the synthesized DEL compound.
  • Step B Providing a modified primer (for example, a crosslinker-modified primer such as a photoreactive crosslinker) having a desired functional site.
  • a modified primer for example, a crosslinker-modified primer such as a photoreactive crosslinker
  • the crosslinker-modified double-stranded DEL compound can further bind the crosslinker to the target protein, resulting in remarkable detection sensitivity.
  • Non-Patent Documents 7, 11, etc. In the practice of DEL technology for evaluating a very large number of library compounds, it is very useful to enhance the affinity of the library compounds and improve the detection sensitivity.
  • INDUSTRIAL APPLICABILITY The present invention provides a novel and highly efficient method for producing a crosslinker-modified double-stranded DEL compound, and is extremely useful.
  • the present invention provides suitable conditions in a method of deriving and evaluating a DEL containing a cleavable site in its DNA strand into a cross-linker-modified double-stranded DEL.
  • the crosslinker-modified double-stranded DEL compound preferably has a crosslinker linked to an oligonucleotide having a coding sequence via a covalent bond.
  • Such a "crosslinker-modified double-stranded DEL compound” forms a covalent bond between the target and the coding sequence after screening, and for the removal of non-specific binding agents, etc., stronger separation and separation than before. It is very useful because it is resistant to elution conditions.
  • crosslinker in one embodiment, means a reactive group capable of forming a covalent bond through reaction with a biological target such as a protein or nucleic acid molecule.
  • a biological target such as a protein or nucleic acid molecule.
  • crosslinkers as described in Thermo Scientific Crosslinking Technical Handbook are known.
  • the crosslinker used in the present invention is preferably a reactive group containing at least one azide group, diazirine group, sulfonyl fluoride group, diazo group, cinnamoyl group, or acrylate group, more preferably an azide group, It is a reactive group containing at least one diazirine group or sulfonyl fluoride group.
  • crosslinker and “crosslinker modification” mean having a partial structure containing a crosslinker as a substituent.
  • crosslinker-modified double-stranded DEL is the five of "double-stranded DEL", “DNA”, and “primer”, respectively.
  • a crosslinker is directly attached to the ' end or is attached via a bifunctional spacer.
  • the crosslinker has the following formulas (AA) to (AE) or (BA) or (BB ) is preferred. (Wherein, * means the 5' end of "double-stranded DEL", “DNA”, or “primer”, or the binding position with the bifunctional spacer side bound to the 5' end)
  • the crosslinker used in the present invention is a photoreactive crosslinker.
  • the photoreactive crosslinker is a reactive group that changes to a reactive group with high reactivity (e.g., nitrene and carbene) by light irradiation and forms a covalent bond with a nearby biological target.
  • a reactive group with high reactivity e.g., nitrene and carbene
  • an azide group and a diazirine group are known, and the structures of the above formulas (AA) to (AE) are known.
  • the crosslinker used in the present invention is a reactive group containing at least one sulfonyl fluoride group.
  • Sulfonyl fluoride groups for example, react with residues such as serine, threonine, tyrosine, lysine, cysteine, and histidine in biological target proteins to form covalent bonds.
  • residues such as serine, threonine, tyrosine, lysine, cysteine, and histidine in biological target proteins to form covalent bonds.
  • the structures of the above formulas (BA)-(BB) are known.
  • the cross-linking reaction between the crosslinker and the biological target is preferably carried out in a temperature range in which the desired higher-order structure of the biological target does not change significantly.
  • Preferred temperatures are, for example, in the range of 4-40°C.
  • a bifunctional spacer is a spacer moiety that has at least two reactive groups that allow bonding of the substructure An of the compound library to the headpiece. Further in the context of the present invention, a bifunctional spacer is a spacer moiety that has two reactive groups that allow binding of a crosslinker to a "double stranded DEL", "DNA” or "primer”. This embodiment of the bifunctional spacer is sometimes referred to as a "crosslinker bifunctional spacer".
  • bifunctional spacers that bind to the aforementioned compound library are sometimes referred to as “compound library bifunctional spacers.”
  • preferred aspects of the "crosslinker bifunctional spacer” are the same as the preferred aspects of the aforementioned "compound library bifunctional spacer”.
  • the "crosslinker bifunctional spacer” 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”.
  • the “coding sequence” is a sequence portion of an oligonucleotide having a sequence that allows the structure of the library molecule to be identified among the sequences of the oligonucleotides contained in the DEL.
  • the "reactive group for crosslinker modification” is not particularly limited as long as it is a reactive group capable of reacting with the crosslinker unit described below.
  • the "reactive group for crosslinker modification” is a reactive group having reactivity selectivity with the crosslinker. Having reaction selectivity with the crosslinker makes it possible to apply to the present invention reaction conditions to which the crosslinker cannot be applied, such as the crosslinker reacting first and undergoing structural conversion. That is, a unit having a reactive group for crosslinker modification is introduced into the process of the present invention, reaction conditions to which the crosslinker cannot be applied are used in the process of the present invention, and then reacted with the crosslinker unit to obtain the crosslinker of the present invention. can be introduced into the crosslinker-modified DEL of the invention.
  • 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 the binding reaction. When two compounds each having this pair bind, the pair preferentially reacts to form a bond with high selectivity, even if there are various other functional groups in the compounds.
  • a “click response” is understood as one aspect as follows.
  • a “click reaction” has at least the following characteristics: (1) it exhibits orthogonality of functional groups (i.e., the functional moiety reacts complementary to it without reacting with other reactive sites); and (2) the resulting bond is irreversible (i.e., once reactants react to form products, decomposition of the products into reactants becomes difficult).
  • reaction in which the resulting bond may be reversible (ie, under appropriate conditions, reverts to the reactants).
  • "click" chemistry is characterized by: (1) stereospecificity; (2) reaction conditions without rigorous purification, atmosphere control, etc.; (3) readily available starting materials and reagents; (4) the availability of harmless solvents or no solvent at all; (5) isolation of the product by crystallization or distillation; (6) physiological stability; (8) a single reaction product; and (9) high chemical yield (eg, greater than 50%), or You can have more than one.
  • 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, and more preferably, It is an alkynyl group, an alkenyl group, an azide group or a tetrazinyl group, more preferably any one of formulas (CA) to (CL).
  • the pair of "reactive group for crosslinker modification” and “reactive group paired with the reactive group for crosslinker modification” is preferably an azide group for an alkynyl group or a tetrazinyl group for an alkenyl group. mentioned. These pairs have 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". Their selection is well known to those skilled in the art.
  • the "reactive group for crosslinker modification” and the “reactive group paired with the reactive group for crosslinker modification” include the above (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).
  • crosslinker unit is not particularly limited as long as it is a unit having the aforementioned "reactive group paired with the reactive group for crosslinker modification” and a crosslinker.
  • the "crosslinker unit” is composed of a "reactive group paired with a reactive group for crosslinker modification", a “bifunctional spacer” and a “crosslinker”.
  • the embodiment of the "bifunctional spacer” is as described above.
  • 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”.
  • 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.
  • crosslinker modified primer For the aspect of "modified primer having a reactive group for crosslinker modification", the aforementioned “crosslinker modified primer” is referred to. However, “crosslinker” is referred to as "reactive group for crosslinker modification”.
  • nucleic acids with various sequences in the examples can be prepared according to standard methods, for example, using an automatic nucleic acid synthesizer.
  • automatic nucleic acid synthesizers include nS-8II (manufactured by Genedesign).
  • outsourced synthesis or contract laboratories can be used. Contract labs well known to those skilled in the art include Genedesign, LGC Biosearch Technologies, and the like. In general, these contract laboratories prepare nucleic acids with sequences specified by the consignor under confidentiality agreements and deliver them to the consignor.
