Modified nucleobases with uniform hydrogen-bonding interactions, homo- and hetero-base pair bias, and mismatch discrimination

Abstract

To provide nucleobases, polymer monomers comprising the nucleobases, nucleic acids and analogs thereof comprising the nucleobases, and methods of use thereof.SOLUTION: Described herein are divalent nucleobases that each bind two nucleic acid strands, matched or mismatched when incorporated into a nucleic acid backbone or nucleic acid analog backbone, e.g., in a γ-peptide nucleic acid (γPNA). Also provided are genetic recognition reagents comprising one or more of the divalent nucleobases and a nucleic acid backbone or nucleic acid analog backbone, such as a γPNA backbone. Uses of the divalent nucleobases and monomers and genetic recognition reagents containing the divalent nucleobases also are provided.SELECTED DRAWING: Figure 1

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 763,299, filed June 8, 2018, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Described herein are nucleobases, polymer monomers comprising said nucleobases, and nucleic acids and analogs thereof comprising said nucleobases. Also described herein are methods of using the nucleobases, polymer monomers comprising said nucleobases, and nucleic acids and analogs thereof comprising said nucleobases.

[0003] For most organisms, genetic information is encoded in double-stranded DNA in the form of Watson-Crick base pairing, where adenine (A) pairs with thymine (T) and cytosine (C) pairs with guanine (G). Which set of this genetic information is decoded through transcription and translation determines developmental programs and physiological states. The development of molecules that can be tailored to bind sequence-specifically to any portion of this genetic biopolymer (DNA or RNA), thereby enabling control of genetic information flow and assessment and manipulation of genome structure and function, is important for biological and biomedical research in the effort to elucidate the molecular basis of life, including as molecular tools for basic biological research. This effort is also important for medical and therapeutic applications for the treatment and detection of genetic diseases.

[0004] Oligonucleotides are versatile in their use as molecular tools for basic biological research and as molecular reagents for therapeutic and diagnostic applications, such as the treatment and detection of genetic diseases. Generally, oligonucleotide molecules are short fragments (10–30 nucleotides in length) of single-stranded DNA or RNA or their derivatives. Oligonucleotide molecules may contain a sugar phosphodiester backbone linked to the nucleobases adenine (A), cytosine (C), guanine (G), and thymine (T) or uridine (U), or other backbones. Oligonucleotide molecules are designed to bind to DNA or RNA targets via Watson-Crick base pairing, where A pairs with T (or U) and C pairs with G. Oligonucleotide molecules have been used in a wide range of applications, including, for example, interrogating nucleic acid sequence information, manipulating RNA structure, and modulating gene expression. The success of many of these applications is due to the oligomer's ability to bind to DNA or RNA targets in a strict sequence-specific manner. Additional requirements for intracellular and in vivo gene targeting include enzyme stability and cell permeability. Summary of the Invention

[0005] comprising a plurality of nucleobase moieties attached to a nucleic acid backbone or nucleic acid analog backbone, wherein at least one nucleobase moiety is: [ka] (wherein X1 is =O (= represents a double bond), =S, =Se, or CH3; X2 is H, CH3, CN, NC, N3, C(O)OH, or C(O)NH2; X3 is O or S; X4 is H, C(O)CH3, or C(O)OCH3; and Y is N or CH, and in (1), when X1 and X3 are O, X2 is not H or methyl.) A gene recognition reagent is provided, wherein:

[0006] structure: [ka] (wherein X1 is =O, =S, =Se, or CH3; X2 is H, CH3, CN, NC, N3, C(O)OH, or C(O)NH2; X3 is O or S; X4 is H, C(O)CH3, or C(O)OCH3; and Y is N or CH, and in (I), when X1 and X3 are O, X2 is not H or methyl.) Also provided are compounds comprising a nucleic acid backbone monomer or a nucleic acid analog backbone monomer having the formula: [Brief explanation of the drawings]

[0007] [Figure 1] FIG. 1 shows hydrogen bonding interactions between (A) modified nucleobase and modified nucleobase (homoduplex), (B) natural and natural, and (C) modified and natural (heteroduplex). [Figure 2] Figure 2 is a schematic diagram illustrating the distinct advantages of newly designed oligonucleotide molecules that can selectively target RNA secondary structures. (A) Despite (a' / a) sequence complementarity, PNA oligomers (chiral or nonchiral) containing modified (u, c, a, and g) nucleobases cannot adopt a hairpin structure and can hybridize to their complementary stem-loop RNA targets. (B) While tightly binding oligonucleotide molecules such as LNA or yPNAs can penetrate stem-loop structures, their application in therapeutic and diagnostic applications poses significant risks due to nonspecific binding. (C) Medium-avidity oligonucleotides, the category that most oligonucleotide molecules fall into, cannot open the stem-loop structure due to a lack of binding free energy or as a result of the formation of a dynamic (hairpin) trap. [Figure 3] FIG. 3 shows schematic examples of RNA (A) secondary and (B) tertiary structures that allow the oligonucleotide molecules described herein to selectively target something that would otherwise be difficult to achieve with existing nucleic acid systems. [Figure 4] FIG. 4 shows exemplary nucleobase structures. [Figure 5] FIG. 5 shows exemplary nucleic acid analog residues for nucleic acid analogs, including phosphorothioate DNA (PS DNA), α,β-constrained nucleic acid (α,β-CNA), 2′-methoxyl RNA, 2′-fluoro RNA, phosphorodiamidate morpholino oligomer (PMO), locked nucleic acid (LNA), 2′,4′-constrained ethyl nucleic acid ((S)-cEt), 2′,4′-bridged nucleic acid NC(NH) (BNA-NC(NH)), 2′,4′-bridged nucleic acid NC(N-methyl) (BNA-NC(N-Me)), ((S)-5′-C-methyl DNA(RNA)), and 5′-E-vinylphosphonate nucleic acid (E-VP) (where R is H, OH, F, OMe, or O(CH)OMe). [Figure 6] FIG. 6 shows exemplary synthetic schemes for selected nucleobases. [Figure 7] FIG. 7 shows an exemplary synthesis scheme for selected cell-permeable γPNA monomers. Specific Description of the Invention

[0008] The use of numerical values ​​in the various ranges set forth herein, unless otherwise expressly stated, is described as an approximation, as if the word "about" preceded both the minimum and maximum values ​​within the stated range. In this manner, slight variations above and below the stated range can be used to achieve substantially the same results as values ​​within the range. Also, unless otherwise noted, the disclosure of a range is intended as a continuous range, including all values ​​between the minimum and maximum values. As used herein, "a" and "an" refer to one or more.

[0009] As used herein, the term "comprising" is open-ended and can be synonymous with "including," "containing," or "characterized by." As used herein, embodiments that "comprise" one or more stated elements or steps also include, but are not limited to, embodiments that "consist essentially of" and "consist of" those stated elements or steps.