  • Example 1 [Verification of Cleavage Reaction of 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 automatic nucleic acid synthesizer nS-8II (manufactured by Genedesign).
  • nS-8II automatic nucleic acid synthesizer
  • each sequence unit is linked by a phosphodiester bond
  • A means deoxyadenosine
  • T means thymidine.
  • U-DEL1-sh, U-DEL5-HP, U-DEL6-HP, U-DEL7-HP, U-DEL8-HP, U-DEL9-HP, and U-DEL10-HP are 20 ⁇ L each after 20 hours sampled.
  • U-DEL8-HP and U-DEL9-HP were further incubated at 90° C. for 1 hour, and then sampled at 20 ⁇ L each.
  • U-DEL1-sh, U-DEL2-sh, U-DEL3-sh, and U-DEL4-sh were analyzed under the following analysis conditions 1, U-DEL5-HP, U- DEL6-HP, U-DEL7-HP, U-DEL8-HP, U-DEL9-HP, and U-DEL10-HP were analyzed under analysis condition 2 shown below.
  • Tables 2 and 3 show the sequences and theoretical molecular weights of the products assumed in each reaction solution (the abasic form of the deoxyuridine moiety and the cleaved fragment), and the molecular weights detected in each reaction solution. Note that the notation of each column in Tables 2 and 3 is as follows.
  • Entry Experiment numbers are shown, and 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
  • No. 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 are substrates of each reaction solution; 11, 14, 17, 20, 22, 25, 29, 31, 33, and 35 are abasic versions of the deoxyuridine portion of each substrate, and the remaining SEQ ID NOs are fragments of each substrate cleaved.
  • the conversion rate of the abasic reaction and the cleavage reaction was calculated from the area ratio of the peak corresponding to each detected sequence.
  • the abasic reaction showed more than 99% conversion for all substrates at 37° C. for 1 hour (substrate peak was less than 1%, remaining peaks were abasic and cleaved fragments only).
  • FIG. 10 shows a graph showing the conversion rate of the cleavage reaction.
  • the compound (hairpin DEL) having the sequence shown in Table 4 was synthesized by the following procedure.
  • "S” is the following formula (7) means a group represented by and other notations are the same as in Table 1.
  • the name of the compound corresponding to each sequence number (No.) is 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 raw material headpieces for synthesizing each hairpin DEL are as follows.
  • the raw material headpieces shown in Table 5 were prepared in the same manner as in Example 1 using an automatic nucleic acid synthesizer nS-8II (manufactured by Genedesign).
  • a PCR tube 2.0 ⁇ L of 1 mM aqueous solution of various raw material headpieces; 2.4 ⁇ L of 1 mM aqueous solution of Pr_TAG (prepared by annealing Pr_TAG_a and Pr_TAG_b synthesized as in Example 1, sequences are shown in Table 6); 0.8 ⁇ L 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 ⁇ L deionized water were added.
  • 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
  • reaction solution was treated with 0.8 ⁇ L of 5 M sodium chloride aqueous solution and 17.6 ⁇ L of cold ( ⁇ 20° C.) ethanol and left at ⁇ 78° C. for 2 hours. After centrifugation, the supernatant was removed and the resulting pellet was air-dried. A solution was prepared by adding 2.0 ⁇ L deionized water to each pellet.
  • reaction solution was treated with 0.8 ⁇ L of 5 M sodium chloride aqueous solution and 17.6 ⁇ L of cold ( ⁇ 20° C.) ethanol and left at ⁇ 78° C. for 2 hours. After centrifugation, the supernatant was removed and the resulting pellet was air-dried. 10 ⁇ L of deionized water was added to the pellet to form a solution.
  • 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 efficiencies of various hairpin DELs before and after treatment with USER® enzyme.
  • DS-DEL shown in Table 7 (prepared by annealing the compounds of sequence Nos. 47 and 48) was used.
  • "(amino-C6-L)" is the following formula (8) means a group represented by and other notations are the same as in Table 1.
  • the Ct values of the various DEL samples obtained above were measured by real-time PCR, and the PCR efficiencies were compared. The conditions are as follows, and the results are shown in FIG.
  • the Ct value is the number of cycles at which the fluorescent signal generated by DNA amplification reaches an arbitrary threshold value in real-time PCR. That is, the higher the PCR efficiency, the lower the Ct value for the same initial number of DNA molecules.
  • Apparatus 7500 real-time PCR system (manufactured by Applied Biosystems) Plate: MicroAmp 96-well plate (manufactured by Applied Biosystems, catalog number N8010560) PCR reaction solution: ⁇ TB Green Premix Ex taqII (manufactured by Takara Bio Inc., catalog number RR820): 10 ⁇ L ⁇ Forward primer (Table 9, SEQ ID NO: 55): 0.80 ⁇ L ⁇ Reverse primer (Table 9, SEQ ID NO: 56): 0.80 ⁇ L ⁇ ROX Reference Dye II (manufactured by Takara Bio Inc., catalog number RR39LR): 0.40 ⁇ L ⁇ Aqueous solutions of various DEL samples (0.05 pM, 0.5 pM, 5 pM) * 1: 2.0 ⁇ L ⁇ Deionized water: 6.0 ⁇ L *1: The number of moles of the DEL sample is 0.1 amol, 1 amol, and 10 amol. Temperatur
  • the conventional hairpin DEL had no change in Ct values before and after USER enzyme treatment
  • the deoxyuridine-containing cleavable hairpin DEL U-DEL1, U- DEL2, U-DEL4, U-DEL7, U-DEL8, U-DEL9, and U-DEL10
  • the deoxyuridine-containing cleavable hairpin DEL U-DEL1, U- DEL2, U-DEL4, U-DEL7, U-DEL8, U-DEL9, and U-DEL10
  • Example 3 [Verification of cleavage reaction of hairpin DEL containing deoxyuridine by USER (registered trademark) enzyme] ⁇ Synthesis of four hairpin DELs (U-DEL5, U-DEL11, U-DEL12, and U-DEL13)> A compound (hairpin DEL) having the sequence shown in Table 10 was synthesized by the following procedure.
  • “[mdC (TEG-amino)]” is the following formula (9) means a group represented by and other notations are the same as in Table 4.
  • the name of the compound corresponding to each sequence number (No.) is as follows. No. 57: U-DEL5 No. 58: U-DEL11 No.
  • U-DEL12-HP and U-DEL13-HP were prepared in the same manner as in Example 1 using an automatic nucleic acid synthesizer nS-8II (manufactured by Genedesign).
  • U-DEL11-HP was similarly prepared according to the standard method.
  • a portion of the obtained solution was sampled, diluted with deionized water, and subjected to mass spectrometry by ESI-MS under the following analysis conditions 3 to identify the target product (theoretical molecular weight of each sequence, and The molecular weights detected are shown in Table 10). After the remainder of the solution was lyophilized, deionized water was added to each to adjust to 20 ⁇ M.
  • the MS of the substrate was not detected in any sample, and the MS of the product after cleavage was observed as the main peak.
  • Lane 1 20 bp DNA ladder (manufactured by Lonza, Lonza 20 bp DNA Ladder, catalog number 50330) Lane 2: U-DEL5 Lane 3: After the cleavage reaction of U-DEL5 Sample lane 4: U-DEL7 Lane 5: after the cleavage reaction of U-DEL7 Sample lane 6: U-DEL9 Lane 7: after the cleavage reaction of U-DEL9 Sample 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 denaturing polyacrylamide gel electrophoresis after U-DEL13 cleavage reaction: Gel: NovexTM 10% TBE-urea gel (manufactured by Invitrogen by ThermoFisher SCIENTIFIC, catalog number EC68755BOX) Loading Buffer: Novex
  • Example 4 [Verification of cleavage reaction of hairpin DEL containing deoxyinosine by endonuclease V] ⁇ Synthesis of Hairpin DEL (I-DEL1, I-DEL2, I-DEL3, and I-DEL4) Containing Four Deoxyinosines>
  • a compound (hairpin DEL) having the sequence shown in Table 13 was synthesized by the following procedure.