[0010] The term "polymer composition" refers to a composition comprising one or more polymers. As a class, "polymers" include, but are not limited to, homopolymers, heteropolymers, copolymers, block polymers, and block copolymers, both natural and synthetic. Homopolymers contain one type of building block or monomer, while copolymers contain two or more types of monomer. An "oligomer" is a polymer comprising a small number of monomers, e.g., 3 to 100 monomer residues. Thus, the term "polymer" includes oligomers. The terms "nucleic acid" and "nucleic acid analog" include nucleic acid and nucleic acid polymers and oligomers.

[0011] When a polymer incorporates a recited monomer, the polymer "comprises" or "derived from" that monomer. Thus, the incorporated monomer that the polymer contains is not the same as the monomer prior to its incorporation into the polymer, at least insofar as certain crosslinking groups have been incorporated into the polymer backbone or certain groups have been removed during polymerization. When a particular type of bond is present within a polymer, the polymer is said to comprise that bond. The incorporated monomer is a "residue." Typical monomers of nucleic acids or nucleic acid analogs, when incorporated into a polymer, are referred to as nucleotides or nucleotide residues.

[0012] A "moiety" is a part of a chemical compound and comprises a group, such as a functional group. Thus, a nucleobase moiety is a nucleobase that has been modified by attachment to another chemical moiety, such as a polymer monomer, e.g., a nucleic acid or nucleic acid analog monomer described herein, or a polymer, such as a nucleic acid or nucleic acid analog described herein.

[0013] "Alkyl" refers to a straight-chain, branched-chain, or cyclic hydrocarbon group containing 1 to about 20 carbon atoms, including, but not limited to, C 1-3 , C 1-6 , C 1-10The term "alkyl" refers to groups such as, but not limited to, straight chain, branched chain alkyl groups such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, etc. Alkyl groups include, for example, substituted or unsubstituted C1, C2, C3, C4, C5, C6, C7, C8, C9, C 10 , C 11 , C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C 18 , C 19 , C 20 , C 21 , C 22 , C 23 , C 24 , C 25 , C 26 , C 27 , C 28 , C 29 , C 30 , C 31 , C 32 , C 33 , C 34 , C 35 , C 36 , C 37 , C 38 , C 39 , C 40 , C 41 , C 42 , C 43 , C 44 , C 45 , C 46 , C 47 , C 48 , C 49 , or C 50It can be a group. Non-limiting examples of straight-chain alkyl groups include methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, and decyl. Branched-chain alkyl groups include any straight-chain alkyl group substituted with any number of alkyl groups. Non-limiting examples of branched-chain alkyl groups include isopropyl, isobutyl, sec-butyl, and t-butyl. Non-limiting examples of cyclic alkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups. Cyclic alkyl groups also include fused bicyclic, bridged bicyclic, and spiro bicyclic rings, as well as higher-order fused, bridged, and spiro systems. Cyclic alkyl groups can be substituted with any number of straight-chain, branched, or cyclic alkyl groups.

[0014] "Substituted alkyl" refers to an alkyl substituted at one or more positions (e.g., 1, 2, 3, 4, 5, or 6 positions) with a substituent as described herein, wherein the substituent is attached at any available atom to result in a stable compound. "Optionally substituted alkyl" refers to an alkyl or substituted alkyl. "Halogen," "halide," and "halo" refer to -F, -CI, -Br, and / or -I. "Alkylene" and "substituted alkylene" refer to divalent alkyl and divalent substituted alkyl, respectively, including, but not limited to, ethylene (-CH-CH-). "Optionally substituted alkylene" refers to alkylene or substituted alkylene.

[0015] "Alkene or alkenyl" refers to a straight-chain, branched-chain, or cyclic hydrocarbyl group containing from 2 to about 20 carbon atoms, including, but not limited to, C 1 , C 2 , C 3 , C 4 , C 5 , C 6 , C 7 , C 8 , C 9 , C 10 , C 11 , C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C 18 , C 19 , C 20 , C 21 , C 22 , C 23 , C 24 , C 25 , C 26 , C 27 , C 28 , C 29 , C 30 , C 31 , C 32 , C 33 , C 34 , C 35 , C 36 , C 2-3 , C 2-6 , C 2-10The olefin(s) of the alkenyl group can be, for example, E, Z, cis, trans, terminal, or exo-methylene. The alkenyl or alkenylene group can be, for example, a substituted or unsubstituted C2, C3, C4, C5, C6, C7, C8, C9, C 10 , C 11 , C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C 18 , C 19 , C 20 , C 21 , C 22 , C 23 , C 24 , C 25 , C 26 , C 27 , C 28 , C 29 , C 30 , C 31 , C 32 , C 33 , C 34 , C 35 , C 36 , C 37 , C 38 , C 39 , C 40 , C 41 , C 42 , C 43 , C 44 , C 45 , C 46 , C 47 , C 48 , C 49 , or C 50 A haloalkenyl group may be any alkenyl group substituted with any number of halogen atoms.

[0016] "Substituted alkene" refers to an alkene substituted at one or more positions (e.g., 1, 2, 3, 4, or 5 positions) with a substituent as described herein, and the substituents are attached at any available atom to result in a stable compound. "Optionally substituted alkene" refers to an alkene or a substituted alkene. Similarly, "alkenylene" refers to a divalent alkene. Examples of alkenylene include, but are not limited to, ethenylene (-CH=CH-) and all stereoisomeric and conformational isomeric forms thereof. "Substituted alkenylene" refers to a divalent substituted alkene. "Optionally substituted alkenylene" refers to alkenylene or substituted alkenylene.

[0017] Alkyne or "alkynyl" refers to a straight-chain, branched-chain, or cyclic unsaturated hydrocarbon having the indicated number of carbon atoms and at least one triple bond. The triple bond of the alkyne or alkynyl group can be internal or terminal. Examples of (C2-C8)alkynyl groups include, but are not limited to, acetylene, propyne, 1-butyne, 2-butyne, 1-pentyne, 2-pentyne, 1-hexyne, 2-hexyne, 3-hexyne, 1-heptyne, 2-heptyne, 3-heptyne, 1-octyne, 2-octyne, 3-octyne, and 4-octyne. Alkyne or alkynyl groups can be unsubstituted or optionally substituted with one or more substituents as described herein below. Alkyne or alkynyl groups can be, for example, substituted or unsubstituted C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, C25, C26, C27, C28, C29, C30, C31, C32, C33, C34, C35, C36, C37, C38, C39, C40, C41, C42, C43, C44, C45, C46, ​​C47, C48, C49, C50, C51, C52, C53, C54, C55, C56, C57, C58, C59, C60, C61, C62, C63, C64, C65, C66, C78, ​​C79, C80, C81, C 10 , C 11 , C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C 18 , C 19 , C 20 , C 21 , C 22 , C 23 , C 24 , C 25 , C 26 , C 27 , C 28, C 29 , C 30 , C 31 , C 32 , C 33 , C 34 , C 35 , C 36 , C 37 , C 38 , C 39 , C 40 , C 41 , C 42 , C 43 , C 44 , C 45 , C 46 , C 47 , C 48 , C 49 , or C 50 A haloalkynyl group may be any alkenyl group substituted with any number of halogen atoms.