  • "I” means deoxyinosine, and other notations are the same as in Table 2.
  • the name of the compound corresponding to each sequence number (No.) is as follows. No. 73: I-DEL1 No. 74: I-DEL2 No. 75: I-DEL3 No.
  • the raw material headpiece shown in Table 14 was prepared according to a standard method.
  • the MS of the substrate was not detected in any sample, and the MS of the product after cleavage was observed as the main peak.
  • Lane 1 20 bp DNA ladder (manufactured by Lonza, Lonza 20 bp DNA Ladder, catalog number 50330) Lane 2: I-DEL1 Lane 3: Sample lane 4 after I-DEL1 cleavage reaction: I-DEL2 Lane 5: After I-DEL2 cleavage reaction Sample lane 6: I-DEL3 Lane 7: After I-DEL3 cleavage reaction Sample lane 8: I-DEL4 Lane 9: Sample after I-DEL4 cleavage reaction
  • endonuclease V cleaves the second phosphodiester bond in the 3' direction from deoxyinosine in hairpin-type DELs containing various deoxyinosines.
  • Example 5 [Verification of cleavage reaction by RNase HII of hairpin DEL containing ribonucleoside] ⁇ Synthesis of Hairpin DEL (R-DEL1) Containing Ribonucleoside>
  • a compound (hairpin DEL) having the sequence shown in Table 16 was synthesized by the following procedure.
  • "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 material headpieces for synthesizing each hairpin DEL are as follows.
  • Hairpin DEL Raw headpiece R-DEL1: R-DEL1-HP
  • SEQ ID NO "No.” and sequence "Seq” of R-DEL1-HP are shown in Table 17 below. Note that the notations in Table 17 are the same as those in Table 16.
  • the raw material headpiece shown in Table 17 was prepared according to a standard method.
  • the MS of the substrate was not detected in any sample, and the MS of the product after cleavage was observed as the main peak.
  • ⁇ U-DEL9-HP - 3 building blocks (BB1, BB2, and BB3): - 10 types of double-stranded oligonucleotide tags (tag numbers in Table 19: Pr, A1, A2, A3, B1, B2, B3, C1, C2, and C3)
  • each double-stranded oligonucleotide tag was prepared by annealing two oligonucleotides of SEQ ID NOs corresponding to each tag number.
  • the above solution was treated with 295 ⁇ L of 5 M sodium chloride aqueous solution and 9.7 mL of cooled ( ⁇ 20° C.) ethanol, respectively, and allowed to stand at ⁇ 78° C. overnight. After centrifugation, the supernatant was removed and the resulting pellet was air-dried. Each pellet was dissolved by adding 2.75 mL of deionized water, added with 306 ⁇ L of piperidine at 0° C., and shaken at 10° C. for 3 hours. After centrifuging the mixture, the precipitate was removed by filtration and washed twice with 1.47 mL of deionized water.
  • the resulting filtrates were each treated with 600 ⁇ L of 5 M aqueous sodium chloride solution and 19.8 mL of chilled ( ⁇ 20° C.) ethanol, and left at ⁇ 78° C. overnight. After centrifugation, the supernatant was removed and the resulting pellet was air-dried.
  • the reaction solution was treated with 80 ⁇ L of 5 M sodium chloride aqueous solution and 2640 ⁇ L of cold (-20° C.) ethanol and incubated at -78° C. for 2 hours. After centrifugation, the supernatant was removed and 400 ⁇ L of deionized water was added to the resulting pellet. The resulting solution was concentrated with an Amicon® Ultra Centrifugal filter (30 kD cutoff). A part of the obtained solution was sampled and subjected to mass spectrometry by ESI-MS under the conditions of analysis condition 2 to identify the target product (the theoretical molecular weight of the compound and the detected molecular weight are shown in Table 21. ).
  • ⁇ Cycle A> In each of three PCR tubes, 20 ⁇ L of a 1 mM solution of the compound “AOP-U-DEL9-HP-Pr” obtained above; 30 ⁇ L of a 1 mM aqueous solution of one of the double-stranded oligonucleotide tags A1-A3; 8.0 ⁇ L 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 ⁇ L of deionized water were added. 0.4 ⁇ L of T4 DNA ligase (Thermo Fisher, Catalog No. EL0013) was added to the solution and the resulting solution was incubated at 16° C. for 18 hours.
  • T4 DNA ligase Thermo Fisher, Catalog No. EL0013
  • reaction solutions were treated with 8.0 ⁇ L of 5 M sodium chloride aqueous solution and 264 ⁇ L of cooled (-20° C.) ethanol and allowed to stand at -78° C. for 30 minutes. After centrifugation, the supernatant was removed and each pellet obtained was dissolved in 20 ⁇ L of 150 mM sodium borate buffer (pH 9.4).
  • reaction solutions were treated with 3.2 ⁇ L of 5 M sodium chloride aqueous solution and 106 ⁇ L of cooled (-20°C) ethanol and allowed to stand at -78°C for 30 minutes. After centrifugation, the supernatant was removed, 18 ⁇ L of deionized water was added to each pellet obtained, and the three solutions were mixed in one PCR tube.
  • ⁇ Cycle B> For each of three PCR tubes, 13.7 ⁇ L of a 1 mM solution of the starting material obtained in cycle A; 20.6 ⁇ L of a 1 mM aqueous solution of one of the double-stranded oligonucleotide tags B1-B3; 5.5 ⁇ L of 10X ligase. Buffer (500 mM Tris-HCl, pH 7.5; 500 mM sodium chloride; 100 mM magnesium chloride; 100 mM dithiothreitol; 20 mM adenosine triphosphate) and 14.8 ⁇ L of deionized water were added. 0.3 ⁇ L of T4 DNA ligase (Thermo Fisher, Catalog No. EL0013) was added to the solution and the resulting solution was incubated at 16° C. for 16 hours.
  • T4 DNA ligase Thermo Fisher, Catalog No. EL0013
  • reaction solutions were treated with 5.5 ⁇ L of 5 M sodium chloride aqueous solution and 181 ⁇ L of cooled (-20°C) ethanol, and allowed to stand at -78°C for 30 minutes. After centrifugation, the supernatant was removed and each pellet obtained was dissolved in 13.7 ⁇ L of 150 mM sodium borate buffer (pH 9.4).
  • each tube To each tube, add 80 equivalents of one of building blocks BB1-BB3 (5.5 ⁇ L, 200 mM N,N-dimethylacetamide solution) followed by 80 equivalents of DMTMM (5.5 ⁇ L, 200 mM aqueous solution), Shake at 10°C for 1 hour. Further, 40 equivalents of building block (2.3 ⁇ L, 200 mM N,N-dimethylacetamide solution) and then 40 equivalents of DMTMM (2.3 ⁇ L, 200 mM aqueous solution) were added to each tube and shaken at 10°C for 2 hours. I did.
  • reaction solutions were treated with 2.5 ⁇ L of 5 M sodium chloride aqueous solution and 81.4 ⁇ L of cooled (-20°C) ethanol, and allowed to stand at -78°C for 30 minutes. After centrifugation, the supernatant was removed, 12.3 ⁇ L of deionized water was added to each pellet obtained, and the three solutions were mixed in one PCR tube.
  • ⁇ Cycle C> For each of three PCR tubes, 14.5 ⁇ L of a 0.48 mM solution of the starting material obtained in Cycle B; 10.5 ⁇ L of a 1 mM aqueous solution of one of the double-stranded oligonucleotide tags C1-C3; and 2.8 ⁇ L. 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) was added. 0.14 ⁇ L of T4 DNA ligase (Thermo Fisher, Catalog No. EL0013) was added to the solution and the resulting solution was incubated at 16° C. for 16 hours.
  • 10X ligase buffer 500 mM Tris-HCl, pH 7.5; 500 mM sodium chloride; 100 mM magnesium chloride; 100 mM dithiothrei
  • reaction solutions were treated with 2.8 ⁇ L of 5 M sodium chloride aqueous solution and 92 ⁇ L of cooled (-20° C.) ethanol and allowed to stand at -78° C. for 30 minutes. After centrifugation, the supernatant was removed and each pellet obtained was dissolved in 7.0 ⁇ L of 150 mM sodium borate buffer (pH 9.4).
  • reaction solutions were treated with 1.3 ⁇ L of 5 M sodium chloride aqueous solution and 41.4 ⁇ L of cooled (-20°C) ethanol, and allowed to stand at -78°C for 30 minutes. After centrifugation, the supernatant was removed, 6.3 ⁇ L of deionized water was added to each pellet obtained, and the three solutions were mixed in one PCR tube.
  • ⁇ CP ligation> In a PCR tube, 12.2 ⁇ L Cycle C 0.41 mM solution of starting material; 6.0 ⁇ L CP 1 mM in water (same as used in Example 2); 2.1 ⁇ L 10X ligase buffer solution (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 ⁇ L of deionized water were added.
  • 0.1 ⁇ L of T4 DNA ligase (manufactured by Thermo Fisher, catalog number EL0013) was added to the solution and the resulting solution was incubated at 16° C. for 16 hours.
  • reaction solution was treated with 2.1 ⁇ L of 5 M sodium chloride aqueous solution and 69.6 ⁇ L of cooled (-20°C) ethanol and allowed to stand at -78°C for 30 minutes. After centrifugation, the supernatant was removed and 400 ⁇ L of deionized water was added to the resulting pellet. The resulting solution was concentrated by Amicon® Ultra Centrifugal filter (30 kD cutoff) and adjusted to 20 ⁇ M by adding deionized water.
  • Lane 1 AOP-U-DEL9-HP-Pr
  • Lane 2 Sample after cycle A double-stranded oligonucleotide tag A1 ligation
  • Lane 3 Sample after cycle A double-stranded oligonucleotide tag A2 ligation
  • Lane 4 Cycle A after double-stranded oligonucleotide tag A3 ligation
  • Sample lane 5 After double-stranded oligonucleotide tag B1 ligation in cycle B
  • Sample lane 6 After double-stranded oligonucleotide tag B2 ligation in cycle B
  • Sample lane 7 After double-stranded oligonucleotide tag B3 ligation in cycle B
  • Sample lane 8 Sample lane 9 after Cycle C double-stranded oligonucleotide tag C1 ligation: Sample lane 10 after Cycle C double-stranded oligonucleotide tag C2 ligation: Cycle C double-stranded oligonucle
  • FIG. 18 shows the results of chromatograph and mass spectrum. An average molecular weight of 35532.4 was observed by deconvolution of the obtained mass spectrum. This result is consistent with the expected average molecular weight (35514.2) after completion of cycle C, and the reactions for library synthesis (ligation of double-stranded oligonucleotide tags and introduction of building blocks) were achieved with high efficiency. It was shown that
  • model library 20 ⁇ M aqueous solution
  • CutSmart (registered trademark) Buffer manufactured by New England BioLabs, catalog number B7204S
  • USER® enzyme manufactured by New England BioLabs, Catalog No. M5505S
  • Example 7 [Conversion of DEL compound from hairpin DNA to single-stranded DNA and imparting new function] ⁇ Synthesis of DEL compound raw material headpiece (AAZ-DEL-HP)> AAZ-DEL-HP having the sequence shown in Table 22 was synthesized by the following procedure.
  • AAZ-AOP-AminoC7) is the following formula (11) means a group represented by and other notations are the same as in Table 2.
  • the reaction solution was put together in one Violamo centrifuge tube, treated with 800 ⁇ L of 5 M sodium chloride aqueous solution and 26.3 mL of cooled ( ⁇ 20° C.) ethanol, and allowed to stand at ⁇ 78° C. for 30 minutes. After centrifugation, the supernatant was removed and the resulting pellet was air-dried. The pellet was dissolved in deionized water and purified by reverse phase HPLC using a Phenomenex Gemini C18 column. A binary mobile phase gradient profile was used to elute the target using 50 mM triethylammonium acetate buffer (pH 7.5) and acetonitrile/water (100:1, v/v).
  • DEL compounds with the sequences shown in Table 24 were synthesized by the following procedure.
  • “(BIO)” is the following formula (15) and other notations are the same as in Tables 2, 20, 22 and 23.
  • the name of the compound corresponding to each sequence number (No.) is as follows. No. 117: AAZ-BIO-DEL No. 118: SABA-BIO-DEL No. 119: ClSABA-BIO-DEL No.
  • 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
  • the reaction solution was treated with 4 ⁇ L of 5 M sodium chloride aqueous solution and 132 ⁇ L of cooled (-20° C.) ethanol and allowed to stand at -78° C. for 30 minutes. After centrifugation, the supernatant was removed, the resulting pellet was air-dried, and the pellet was dissolved in deionized water. The resulting solution was desalted with an Amicon (registered trademark) Ultra Centrifugal filter (3 kD cutoff).
  • Amicon registered trademark
  • Table 26 The sequence notation in Table 26 is the same as in Table 24, and the five compounds are SEQ ID NOS: 124 and 125, SEQ ID NOS: 126 and 127, SEQ ID NOS: 128 and 129, and SEQ ID NO: 130, respectively. and SEQ ID NO: 131, and SEQ ID NO: 132 and SEQ ID NO: 133.
  • the resulting reaction solution was desalted and concentrated with an Amicon (registered trademark) Ultra Centrifugal filter (3 kD cutoff), ethanol precipitation was performed, and then deionized water was added to each obtained pellet to obtain an aqueous solution. .
  • Amicon registered trademark
  • Ultra Centrifugal filter 3 kD cutoff
  • Example 3 A portion of the resulting solution was sampled, diluted with deionized water, and subjected to mass spectrometry by ESI-MS under the analysis conditions 3 of Example 3 to obtain a DEL compound "DS -AAZ-BIO-DEL", “DS-SABA-BIO-DEL”, “DS-ClSABA-BIO-DEL”, “DS-mSABA-BIO-DEL”, and “DS-Amino-BIO-DEL” respectively identified (theoretical molecular weights of the compounds and the detected molecular weights are shown in Table 26)
  • DEL compounds having the double-stranded nucleic acid obtained above “DS-AAZ-BIO-DEL”, “DS-SABA-BIO-DEL”, “DS-ClSABA-BIO-DEL”, “DS-mSABA-BIO- DEL” and “DS-Amino-BIO-DEL” are treated with streptavidin beads, respectively, and the DEL compounds "SS-AAZ-DEL” and "SS-SABA-DEL” having single-stranded DNA are prepared by the following procedure. ”, “SS-ClSABA-DEL”, “SS-mSABA-DEL”, and “SS-Amino-DEL” were prepared. The five compounds are oligonucleotide chains of SEQ ID NOs: 125, 127, 129, 131 and 133 in Table 26, respectively.