[0018] The term "alkynylene" refers to a divalent alkyne. Examples of alkynylene include, but are not limited to, ethynylene and propynylene. "Substituted alkynylene" refers to a divalent substituted alkyne.

[0019] The term "alkoxy" refers to an -O-alkyl group having the indicated number of carbon atoms. An ether or ether group comprises an alkoxy group. For example, (C1-C6)alkoxy groups include -O-methyl (methoxy), -O-ethyl (ethoxy), -O-propyl (propoxy), -O-isopropyl (isopropoxy), -O-butyl (butoxy), -O-sec-butyl (sec-butoxy), -O-tert-butyl (tert-butoxy), -O-pentyl (pentoxy), -O-isopentyl (isopentoxy), -O-neopentyl (neopentoxy), -O-hexyl (hexyloxy), -O-isohexyl (isohexyloxy), and -O-neohexyl (neohexyloxy). "Hydroxyalkyl" refers to an alkyl group in which one or more of its hydrogen atoms has been replaced with an -OH group (C1-C6). 10) alkyl groups. Examples of hydroxyalkyl groups include, but are not limited to, -CH2OH, -CH2CH2OH, -CH2CH2CH2OH, -CH2CH2CH2CH2OH, -CH2CH2CH2CH2CH2OH, -CH2CH2CH2CH2CH2CH2OH, and branched forms thereof. The term "ether" or "oxygen ether" refers to an alkyl group in which one or more of the carbon atoms of the alkyl group has been replaced with an -O- group (C1-C 10 ) alkyl group. The term ether includes -CH2-(OCH2-CH2) q OP1 compounds, wherein P1 is a protecting group, —H, or (C1-C 10 ) alkyl. Exemplary ethers include polyethylene glycol, diethyl ether, methylhexyl ether, and the like.

[0020] The term "thioether" refers to a thioether in which one or more of the carbon atoms of an alkyl group has been replaced with an -S- group (C1-C 10 ) alkyl group. The term thioether includes -CH2-(SCH2-CH2) q -SP1 compounds, wherein P1 is a protecting group, -H, or (C1-C 10 ) alkyl. Exemplary thioethers include dimethyl thioether or ethyl methyl thioether.

[0021] Protecting groups (e.g., for protecting amines during the synthesis of the compounds herein) are known in the art and include, but are not limited to, 9-fluorenylmethyloxycarbonyl (Fmoc), t-butyloxycarbonyl (Boc), benzhydryloxycarbonyl (Bhoc), benzyloxycarbonyl (Cbz), O-nitroveratryloxycarbonyl (Nvoc), benzyl (Bn), allyloxycarbonyl (alloc), trityl (Trt), dimethoxytrityl (DMT), 1-(4,4-dimethyl-2,6-dioxacyclohexylidene)ethyl (Dde), diathiasuccinoyl (Dts), benzothiazole-2-sulfonyl (Bts), and monomethoxytrityl (MMT) groups.

[0022] "Aryl," alone or in combination, refers to an aromatic monocyclic or bicyclic ring system such as phenyl or naphthyl. "Aryl" also includes aromatic ring systems optionally fused with a cycloalkyl ring. "Substituted aryl" refers to an aryl independently substituted with one or more substituents attached at any available atom to produce a stable compound, the substituents being as described herein. The substituents can be, for example, hydrocarbyl groups, alkyl groups, alkoxy groups, and halogen atoms. "Optionally substituted aryl" refers to an aryl or substituted aryl. An aryloxy group can be, for example, one in which the oxygen atom is substituted with any aryl group, such as phenoxy. An arylalkoxy group can be, for example, one in which the oxygen atom is substituted with an aralkyl group, such as benzyloxy.

[0023] "Arylene" refers to a divalent aryl, and "substituted arylene" refers to a divalent substituted aryl. "Optionally substituted arylene" refers to an arylene or substituted arylene.

[0024] "Heteroatom" refers to N, O, P, and S. Compounds containing an N or S atom may be optionally oxidized to the corresponding N-oxide, sulfoxide, or sulfone compound. "Heterosubstituted" refers to an organic compound, in any embodiment described herein, in which one or more carbon atoms are replaced with N, O, P, or S.

[0025] "Cycloalkyl" refers to a monocyclic, bicyclic, tricyclic, or polycyclic, saturated, unsaturated, or aromatic 3- to 14-membered ring system. The cycloalkyl group may be attached via any atom. Cycloalkyl also contemplates fused rings in which a cycloalkyl is fused to an aryl or heteroaryl ring. Representative examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. A cycloalkyl group may be unsubstituted or optionally substituted with one or more substituents as described herein below. "Cycloalkylene" refers to a divalent cycloalkyl. The term "optionally substituted cycloalkylene" refers to a cycloalkylene substituted with one, two, or three substituents attached at any available atom to result in a stable compound.

[0026] "Carboxyl" or "carboxylic" refers to a group having the indicated number of carbon atoms and terminated by a -C(O)OH group, thus having the structure -RC(O)OH, where R is a divalent organic group containing a straight-chain, branched-chain, or cyclic hydrocarbon. Non-limiting examples of these include C 1-8 Examples of carboxylic acid groups include an ethanoic acid group, a propanoic acid group, a 2-methylpropanoic acid group, a butanoic acid group, a 2,2-dimethylpropanoic acid group, and a pentanoic acid group.

[0027] "(C3-C8)aryl-(C1-C6)alkylene" refers to a C1-C6 alkylene group. refers to a divalent alkylene in which one or more hydrogen atoms are replaced by a (C3-C8) aryl group Examples of (C3-C8)aryl-(C1-C6)alkylene groups include, but are not limited to, 1-phenylbutylene, phenyl-2-butylene, 1-phenyl-2-methylpropylene, phenylmethylene, phenylpropylene, and naphthylethylene. The term "(C3-C8)cycloalkyl-(C1-C6)alkylene" refers to a divalent alkylene in which one or more hydrogen atoms of the C1-C6 alkylene group are replaced with a (C3-C8)cycloalkyl group. Examples of (C3-C8)cycloalkyl-(C1-C6)alkylene groups include, but are not limited to, 1-cyclopropylbutylene, cyclopropyl-2-butylene, cyclopentyl-1-phenyl-2-methylpropylene, cyclobutylmethylene, and cyclohexylpropylene.