  • the resulting particles were respectively labeled "DS-AAZ-BIO-DEL”, “DS-SABA-BIO-DEL”, “DS-ClSABA-BIO-DEL”, “DS-mSABA-BIO-DEL”, or "DS -Amino-BIO-DEL” (700 pmol, 450 ⁇ L, respectively), and 450 ⁇ L 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 ) was added, mixed and shaken at room temperature for 20 minutes.
  • 2 ⁇ binding buffer (20 mM Tris-HCl, pH 7.5; 1 mM ethylenediaminetetraacetic acid; 2 M sodium chloride; 0.1% v/v Tween 20
  • the supernatant was removed from each mixture by magnetic separation, and 900 ⁇ L 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. The washing of the particles used and the removal of the supernatant by magnetic separation were each repeated twice. After that, 900 ⁇ L of denaturation solution (0.1 M sodium hydroxide; 0.1 M sodium chloride) was added to each, and the supernatant was recovered by magnetic separation.
  • 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
  • reaction solution was treated with 23.6 ⁇ L of 5 M sodium chloride aqueous solution and 778.8 ⁇ L of cooled (-20°C) ethanol and allowed to stand overnight at -78°C. After centrifugation, the supernatant was removed and the resulting pellet was air-dried. After adding 180 ⁇ L of deionized water to the pellet to make a solution, 20 ⁇ L of piperidine was added and shaken at 10° C. for 3 hours.
  • the obtained solution was treated with 20 ⁇ L of 5 M sodium chloride aqueous solution and 660 ⁇ L of cold (-20° C.) ethanol and allowed to stand at -78° C. for 30 minutes. After centrifugation, the supernatant was removed, and 200 ⁇ L of deionized water was added to the obtained pellet to make a 1 mM solution.
  • the obtained solution was treated with 20 ⁇ L of 5 M sodium chloride aqueous solution and 660 ⁇ L of cold (-20° C.) ethanol and allowed to stand at -78° C. for 30 minutes. After centrifugation, the supernatant was removed, and 100 ⁇ L of deionized water was added to the resulting pellet, followed by 75 ⁇ L of triethylamine hydrochloride buffer (500 mM, pH 10) and 50 equivalents of Sulfo-SDA (25 ⁇ L, 200 mM aqueous solution). , 37° C. for 1 hour and 20 minutes. An additional 50 equivalents of Sulfo-SDA (25 ⁇ L, 200 mM aqueous solution) was added and shaken at 37° C. for 40 minutes.
  • the resulting solution was treated with 22.5 ⁇ L of 5 M sodium chloride aqueous solution and 743 ⁇ L of chilled (-20°C) ethanol and allowed to stand overnight at -78°C. After centrifugation, the supernatant was removed, and 100 ⁇ L of deionized water was added to the resulting pellet, followed by 75 ⁇ L of triethylamine hydrochloride buffer (500 mM, pH 10), followed by 50 equivalents of Sulfo-SDA (25 ⁇ L, 200 mM aqueous solution). ) was added and shaken at 37° C. for 3 hours.
  • the resulting solution was treated with 20 ⁇ L of 5 M sodium chloride aqueous solution and 660 ⁇ L of chilled (-20° C.) ethanol and allowed to stand at -78° C. overnight. 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. A binary mobile phase gradient profile was used to elute the target using 50 mM triethylammonium acetate buffer (pH 7.5) and acetonitrile/water (100:1, v/v).
  • a part of the obtained solution was sampled, diluted with deionized water, and then subjected to mass spectrometry by ESI-MS under the analysis conditions 3 of Example 3 to obtain the desired photoreactive crosslinker.
  • a modified primer 'PXL-Pr' was identified (compound theoretical and detected molecular weights are shown in Table 27).
  • Table 29 The sequence notation in Table 29 is the same as in Tables 26 and 27, and the five compounds are SEQ ID NOS: 136 and 125, SEQ ID NOS: 136 and 127, and SEQ ID NOS: 136 and 129, respectively. , SEQ ID NO: 136 and SEQ ID NO: 131, and SEQ ID NO: 136 and SEQ ID NO: 133.
  • the resulting solution was desalted with an Amicon (registered trademark) Ultra Centrifugal filter (3 kD cutoff). Deionized water was added to the obtained supernatant to make a solution of 60 ⁇ L, which was then treated with 6 ⁇ L of 5 M sodium chloride aqueous solution and 198 ⁇ L of cold ( ⁇ 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 ⁇ L of deionized water was added to the pellet to form a solution.
  • Amicon registered trademark
  • Lane 1 20 bp DNA ladder (manufactured by 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-S
  • Polyacrylamide gel electrophoresis Gel: SuperSep (trademark) DNA 15% TBE gel (manufactured by Fujifilm Wako Pure Chemical Industries, catalog number 190-15481)
  • Loading buffer 6x Loading Buffer (manufactured by Takara Bio Inc., catalog number 9156)
  • Electrophoresis time 50 minutes
  • Staining reagent SYBER (trademark) Green II Nucleic Acid Gel Stain (manufactured by Takara Bio Inc., catalog number 5770A)
  • Example 8 [Comparison of binder recovery efficiency of photoreactive crosslinker-modified double-stranded DEL with and without photocrosslinking reaction] ⁇ Preparation of DEL sample> Each of the five photoreactive crosslinker-modified double-stranded DELs obtained above was diluted with deionized water to prepare a 50 nM DEL sample.
  • UVP Ultraviolet Crosslinker
  • Reaction tube bottom vial for 96 holes (manufactured by Techno Lab Bossy Co., Ltd., catalog number 96-V050FB)
  • Photocrosslinking reaction solution - Salmon Sperm DNA, sheared (Invitrogen, catalog number AM9680): 1.6 ⁇ L ⁇ 1 M NaCl aqueous solution: 5.0 ⁇ L ⁇ D-PBS (-) (9 manufactured by FUJIFILM, catalog number 045-29795): 32.4 ⁇ L ⁇ Carbonic Anhydrase IX/CA9 (Sino Biology cal, catalog number 10107-H08H): 10.0 ⁇ L ⁇ 50 nM aqueous solution of various DEL samples: 1.0 ⁇ L Reaction conditions: A mixture of the CA9 protein having the above composition and the DEL solution was incubated on ice for 1 hour. A portion of the solution was then collected
  • the reaction solution after UV irradiation was mixed with dyanebeads his-tag pulldown and incubated at room temperature for 30 minutes. It was fixed on a magnetic stand, allowed to stand for 2 minutes, the supernatant was removed, and 200 ⁇ L of wash buffer was added to suspend the Dynabeads. Furthermore, this washing operation was repeated five times. 80 ⁇ L of D-PBS( ⁇ ) was added to the washed Dybabeads and allowed to react at 95° C. for 10 minutes. After the reaction, the Dynabeads were placed on a magnetic stand, and after 2 minutes the supernatant was collected (solution E).
  • Apparatus 7500 real-time PCR system (manufactured by Applied Biosystems) Plate: MicroAmp 96-well plate (manufactured by Applied Biosystems, catalog number N8010560) PCR reaction solution: ⁇ TaqMan Gene Expression Master Mix (manufactured by Applied Biosystems, catalog number 4369016): 10.0 ⁇ L - Forward primer 1 (Table 30, SEQ ID NO: 137): 1.0 ⁇ L - Reverse primer 1 (Table 30, SEQ ID NO: 138): 1.0 ⁇ L -Taqman MGB probe (manufactured by Thermo Fisher, product number 4316034, TaqMan (registered trademark) labeled with FAM (registered trademark) fluorescent dye at the 5' end of the nucleotide sequence of SEQ ID NO: 139 in Table 30, and NFQ and MGB at the 3' end ) probe): 0.50 ⁇ L ⁇ Aqueous solutions of various DEL samples (S, E samples)
  • Example 9 [Preparation of DEL with single-stranded DNA using Lambda Exonuclease]
  • the DEL compound "DS-Amino-BIO-DEL” having a double-stranded nucleic acid was treated with Lambda Exonuclease to convert it to a DEL compound "SS-Amino-DEL” having a single-stranded DNA according to the following procedure.