[0028] A moiety such as a nucleobase moiety, a functional group, a guanidine-containing group, or a PEG-containing group within a larger molecule, such as a nucleic acid or nucleic acid polymer chain as described herein, is "linked" to the rest of the molecule, meaning that it is covalently attached to the rest of the molecule directly or via an inert bridging moiety. "Inert" means that the linker does not substantially affect the function of the nucleic acid or nucleic acid analog for its intended use. Non-limiting examples of inert linkers include linear, branched, and / or cyclic hydrocarbyl or substituted hydrocarbyl moieties having, for example, less than 15, 10, or 6 carbon atoms, e.g., alkylene moieties such as methylene, ethylene, propylene, or butylene groups, or aryl groups, and optionally containing heteroatoms such as S (e.g., thioether), N (tertiary amine), or O (e.g., ether) atoms, and / or bridging bonds, e.g., but not limited to, ester, amide, carbamate, or carbonate bonds. A linker can act as a spacer to physically or spatially separate two components of a molecule.

[0029] Provided herein are nucleic acids and their analogs, collectively "gene recognition reagents," that specifically bind to nucleic acid strands under physiological conditions, such as saline (0.9% by weight NaCl) or another isotonic solution, such as Tris-buffered saline or PBS, at, for example, 37° C. The gene recognition reagent comprises multiple nucleobase moieties, each attached to a nucleic acid backbone or nucleic acid analog backbone monomer residue, e.g., in the case of DNA or RNA, to form a nucleoside or, together with a phosphate group, to form a nucleotide, and forming part of a larger gene recognition reagent comprising at least two nucleic acid or nucleic acid monomer residues, and thus at least two nucleobase moieties.

[0030] In one aspect, all the nucleic acid bases of gene recognition reagent are modified nucleic acid bases as described herein.In another embodiment, the gene recognition reagent described herein comprises at least one modified nucleic acid base as described herein, and other nucleic acid bases are natural nucleic acid bases (for example, adenine, guanine, cytosine, thymine or uracil) or are different from these modified bases.

[0031] Thus, in one embodiment, modified nucleobases are provided. These nucleobases can be incorporated into nucleic acid or nucleic acid analog monomers (e.g., nucleotides), which can then be incorporated into oligomers or polymers of the monomers having the desired nucleobase sequence. The structure of the modified nucleobase is shown below: [ka] (In the formula, X1 is =O (= represents a double bond), =S, =Se, or CH3; X2 is H, CH3, CN, NC, N3, C(O)OH, or C(O)NH2; X3 is O or S; X4 is H, C(O)CH3, or C(O)OCH3; and Y is N or CH; In (1), when X1 and X3 are O, X2 is not H or methyl. Non-limiting examples of such nucleobases that form a complete set of nucleobases that can combine with A, T, G, and C in natural DNA or RNA include: [ka] Includes:

[0032] Further examples of such nucleobases are the following fluorescent bases: [ka] is.

[0033] Any, some, or all of the above nucleic acid bases can be incorporated into nucleotide monomers and gene recognition reagents.

[0034] In one embodiment, the gene recognition reagents described herein can self-assemble onto a nucleic acid template comprising the target sequence of the gene recognition reagent. For example, a first gene recognition reagent can hybridize to a first portion of the nucleic acid template. A second self-assembling gene recognition reagent can hybridize to a second portion adjacent to the first portion of the nucleic acid template. The first gene recognition reagent and the second gene recognition reagent can be covalently linked or non-covalently linked to each other using various functional end groups to form a continuous structure. The gene recognition reagent can comprise a first portion linked to a first end of the nucleic acid backbone or nucleic acid analog backbone by a linker and a second portion linked to a second end of the nucleic acid backbone or nucleic acid analog backbone by a linker. The second portion can be the same as the first portion. The second portion can be different from the first portion. In one example, International Patent Application No. PCT / US18 / 67096, which is incorporated herein by reference, includes amino acids such as TTC, TTCTTC, TCT, TCTTCT, CTT, CTTCTT, CCG, CCGCCG, CGC, CGCCGC, GCC, GCCGCC, CGG, CGGCGG, GCG, GCGGCG, GGC, GGCGGC, CTG, CTGCTG, TGC, TGCTGC, GCT, GCTGCT, CAG, CAGCAG, AGC, AGCAGC, GCA, GCAGCA, CAGG, CA Self-assembling gene recognition reagents have been disclosed that contain terminal aryl groups that form strong non-covalent bonds between unit gene recognition reagents by pi-stacking when adjacent to target sequences, such as extended repeats, such as GGCAGG, AGGC, AGGCAGGC, GGCA, GGCAGGCA, GCAG, GCAGGCAG, AGAAT, GAATA, AATAG, ATAGA, TAGAA, GGCCCC, GCCCCG, CCCCGG, CCCGGC, CCGGCC, and CGGCCC. Another example of a self-assembling gene recognition reagent is described in International Patent Application Publication No. WO2014 / 169216, the entire contents of which are incorporated herein by reference, which self-assembles and covalently bonds in a reducing environment due to terminal thioester and sulfhydryl groups.Covalent bonds form between the unit self-assembling gene recognition reagents when adjacently juxtaposed with a target sequence, e.g., a nucleic acid comprising an expanded repeat as described above. Other terminal groups, such as affinity binding partners or reactive groups, can also be attached to the termini of the gene recognition reagents described herein to allow them to self-assemble onto complementary nucleic acid templates.

[0035] In one embodiment, a compound comprising a nucleobase and a nucleic acid backbone or nucleic acid analog backbone monomer is provided herein. In the context of this disclosure, "nucleotide" refers to a compound or a residue of a gene recognition reagent comprising at least one nucleobase and a backbone element (ribose or deoxyribose in the case of nucleic acids such as RNA or DNA). The nucleotide monomer also comprises a reactive group that allows polymerization under certain conditions. In natural DNA and RNA, these reactive groups are a 5' phosphate group and a 3' hydroxyl group. For the chemical synthesis of nucleic acids and their analogs, the base and backbone monomer may contain modified groups such as blocked amines, as is known in the art. A "nucleotide residue" refers to a single nucleotide incorporated into an oligonucleotide or polynucleotide. Similarly, a "nucleobase residue" refers to a nucleobase incorporated into a nucleotide or nucleic acid or its analog. A "gene recognition reagent" generally refers to a nucleic acid or nucleic acid analog comprising a sequence of nucleobases that can hybridize to a complementary nucleic acid sequence on a nucleic acid by cooperative base pairing (e.g., Watson-Crick base pairing or Watson-Crick-like base pairing) (see Figure 1). Intramolecular base pairing does not occur between the modified bases described herein, where steric clashes occur or there are insufficient hydrogen bonds between bases that normally base pair in natural nucleic acids (Figure 1 panel (A) ("Figure 1(A)")). As can be seen in Figure 1(B), normal base pairing is asymmetric, with two hydrogen bonds forming between nucleobases A and T / U and three hydrogen bonds forming between nucleobases G and C. Figure 1(C) illustrates a substantial benefit of using the modified nucleobases described herein in that binding is symmetric, with two hydrogen bonds forming between all combinations of the modified nucleobases described herein and their natural base pairing partners. Not only does the nearly equal distribution of base pair binding "weights" greatly simplify probe design, but the stronger dependence of probe binding strength on length rather than length and sequence composition confers greater sequence discrimination against base pair mismatches than is achievable with natural nucleobases.

[0036] Figure 2 illustrates the benefits of the modified nucleobases described herein. Figure 2(A) shows that despite (a' / a) sequence complementarity, PNA oligomers (chiral or non-chiral) containing modified (u, c, a, and g) nucleobases cannot adopt a hairpin structure but can hybridize to their complementary stem-loop RNA targets. Figure 2(B) shows that tightly binding oligonucleotide molecules, such as locked nucleic acids (LNAs) or PNAs (e.g., yPNAs), can enter the stem-loop structure, but their application in therapeutic and diagnostic applications poses significant risks due to nonspecific binding. Figure 2(C) shows that medium-avidity oligonucleotides, the category that most oligonucleotide molecules fall into, cannot open the stem-loop structure due to lack of binding free energy or as a result of the formation of a dynamic (hairpin) trap.

[0037] Examples of selectively targetable RNA secondary and tertiary structures are shown in Figure 3. Potential therapeutic and diagnostic targets comprise both coding and non-coding RNA, as well as DNA.

[0038] In some embodiments, modified nucleobases are provided herein. Nucleobases are recognition moieties that specifically bind to one or more of adenine, guanine, thymine, cytosine, and uracil, for example, by Watson-Crick or Watson-Crick-like base pairing through hydrogen bonding. "Nucleobases" include basic (natural) nucleobases: adenine, guanine, thymine, cytosine, and uracil, as well as modified purine and pyrimidine bases, such as, but not limited to, hypoxanthine, xanthene, 7-methylguanine, 5,6, dihydrouracil, 5-methylcytosine, and 5-hydroxymethylcytosine. Figure 4 also shows non-limiting examples of nucleobases, including monovalent nucleobases (e.g., adenine, cytosine, guanine, thymine, or uracil that bind to one strand of a nucleic acid or nucleic acid analog), and "clamp" nucleobases, such as "G-clamps," that bind to complementary nucleobases with enhanced strength. Additional purine, purine-like, pyrimidine and pyrimidine-like nucleobases are also known in the art, as disclosed, for example, in U.S. Patent Nos. 8,053,212, 8,389,703, and 8,653,254. Divalent nucleobases are described in more detail in U.S. Patent Application Publication No. 2016 / 0083434A1 and International Patent Application Publication No. WO / 2018 / 058091, all of which are incorporated herein by reference, and can bind to two nucleobases instead of one, thus forming complex trimeric structures with matched or mismatched nucleic acids.

[0039] In one example, the backbone monomer is a ribose monophosphate, diphosphate, or triphosphate or a deoxyribose monophosphate, diphosphate, or triphosphate, e.g., the 5' monophosphate, diphosphate, or triphosphate of ribose or deoxyribose. Backbone monomers include structural "residue" components, such as ribose in RNA, and active groups that are modified when interconnecting monomers, e.g., both the 5' triphosphate and 3' hydroxyl groups of a ribonucleotide, which are modified to leave a phosphodiester bond upon polymerization into RNA. Similarly, with respect to PNA, the C-terminal carboxyl and N-terminal amine active groups of an N-(2-aminoethyl)glycine backbone monomer condense upon polymerization to leave a peptide (amide) bond. In another embodiment, the active group is a phosphoramidite group, useful for phosphoramidite oligomer synthesis, as is widely known in the art. Nucleotide monomers may also optionally comprise one or more protecting groups, such as 4,4'-dimethoxytrityl (DMT), as described herein, as are known in the art. Several additional methods for producing synthetic gene recognition reagents are known, and vary depending on the specific chemistry of the backbone structure and base addition step. The determination of which active groups to use for linking nucleotide monomers and which groups in the bases to protect, as well as the steps required in producing the oligomer, are well within the capabilities of those skilled in the art of chemistry and the specific field of nucleic acid and nucleic acid analog oligomer synthesis.

[0040] As used herein, the term "nucleic acid" refers to deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Nucleic acid analogs include, but are not limited to (see, e.g., Figure 5): phosphorothioate DNA (PS DNA), α,β-constrained nucleic acid (α,β-CNA), 2'-methoxyl RNA, 2'-fluoro RNA, phosphorodiamidate morpholino oligomer (PMO), locked nucleic acid (LNA), 2',4'-constrained ethyl nucleic acid ((S)-cEt), 2',4'-bridged nucleic acid NC(NH) (BNA-NC(NH)), 2',4'-bridged nucleic acid NC(N-methyl) (BNA-NC(N-Me)), ((S)-5'-C-methyl DNA(RNA)), and 5'-E-vinylphosphonate nucleic acid (E-VP) (where R is H, OH, F, OMe, or O(CH2)2OMe), as well as combinations thereof, optionally containing ribonucleotide or deoxyribonucleotide residues. An "oligonucleotide" is a short, single-stranded gene recognition reagent. Oligonucleotides can be designated by the designation "mer" depending on the length of the chain (i.e., the number of nucleotides or nucleobases). For example, an oligonucleotide of 22 nucleotides is designated a 22-mer.

[0041] "Peptide nucleic acid" refers to a DNA or RNA analog or mimetic in which the sugar phosphodiester backbone of DNA or RNA is replaced with N-(2-aminoethyl)glycine units. In one embodiment, a peptide nucleic acid has the following structure: [ka] (wherein n is 1 or more, and R1, R2, R3, R4, R5, and R6 independently represent H; CH3, CH2OH, CH(CH3)OH, CH2SH, CH(CH3)CH3, CH2CH(CH3)CH3, CH(CH3)CH2CH3, CH2CH2SCH3, CH2CH3, CH2-C6H5, CH2-C6H4OH, 1H-indol-3-ylmethyl, CH2C(O)OH, CH2CH2C(O)OH, CH2C(O)NH2, CH2CH2C(O)NH 2, 1H-imidazol-4-ylmethyl, CH2CH2CH2CH2NH2, or CH2CH2CH2NHC(NH)NH2; straight-chain or branched (C3-C8) alkyl, (C2-C8) alkenyl, (C2-C8) alkynyl, (C3-C8) aryl, (C3-C8) cycloalkyl, (C3-C8) aryl(C1-C6) alkylene, (C3-C8) cycloalkyl(C1-C6) alkylene, guanidine-containing groups, CH2-(OCH2-CH2) n -OH, CH2-(OCH2-CH2) n -NH2, CH2-(OCH2-CH2) n -SH, CH2-(OCH2-CH2) n -NHC(NH)NH2, CH2-(OCH2-CH2) n -morpholine, CH2-(OCH2-CH2) n -piperazine, [ka] wherein X is a linker, such as a linear linker, and R1 and R2 together form a 1,3-propylene bond, R3 and R4 together form a 1,3-propylene bond, or R5 and R6 together form a 1,3-propylene bond, and each occurrence of R7 is independently a nucleobase, and when n is 2 or greater, forms a nucleobase sequence. It has.