  • DS-Amino-BIO-DEL 500 pmol aqueous solution; 5 ⁇ L of 10 ⁇ Lambda Exonuclease Reaction Buffer (New England BioLabs, catalog number B0262); Grand BioLabs, catalog number M0262) was added, and deionized water was added to prepare a solution with a total volume of 50 ⁇ L. The resulting solution was incubated at 37°C for 30 minutes.
  • the MS of one oligonucleotide strand (SEQ ID NO: 132) contained in DS-Amino-BIO-DEL was not detected, and the MS of the product after single-strand formation was observed as the main peak. It was confirmed that the chain reaction was progressing.
  • Example 10 Synthesis of Photoreactive Crosslinker-Modified Double-Stranded DEL with Different Linker Structure from Photoreactive Crosslinker-Modified Double-Stranded DEL Used in Example 8] ⁇ Preparation of DEL having 5 types of single-stranded DNA>
  • Example 7 DEL compounds having five types of 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 ⁇ Synthesis of five DEL compounds having biotin at the 3'end> described in Example 7, Pr_TAG2_CP (Example Prepared by annealing Pr_TAG2_CP_a and Pr_TAG2_CP_b synthesized in the same manner as in Example 1, the sequences are shown in Table 31), instead of the ⁇ Preparation of DEL with single-stranded DNA using streptavidin beads> step, Example 9 Preparation of DEL having single-stranded DNA was carried out in the same procedure as in. The sequence notation in Table 31 is the same as in Table 25. The name of the compound corresponding to each sequence number (No.) is They are as follows. No. 122: Pr_
  • 3-(3-methyl-3H-diazilin-3-yl)propanoic acid (5 ⁇ L, 0.2 M N,N-dimethylacetamide solution) was added to the PCR tube.
  • 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (2.5 ⁇ L, 0.4M N,N- dimethylacetamide solution), followed by N,N-diisopropylethylamine (2.5 ⁇ L, 0.4M N,N-dimethylacetamide solution), and the resulting solution was shaken at 4° C. for 10 minutes.
  • the above solution was treated with 11 ⁇ L of 5 M sodium chloride aqueous solution and 363 ⁇ L 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. After dissolving the resulting pellet in deionized water, the solution was desalted through an Amicon® Ultra Centrifugal filter (3 kD cutoff).
  • a part of the obtained supernatant was sampled, diluted with deionized water, and subjected to mass spectrometry by ESI-MS under the analysis conditions 3 of Example 3 to obtain the desired photoreactive crosslinker.
  • a modified primer "PXL-Pr2" was identified (theoretical and detected molecular weights for each compound are shown in Table 32).
  • Table 33 The sequence notation in Table 33 is the same as in Tables 26 and 32, and the five compounds are SEQ ID NOS: 142 and 125, SEQ ID NOS: 142 and 127, and SEQ ID NOS: 142 and 129, respectively. , SEQ ID NO: 142 and SEQ ID NO: 131, and SEQ ID NO: 142 and SEQ ID NO: 133.
  • Double-stranded DEL “PXL-DS-AAZ-DEL2”, “PXL-DS-SABA-DEL2”, “PXL-DS-ClSABA-DEL2”, “PXL-DS-mSABA-DEL2”, and "PXL-DS- Amino-DEL2” was identified (compound theoretical molecular weight and detected molecular weight are shown in Table 33).
  • Lane 1 20 bp DNA ladder (manufactured by Lonza, 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 SS-Amino-DEL primer extension reaction (PXL-DS-
  • DEL compound “SS-Amino-DEL3” having single-stranded DNA was prepared in the same manner as in Example 1 using an automatic nucleic acid synthesizer nS-8II (manufactured by Genedesign).
  • "SS-Amino-DEL3" uses "U-DEL12-HP" (sequence shown in Table 11) as a raw material headpiece, and the procedure is the same as the above ⁇ Preparation of DEL having 5 types of single-stranded DNA>. is the same structure as the oligonucleotide derived from Moreover, the sequence notation in Table 34 is the same as in Table 10.
  • Example 3 A part of the obtained solution was sampled, diluted with deionized water, and then subjected to mass spectrometry by ESI-MS under the conditions of analysis condition 3 in Example 3 to obtain the desired single-stranded DNA.
  • the DEL compound 'SS-AAZ-DEL3' was identified (the compound's theoretical and detected molecular weights are shown in Table 35).
  • the above solution was treated with 5.8 ⁇ L of 5 M sodium chloride aqueous solution and 192 ⁇ L of cooled (-20° C.) ethanol and allowed to stand at -78° C. for 30 minutes. After centrifugation, the supernatant was removed and the resulting pellet was air-dried.
  • the resulting pellet was dissolved in 50 mM triethylammonium acetate buffer (pH 7.5) and purified by reversed-phase HPLC using a Phenomenex Gemini C18 column. Targets were eluted using a binary mobile phase gradient profile using 50 mM triethylammonium acetate buffer (pH 7.5) and acetonitrile/500 mM triethylammonium acetate buffer (9:1, v/v). Fractions containing the desired product were collected, combined and concentrated. The obtained solution was desalted with an Amicon (registered trademark) Ultra Centrifugal filter (3 kD cutoff), ethanol precipitation was performed, and then deionized water was added to the pellet to make a solution.
  • Amicon registered trademark
  • a part of the obtained solution was sampled, diluted with deionized water, and subjected to mass spectrometry by ESI-MS under the conditions of analysis condition 3 in Example 3 to obtain the desired photoreactive crosslinker modification.
  • a primer "PXL-Pr3" was identified (theoretical and detected molecular weights of each compound are shown in Table 37).
  • DEL compounds (“PXL-DS-AAZ-DEL3”, “PXL-DS-SABA-DEL3”, “PXL-DS-ClSABA-DEL3”, “PXL-DS-mSABA-DEL3”, and “PXL-DS-Amino -DEL3”) were synthesized.
  • the sequence notation in Table 39 is the same as in Tables 34 to 37, and the five compounds are respectively SEQ ID NOS: 150 and 144, SEQ ID NOS: 150 and 145, SEQ ID NOS: 150 and 146, and the sequence It means formed by the double strands of the oligonucleotide strands of No. 150 and SEQ ID No. 147 and SEQ ID No. 150 and SEQ ID No. 143.
  • 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 respectively (the theoretical molecular weight of the compound and the detected molecular weight are shown in Table 39).
  • Lane 1 20 bp DNA ladder (manufactured by Lonza, 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-Amin
  • Example 11 [Comparison of binder recovery efficiency of photoreactive crosslinker-modified double-stranded DEL synthesized in Example 10 with and without photocrosslinking reaction] ⁇ Preparation of DEL sample>
  • Four photoreactive crosslinker-modified double-stranded DELs obtained in Example 10 (PXL-DS-mSABA-DEL2, PXL-DS-Amino-DEL2, PXL-DS-mSABA-DEL3, and PXL-DS- Amino-DEL 3) was each diluted with deionized water to prepare 50 nM DEL samples.