[0042] A linker is a moiety within a compound that covalently connects one moiety to another. The linker does not substantially adversely affect the activity of the overall compound, e.g., in the context of the present invention, the ability of a gene recognition reagent to function in its intended use. In addition to providing a covalent link between two moieties, a linker may have beneficial effects, such as physical separation of the moieties to which it is attached, for example, to optimize spacing to avoid steric effects. A linker can also provide additional functionality, such as providing an additional site (e.g., an amine protected by a protecting group) for linking additional moieties of the compound, or modifying the hydrophobicity / hydrophilicity of the overall molecule to rigidify the entire molecule. The linker is attached to the remainder of the compound by any suitable linking moiety ("bond"), for example, by a carbon-carbon bond, ester bond, thioester bond, amine bond, ether bond, amide bond, carbonate bond, or carbamate bond to the additional moiety of the compound. The linker may be a hydrocarbyl containing only carbon and hydrogen (e.g., 1-10 methylene groups), optionally containing one or more heteroatoms such as N, O, and / or S. In one embodiment, for the present invention, one suitable linker is the PEG group -(O-CH-CH) n -, wherein n is in the range of 2 to 100, for example, 2 to 10 (PEG 2-10 ), or 2-5 (PEG 2-5 ), for example, 2 (PEG2), 3, 4, 5, 6, 7, 8, 9, or 10. The PEG linker may comprise one or more methylene groups at either end in addition to a suitable bond attaching the PEG group to the connecting moiety.

[0043] In another embodiment, the PNA is a gamma PNA (γPNA), which is an oligomer or polymer of gamma-modified N-(2-aminoethyl)glycine monomers in which the γ carbon of formula (I) above is a chiral center; generally, one of R1 or R2 attached to the gamma carbon is H and the other is not hydrogen, or R1 and R2 are different, making the gamma carbon a chiral center. When R1 and R2 are both hydrogen (N-(2-aminoethyl)-glycine backbone) or the same, there is no such chirality about the gamma carbon. An alpha PNA (αPNA) is an oligomer or polymer of alpha-modified N-(2-aminoethyl)glycine monomers in which the α carbon of formula (I) above is a chiral center. A beta PNA (βPNA) is an oligomer or polymer of beta-modified N-(2-aminoethyl)glycine monomers in which the β carbon of formula (I) above is a chiral center. The following discussion of γPNAs applies equally to αPNAs and βPNAs, and any two combinations of the α, β, and γ carbons (α,β, α,γ, or β,γ) or all three (α,β,γ) can form chiral centers.

[0044] In various embodiments, for example, the backbone in one or more of R1, R2, R3, R4, R5, and R6 is pegylated with 1 to 50 oxyethylene residues, i.e., [—O—CH2—CH2—] where n is 1 to 50, inclusive. n The PEG group can be linked by any suitable linking group, for example, C 1-6 It can be linked to the backbone by alkyl groups or and aryl-alkyl groups.

[0045] In other embodiments, the backbone, eg, at one or more of R1, R2, R3, R4, R5, and R6, comprises one or more guanidine-containing groups, eg, an alkyl or aryl-alkyl moiety terminating in a guanidine moiety.

[0046] In other embodiments, to promote cellular uptake and endosomal escape, one or more of R1, R2, R3, R4, R5, and R6 is S1A, S1B, S1C, S1D, S1E, S1F, S1G, S1H, S1I, S1J, S1K, or S1L.

[0047] An "amino acid side chain" is the side chain of an amino acid. An amino acid has the structure: [ka] (wherein "Side" is an amino acid side chain). Non-limiting examples of amino acid side chains include CH3(Ala), CH2OH(Ser), CH(CH3)OH(Thr), CH2SH(Cys), CH(CH3)CH3(Val), CH2CH(CH3)CH3(Leu), CH(CH3)CH2CH3(Ile), CH2CH2SCH3(Met), 4-CH2-C6H4OH(Tyr), CH2-C6H5(Phe), 1H-indole-3 Examples include 1H-imidazol-4-ylmethyl (Trp), CHC(O)OH(Asp), CHCHC(O)OH(Glu), CHC(O)NH(Asn), CHCHC(O)NH(Gln), 1H-imidazol-4-ylmethyl (His), CHCHCHCHNH(Lys), or CHCHCHNHC(NH)NH(Arg comprising a guanidine / guanidinium group). Glycine is not represented in this embodiment due to the absence of its side chain (Side is H).

[0048] The γPNA monomers incorporated into a γPNA oligomer or polymer are referred to herein as "γPNA monomer residues," each of which has the same or a different nucleobase, e.g., a modified nucleobase described herein; thus, as in DNA or RNA, the order of the bases in the γPNA is its "sequence." The nucleobase sequence of a nucleic acid or nucleic acid analog oligomer or polymer, e.g., a γPNA oligomer or polymer, binds to a complementary sequence of adenine, guanine, cytosine, thymine, and / or uracil residues in a nucleic acid strand by cooperative bonding essentially similar to the Watson-Crick bonding of complementary bases in double-stranded DNA or RNA. "Watson-Crick-like" bonding refers to hydrogen bonding of nucleobases other than G, A, T, C, or U, e.g., bonding of divalent bases designated herein as G, A, T, C, U, or other nucleobases.

[0049] Unless otherwise specified, the nucleic acids and nucleic acid analogs described herein are not described with respect to any specific base sequence. The present disclosure is directed to modified nucleobases, compositions comprising these modified nucleobases, and methods for using these modified nucleobases and compounds containing these nucleobases, and the usefulness of any specific embodiment described herein is generally applicable, although in each case it generally depends on a specific sequence. The nucleobase sequence attached to the backbone of a gamma PNA oligomer can hybridize with the complementary nucleobase sequence of a target nucleic acid or nucleic acid analog through Watson-Crick or Watson-Crick-like hydrogen bonding. Those skilled in the art will understand that the compositions and methods described herein are sequence-independent and describe novel, generalized compositions and related methods comprising bivalent nucleobases.

[0050] Gene recognition reagents can be fabricated as small oligonucleotides, which can be assembled in situ, in vivo, ex vivo, or in vitro, as described, for example, in U.S. Patent Application Publication No. 2016 / 0083433 A1, the entire contents of which are incorporated herein by reference. This method allows small oligomers, which have higher cell or tissue permeability than longer sequences, such as trimers, to be translocated into cells, and these oligomers, once hybridized to a template nucleic acid, can assemble into longer, continuous sequences. This can be accomplished in vitro or ex vivo, for example, to rapidly assemble longer sequences for use in hybridizing to target nucleic acids.

[0051] In one embodiment, gene recognition reagents are provided in an array. Arrays are particularly useful for carrying out high-throughput assays, such as gene detection assays. As used herein, the term "array" refers to a reagent, such as the gene recognition reagents described herein, that is arranged or attached to two or more independent, distinguishable, and / or addressable positions on a substrate (support). In one embodiment, an array is a device with two or more independent and distinguishable reaction chambers, such as, but not limited to, a 96-well dish, in which reactions comprising specified components are carried out. In one embodiment, two or more gene recognition reagents comprising one or more bivalent nucleobases as described herein are immobilized on a substrate in a spatially addressable manner, so that each individual primer or probe is located at a different and (addressable) distinguishable position on the substrate. One or more gene recognition reagents are covalently linked to the substrate, or are otherwise bound or located at addressable positions on the array. Substrates include, but are not limited to, multi-well plates, silicon chips, and beads. In one embodiment, the array comprises two or more sets of beads, each bead having a distinguishable marker, such as a quantum dot or a fluorescent tag, so that the beads can be individually distinguished, for example, but not limited to, using a flow cytometer. In one embodiment, the array is a multi-well plate containing two or more wells containing the gene recognition reagents described above for binding to specific sequences. Thus, reagents such as probes and primers are bound to specific locations on the array, or otherwise attached to or positioned on the surface or interior thereof. The reagents may be in any suitable form, including, but not limited to, solution, dry, lyophilized, or vitrified. When covalently linked to a substrate such as agarose beads or a silicon chip, various linking techniques are known for attaching chemical moieties, such as gene recognition reagents, to such substrates.

[0052] Linkers and spacers for use in linking nucleic acids, peptide nucleic acids and other nucleic acid analogs are widely known in the chemical and array fields, and therefore will not be described herein.As a non-limiting example, the γPNA gene recognition reagent contains a reactive amine that can react with carboxyl-functional, cyanogen bromide-functional, N-hydroxysuccinimide ester-functional, carbonyldiimidazole-functional, or aldehyde-functional agarose beads, for example, available from Thermo Fisher Scientific (Pierce Protein Biology Products), Rockford, Illinois, and various other suppliers.The gene recognition reagents described herein can be attached to substrates in any manner with or without a linker.Devices used for carrying out reactions and for reading arrays are widely known and available, and informatics and / or statistical software or other computer-implemented processes for analyzing array data and / or identifying genetic risk factors from data obtained from patient samples are known in the art.

[0053] Certain modified nucleobases described herein fluoresce due to their ring structure. These compositions can be used as fluorescent dyes, or their intrinsic fluorescence can be used as probes by binding to target sequences, for example, in in situ assays or on gels or blots, allowing visualization of the target sequence.

[0054] According to one aspect of the present invention, a method for detecting a target sequence in a nucleic acid is provided, comprising contacting a gene recognition reagent composition as described herein with a sample comprising a nucleic acid and detecting binding of the gene recognition reagent to the nucleic acid. In one embodiment, the gene recognition reagent is immobilized on a substrate, e.g., an array, and a labeled (e.g., fluorescently or radioactively labeled) nucleic acid sample is contacted with the immobilized gene recognition reagent, and the amount of labeled nucleic acid specifically bound to the gene recognition reagent is measured. In one variation, the gene recognition reagent or a nucleic acid comprising the target sequence of the gene recognition reagent is bound to a substrate, and the labeled nucleic acid or labeled gene recognition reagent comprising the target sequence of the gene recognition reagent binds to the immobilized gene recognition reagent or nucleic acid, respectively, to form a complex. In one embodiment, the nucleic acid of the complex comprises a partial target sequence, such that a nucleic acid comprising the complete target sequence is advantageous in competing for the gene recognition reagent with the complexed nucleic acid. The complex is then exposed to a nucleic acid sample, and the loss of the bound label from the complex can be detected and quantified according to standard methods that aid in the quantification of nucleic acid markers in a nucleic acid sample. These are just two of the many possible analytical assays that can be used to detect or quantify the presence of specific nucleic acids in a nucleic acid sample.

[0055] "Immobilized" with respect to a composition, such as a nucleic acid or gene recognition reagent as described herein, means attached to a substrate of any physical structure or chemical composition. The immobilized composition is immobilized by any method useful for the end use. The composition is immobilized by covalent or non-covalent methods, such as by covalent bonding of an amine group to a linker or spacer, or by non-covalent bonding involving van der Waals forces and / or hydrogen bonding. A "label" is a chemical moiety useful for the detection or purification of a molecule or composition comprising the label. Labels can be, for example, but not limited to, 14 C. 32 P, 35The label may be a radioactive moiety such as S, a fluorescent dye such as fluorescein isothiocyanate or cyanine dye, an enzyme, or a ligand for binding with other compounds, for example, biotin for binding with streptavidin, or an epitope for binding with antibody.Many such labels and their use methods are known to those skilled in the art of immunology and molecular biology.However, since certain nucleic acid bases described herein are fluorescent, these bases can be incorporated into the nucleotide residue of nucleic acid or nucleic acid analog, or covalently linked to a bivalent nucleic acid base and nucleic acid, nucleic acid analog, binding reagent, ligand or other detection reagent, allowing detection and / or quantification of the reagent in sample, reaction mixture, array, etc.

[0056] In another aspect of the present invention, a method for isolating and purifying nucleic acids containing target sequences is provided.In one non-limiting embodiment, gene recognition reagents as described herein are immobilized on a substrate such as beads (for example, but not limited to, agarose beads, beads containing fluorescent markers for selection, or magnetic beads), porous matrices, surfaces, tubes, etc.A nucleic acid sample is contacted with this immobilized gene recognition reagent, and the nucleic acid containing target sequences binds to this gene recognition reagent.The bound nucleic acid is then washed to remove unbound nucleic acid, and then the bound nucleic acid is eluted and precipitated or otherwise concentrated by any useful method, as is widely known in the field of molecular biology.

[0057] In a further aspect, a kit is provided. The kit comprises at least one container in any form, including a cartridge for automated nucleic acid, nucleic acid analog, or PNA synthesis, and may comprise one or more containers in the form of individual, independent, and optionally independently addressable compartments for use in an automated sequence preparation device for producing nucleic acids and / or nucleic acid analogs. The container may be disposable or may contain sufficient contents for multiple uses. The kit may also comprise an array. The kit may optionally comprise one or more additional reagents for use in the preparation or use of the gene recognition reagent in any of the embodiments described herein. The kit comprises a container containing any bivalent nucleobase in any form described herein, or a monomer or gene recognition reagent according to any of the aspects described herein. Different nucleobases, monomers, or gene recognition reagents are generally enclosed in separate containers, which may be separate compartments within the cartridge.