  • UVP Ultraviolet Crosslinker
  • Microtube 1.5 mL siliconized microtube round bottom (manufactured by Watson Co., Ltd., catalog number 131-615CH)
  • Reaction tube Bottom vial for 96 holes (manufactured by Techno Lab Bossy Co., Ltd., catalog number 96-V050FB)
  • Photocrosslinking reaction solution - Salmon Sperm DNA, sheared (Invitrogen, catalog number AM9680): 1.6 ⁇ L ⁇ 1M NaCl (manufactured by FUJIFILM, catalog number 191-01665): 5.0 ⁇ L ⁇ D-PBS (-) (manufactured by FUJIFILM, catalog number 045-29795): 32.4 ⁇ L ⁇ Carbonic Anhydrase IX/CA9 (manufactured by Sino Biological, catalog number 10107-H08H): 10.0 ⁇ L ⁇ Aqueous solution
  • D-PBS(-) was added to the reaction solution after UV irradiation. The remaining solution was then mixed with the Dyanebeads his-tag pulldown and incubated for 30 minutes at room temperature. It was fixed on a magnetic stand, allowed to stand for 2 minutes, the supernatant was removed, and 200 ⁇ L of wash buffer was added to suspend the Dynabeads. Furthermore, this operation was repeated 5 times. 100 ⁇ L of Elution buffer was added to the washed Dynabeads and allowed to stand at room temperature for 10 minutes. After the reaction, the Dynabeads were placed on a magnetic stand, and after 2 minutes the supernatant was collected as a sample.
  • FIG. 25 shows the result of comparing the ⁇ Ct value (difference from the Ct value of the negative control).
  • the ⁇ Ct value is small in all samples without UV irradiation, and similar to the results of Example 8, in DEL screening, when photocrosslinking reaction is not performed, moderate affinity It suggests that obtaining a sexual binder is difficult.
  • Example 12 “Photoreactive crosslinker-modified double-stranded DEL with covalent bond between crosslinker and coding sequence” and “Photoreactive crosslinker-modified double-stranded DEL with no covalent bond between crosslinker and coding sequence”] Comparison of binding agent recovery efficiency (DNA detection sensitivity) with
  • Table 40 The sequence notation in Table 40 is the same as Tables 34, 36, and 37, and the four compounds are SEQ ID NOS: 148 and 145, SEQ ID NOS: 148 and 146, and SEQ ID NO: 148, respectively. It means that it is formed by double strands of oligonucleotide strands of SEQ ID NO: 147 and SEQ ID NO: 148 and SEQ ID NO: 143.
  • UVP Ultraviolet Crosslinker
  • Microtube 1.5 mL siliconized microtube round bottom (manufactured by Watson Co., Ltd., catalog number 131-615CH)
  • Reaction tube Bottom vial for 96 holes (manufactured by Techno Lab Bossy Co., Ltd., catalog number 96-V050FB)
  • Photocrosslinking reaction solution - Salmon Sperm DNA, sheared (Invitrogen, catalog number AM9680): 1.6 ⁇ L ⁇ 1M NaCl (manufactured by FUJIFILM, catalog number 191-01665): 5.0 ⁇ L ⁇ D-PBS (-) (FUJIFILM 9, catalog number 045-29795): 32.4 ⁇ L ⁇ Carbonic Anhydrase IX/CA9 (manufactured by Sino Biological, catalog number 10107-H08H): 10.0 ⁇ L ⁇ Aqueous solution of various DEL samples (50
  • the reaction solution after UV irradiation was mixed with Dynabeads his-tag pulldown and incubated at room temperature for 30 minutes. It was fixed on a magnetic stand, allowed to stand for 2 minutes, the supernatant was removed, and 200 ⁇ L of wash buffer was added to suspend the Dynabeads. Furthermore, this operation was repeated 5 times. 100 ⁇ L 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.
  • FIG. 26 shows the result of comparing the ⁇ Ct value (difference from the Ct value of the negative control).
  • UVP Ultraviolet Crosslinker
  • Reaction tube bottom vial for 96 holes (manufactured by Techno Lab Bossy Co., Ltd., catalog number 96-V050FB)
  • Photocrosslinking reaction solution - Salmon Sperm DNA, sheared (Invitrogen, catalog number AM9680): 1.6 ⁇ L ⁇ 1M NaCl (manufactured by FUJIFILM, catalog number 191-01665): 5.0 ⁇ L ⁇ D-PBS (-) (FUJIFILM 9, catalog number 045-29795): 39.4 ⁇ L ⁇ Carbonic Anhydrase IX/CA9 (manufactured by Sino Biological, catalog number 10107-H08H): 3.0 ⁇ L ⁇ Aqueous solution of various DEL samples (50 nM): 1.0 ⁇ L Reaction conditions: A mixture of the CA9 protein having the above composition and the DEL solution was incuba
  • the reaction solution after UV irradiation was mixed with Dynabeads his-tag pulldown and incubated at room temperature for 30 minutes. It was fixed on a magnetic stand, allowed to stand for 2 minutes, then the supernatant was removed, 200 ⁇ L of wash buffer was added to suspend the Dynabeads, and reacted at 90° C. for 10 minutes. Furthermore, this operation was repeated 3 times. 30 ⁇ L of a 200 mM imidazole solution 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.
  • FIG. 27 shows the result of comparing the ⁇ Ct value (difference from the Ct value of the negative control).
  • the present results show that the "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" It is suggested that it is applicable to DEL screening under strong separation conditions and elution conditions for the purpose of removing non-specific binders and the like.
  • Example 14 [Conversion of Hairpin DNA Using U-DEL-13-HP as Raw Material to Single-Strand DNA, and Addition of New Functions] ⁇ Synthesis of DEL compound raw material headpiece (“mSABA-DEL-HP5”)> Using "mSABA-DEL-HP5" having the sequence shown in Table 41 and "U-DEL13-HP" as a starting material, Example 10 ⁇ 3 types of DEL compounds having single-stranded DNA (“SS-SABA-DEL3 ”, “SS-ClSABA-DEL3”, and “SS-mSABA-DEL3”)>. Note that the notation in Table 41 is the same as in Table 36.
  • a hairpin DEL compound (“mSABA-DEL5”) having the sequence shown in Table 42 was synthesized in the same manner as in Example 10 by double-stranded ligation of the starting headpiece “mSABA-DEL-HP5” and Pr_TAG2_CP.
  • the array notation in Table 42 is the same as in Table 36.
  • DEL compound "DS-mSABA-DEL5" was identified (theoretical and detected molecular weights for each compound are shown in Table 43).
  • Lane 1 20 bp DNA ladder (manufactured by Lonza, Lonza 20 bp DNA Ladder, catalog number 50330)
  • Lane 2 mSABA-DEL5
  • Lane 3 Sample lane 4: 20 bp DNA ladder (manufactured by Lonza, Lonza 20 bp DNA Ladder, catalog number 50330) after mSABA-DEL5 was cleaved with USER (registered trademark) enzyme.
  • the DEL compound “DS-mSABA-DEL5" having the double-stranded nucleic acid obtained above was treated with Lambda Exonuclease in the same manner as in Example 9, and the DEL compound "SS-mSABA-DEL5" having a single-stranded DNA was obtained.
  • SS-mSABA-DEL5 is the oligonucleotide chain of SEQ ID NO: 154 in Table 43.
  • a part of the obtained supernatant was sampled, diluted with deionized water, and subjected to mass spectrometry by ESI-MS under the analysis condition 3 of Example 3. A molecular weight of 23825.8 was observed. A DEL compound "SS-mSABA-DEL5" having the desired single-stranded DNA was identified.
  • a part of the obtained solution was sampled, diluted with deionized water, and subjected to mass spectrometry by ESI-MS under the conditions of analysis condition 3 in Example 3 to obtain the desired photoreactive crosslinker modification.
  • a primer "PXL-Pr5" was identified (theoretical and detected molecular weights of each compound are shown in Table 44).
  • a part of the obtained solution was sampled, diluted with deionized water, and subjected to mass spectrometry by ESI-MS under the conditions of analysis condition 3 in Example 3 to obtain the desired photoreactive crosslinker modification.
  • a double-stranded DEL "PXL-DS-mSABA-DEL5" was identified (compound theoretical and detected molecular weights are shown in Table 46).
  • Lane 1 20 bp DNA ladder (manufactured by 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 (manufactured by Lonza, Lonza 20 bp DNA Ladder, catalog number 50330)
  • Example 15 [Synthesis of cross-linker-modified double-stranded DEL] ⁇ Synthesis of three types of crosslinker-modified primers (PA-Pr, TPD-Pr, ACA-Pr)> Various crosslinker-modified primers having the sequences shown in Table 47 were synthesized by the following procedure.
  • “(PA)” is the following formula (22)
  • “(TPD)” means a group represented by the following formula (23)
  • (ACA)” is the following formula (24) means a group represented by and other notations are the same as in Table 2.
  • combining each compound is as follows, respectively.
  • reaction solution was treated with 12 ⁇ L of 5 M sodium chloride aqueous solution and 396 ⁇ L of cooled (-20°C) ethanol and allowed to stand at -78°C for 30 minutes. After centrifugation, the supernatant was removed and the resulting pellet was air-dried.
  • the resulting pellet was dissolved in 50 mM triethylammonium acetate buffer (pH 7.5) and purified by reversed-phase HPLC using a Phenomenex Gemini C18 column. Targets were eluted using a binary mobile phase gradient profile using 50 mM triethylammonium acetate buffer (pH 7.5) and acetonitrile/500 mM triethylammonium acetate buffer (9:1, v/v). Fractions containing the desired product were collected, combined and concentrated. The obtained solution was desalted with an Amicon (registered trademark) Ultra Centrifugal filter (3 kD cutoff), ethanol precipitation was performed, and then deionized water was added to the pellet to make a solution.
  • Amicon registered trademark
  • reaction solution was treated with 23.6 ⁇ L of 5 M sodium chloride aqueous solution and 778.8 ⁇ L of cooled (-20°C) ethanol and allowed to stand overnight at -78°C. After centrifugation, the supernatant was removed and the resulting pellet was air-dried. After adding 180 ⁇ L of deionized water to the pellet to make a solution, 20 ⁇ L of piperidine was added and shaken at 10° C. for 3 hours.
  • the obtained solution was treated with 20 ⁇ L of 5 M sodium chloride aqueous solution and 660 ⁇ L of cold (-20° C.) ethanol and allowed to stand at -78° C. for 30 minutes. After centrifugation, the supernatant was removed, and 200 ⁇ L of deionized water was added to the obtained pellet to make a 1 mM solution.
  • a solution of AOP-L-Pr 40 ⁇ L, 0.5 mM in sodium phosphate buffer (125 mM, pH 9.4) cooled to 10°C was added to the PCR tube.
  • Fifty equivalents of N-succinimidyl 3-maleimidopropionate (5 ⁇ L, 0.2 M dimethylsulfoxide solution) were added to the tube, and the resulting mixture was shaken at 10° C. for 40 minutes. 20 ⁇ L of dimethylsulfoxide was then added and the resulting mixture was further shaken at 10° C. for 25 minutes.
  • reaction solution was treated with 5 ⁇ L of 5 M sodium chloride aqueous solution and 215 ⁇ L of cold (-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. After dissolving the resulting pellet in deionized water, the solution was desalted through an Amicon® Ultra Centrifugal filter (3 kD cutoff).
  • Table 50 The sequence notation in Table 50 is the same as in Tables 26 and 47, and "TPD-DS-mSABA-DEL" is formed by two strands of the oligonucleotide strands of SEQ ID NO: 163 and SEQ ID NO: 131. indicate that
  • Example 3 A part of the obtained solution was sampled, diluted with deionized water, and subjected to mass spectrometry by ESI-MS under the analysis conditions 3 of Example 3 to obtain the desired crosslinker-modified double strand.
  • the DEL "TPD-DS-mSABA-DEL" was identified (the theoretical molecular weight of the compound and the detected molecular weight are shown in Table 50).
  • Example 3 A part of the obtained solution was sampled, diluted with deionized water, and subjected to mass spectrometry by ESI-MS under the analysis conditions 3 of Example 3 to obtain the desired crosslinker-modified double strand.
  • the DEL "ACA-DS-ClSABA-DEL" was identified (the compound's theoretical and detected molecular weights are shown in Table 51).
  • Example 16 [Synthesis of crosslinker-modified double-stranded DEL using double-stranded DEL having reactive groups for crosslinker modification] ⁇ Synthesis of a primer (“BCN-Pr”) having a reactive group for crosslinker modification> A primer “BCN-Pr” having a reactive group for crosslinker modification of the sequences shown in Table 52 was synthesized by the following procedure.
  • “(BCN)” is the following formula (27) means a group represented by and other notations are the same as in Table 2.
  • a part of the obtained solution was sampled, diluted with deionized water, and subjected to mass spectrometry by ESI-MS under the conditions of analysis condition 3 in Example 3 to determine the desired crosslinker modification.
  • a double-stranded DEL "BCN-DS-mSABA-DEL" with reactive groups was identified (compound theoretical and detected molecular weights are shown in Table 53).
  • Example 7 A part 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 FIG. 31, it was confirmed that the primer extension reaction resulted in conversion to "BCN-DS-mSABA-DEL" at a high yield.
  • the samples for each lane in FIG. 31 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 (manufactured by 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 (manufactured by Lonza, Lonza 20 bp DNA Ladder, catalog number 50330)
  • BCN-DS-mSABA-DEL solution (2 ⁇ L, 0.1 mM) in sodium phosphate buffer (250 mM, pH 7.0) was added to the PCR tube, followed by 1.8 ⁇ L of dimethylsulfoxide. After that, 0.2 ⁇ L of the diluted solution obtained above was added, and the obtained solution was shaken at 25° C. for 2 hours.
  • BCN-DS-mSABA-DEL solution (2 ⁇ L, 0.1 mM) in sodium phosphate buffer (250 mM, pH 7.0) was added to the PCR tube, followed by 1.8 ⁇ L of dimethylsulfoxide. After that, 0.2 ⁇ L of the diluted solution obtained above was added, and the obtained solution was shaken at 25° C. for 2 hours.
  • Example 17 [Conversion of Model Library Using U-DEL9-HP as Raw Material to Single-Strand DNA, and Addition of New Functions] ⁇ Conversion of model library to DEL compound having single-stranded DNA using Lambda Exonuclease> Using the sample (synthesized in Example 6) after the cleavage reaction of the model library with USER (registered trademark) enzyme, the model library was converted to a DEL compound having single-stranded DNA in the same manner as in Example 9. bottom. The obtained solution was used as a starting material for the next step.
  • Lane 1 20 bp DNA ladder (manufactured by Lonza, Lonza 20 bp DNA Ladder, catalog number 50330)
  • Lane 2 After cleavage reaction of model library with USER (registered trademark) enzyme
  • Sample lane 3 Sample lane after single-strand DEL conversion reaction with Lambda
  • Exonuclease of model library 4 Single-strand DEL-converted model library and PXL -Sample lane 5 after primer extension reaction with Pr: Sample lane 6 after primer extension reaction between single-stranded DEL model library and BCN-Pr: 20 bp DNA ladder (manufactured by Lonza, Lonza 20 bp DNA Ladder , catalog number 50330)
  • the present invention provides methods utilizing nucleic acid compounds containing selectively cleavable sites. Furthermore, the present invention provides a method for inducing a DEL containing a cleavable site in the DNA strand to a crosslinker-modified double-stranded DEL and evaluating it. It is possible to perform compound screening that combines "expansion and improvement of evaluation methods".

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