[0058] In several embodiments, the compounds and gene-recognition reagents are used for therapeutic purposes. Accordingly, these compounds and gene-recognition reagents are formulated as pharmaceuticals, pharmaceutical compositions, or dosage forms, including compositions for human and veterinary use, containing a therapeutically effective amount of the compound or gene-recognition reagent and an excipient, such as a vehicle or diluent, for therapeutic delivery, for example, but not limited to, oral, topical, intravenous, intramuscular, or subcutaneous administration. Compositions can be formulated in conventional manners using solid or liquid vehicles, diluents, and additives appropriate for the desired mode of administration. Orally, these compounds can be administered in the form of tablets, capsules, granules, powders, and the like. These compositions optionally comprise one or more additional active agents, as is well known in the pharmaceutical, medicinal, veterinary, or biological arts. The compounds described herein can be administered in any effective manner. Further examples of delivery routes include, but are not limited to, topical delivery, such as transdermal, inhalation, enema, intraocular, intraaural, and nasal delivery; enteral delivery, such as oral, gastric feeding tube or swallowing, and rectal delivery; and parenteral delivery, such as intravenous, intraarterial, intramuscular, intracardiac, subcutaneous, intraosseous, intradermal, intrathecal, intraperitoneal, transdermal, iontophoretic, transmucosal, epidural, and intravitreal delivery. Therapeutic / pharmaceutical compositions are prepared in accordance with accepted pharmaceutical procedures, as is well known.

[0059] Any of the compounds described herein can be formulated or otherwise manufactured into compositions suitable for use, such as pharmaceutical dosage forms or pharmaceutical preparations in which the compound or gene recognition reagent is an active ingredient. By way of example, pharmaceutical preparations described herein include oral tablets, capsules, caplets, liquid-filled or gel-filled capsules, and the like. The compositions can comprise a pharmaceutically acceptable carrier, or excipient. An "excipient" is an inert substance used to carry the active ingredient of a medication. While "inert," excipients can facilitate and assist in increasing the delivery, stability, or bioavailability of the active ingredient in a pharmaceutical preparation. Non-limiting examples of useful excipients include anti-adherents, binders, rheology modifiers, coating agents, disintegrants, emulsifiers, oils, buffers, salts, acids, bases, bulking agents, diluents, solvents, flavors, colors, glidants, lubricants, preservatives, antioxidants, adsorbents, vitamins, sweeteners, and the like, as available in the pharmaceutical / formulation arts. [Example]

[0060] Example - Synthesis of modified nucleobases and cell-permeable γPNA The compounds and gene recognition reagents described herein are synthesized according to methods known in the art of chemical and organic synthesis. Exemplary synthetic schemes are shown in Figures 6 and 7.

[0061] The following numbered paragraphs describe non-limiting embodiments and aspects of the present invention.

[0062] Item 1: Comprises a plurality of nucleobase moieties attached to a nucleic acid backbone or nucleic acid analog backbone, wherein at least one nucleobase moiety is: [ka] (In the formula, X1 is =O, =S, =Se, or CH3; X2 is H, CH3, CN, NC, N3, C(O)OH, or C(O)NH2; X3 is O or S; X4 is H, C(O)CH3, or C(O)OCH3; and Y is N or CH; In (1), when X1 and X3 are O, X2 is not H or methyl. A gene recognition reagent.

[0063] Item 2: At least one nucleobase moiety is: [ka] 2. The gene recognition reagent according to claim 1,

[0064] Item 3: At least one nucleobase moiety is: [ka] 2. The gene recognition reagent according to claim 1,

[0065] Item 4: At least one nucleobase moiety is: [ka] 2. The gene recognition reagent according to claim 1,

[0066] Item 5: At least one nucleobase moiety is: [ka] 2. The gene recognition reagent according to claim 1,

[0067] Item 6: At least one nucleobase moiety is: [ka] 2. The gene recognition reagent according to claim 1,

[0068] Item 7: At least one nucleobase moiety is: [ka] 2. The gene recognition reagent according to claim 1,

[0069] Item 8: At least one nucleobase moiety is: [ka] 2. The gene recognition reagent according to claim 1,

[0070] Item 9: The gene recognition reagent according to any one of items 1 to 8, wherein the backbone is selected from the group consisting of DNA, RNA, peptide nucleic acid (PNA), phosphorothioate DNA (PS DNA), α,β-constrained nucleic acid (α,β-CNA), 2'-methoxyl RNA, 2'-fluoro RNA, locked nucleic acid (LNA), 2',4'-constrained ethyl nucleic acid ((S)-cEt), 2',4'-bridged nucleic acid NC(NH) (BNA-NC(NH)), 2',4'-bridged nucleic acid NC(N-methyl) (BNA-NC(N-Me)), 2'-(R)-(S)-5'-C-methyl DNA, and 2'-R-5'-E-vinylphosphonate nucleic acid (E-VP), wherein R is H, OH, F, OMe, or O(CH2)2OMe backbone.

[0071] Item 10: The gene recognition reagent according to item 1, wherein the backbone is a peptide nucleic acid (PNA) backbone.

[0072] Item 11: The gene recognition reagent according to item 10, wherein the backbone is PEGylated with one or more PEG moieties of 2 to 50 (-O-CH2-CH2-) residues linked to the backbone.

[0073] Item 12: The gene recognition reagent according to item 10, wherein the backbone comprises one or more guanidine moieties linked to the backbone.

[0074] Item 13: The gene recognition reagent according to item 1, wherein the backbone is a gamma peptide nucleic acid (γPNA) backbone.

[0075] Item 14: The skeleton is a residue [ka] (wherein n is 1 or more, and R1, R2, R3, R4, R5, and R6 are independently H; CH3, CH2OH, CH(CH3)OH, CH2SH, CH(CH3)CH3, CH2CH(CH3)CH3, CH(CH3)CH2CH3, CH2CH2SCH3, CH2CH3, CH2-C6H5, CH2-C6H4OH, 1H-indol-3-ylmethyl, CH2C(O)OH, CH2CH2C(O)OH, CH2C(O)NH2, CH2CH2C(O)NH 2, 1H-imidazol-4-ylmethyl, CH2CH2CH2CH2NH2, or CH2CH2CH2NHC(NH)NH2; straight-chain or branched (C3-C8) alkyl, (C2-C8) alkenyl, (C2-C8) alkynyl, (C3-C8) aryl, (C3-C8) cycloalkyl, (C3-C8) aryl(C1-C6) alkylene, (C3-C8) cycloalkyl(C1-C6) alkylene, guanidine-containing groups, CH2-(OCH2-CH2) n -OH, CH2-(OCH2-CH2) n -NH2, CH2-(OCH2-CH2) n -SH, CH2-(OCH2-CH2) n -NHC(NH)NH2, CH2-(OCH2-CH2) n -morpholine, CH2-(OCH2-CH2) n -piperazine, [ka] wherein X is a linker, R1 and R2 together form a 1,3-propylene bond, R3 and R4 together form a 1,3-propylene bond, or R5 and R6 together form a 1,3-propylene bond, and each occurrence of R7 is independently a nucleobase. 2. The gene recognition reagent according to claim 1, wherein the PNA backbone comprises:

[0076] Item 15: The gene recognition reagent described in item 14, wherein at least one of R1, R2, R3, R4, R5, and R6 is S1A, S1B, S1C, S1D, S1E, S1F, S1G, S1H, S1I, S1J, S1K, or S1L.

[0077] Item 16: The gene recognition reagent according to item 14 or 15, wherein the α-carbon, β-carbon, or γ-carbon is a chiral center.

[0078] Item 17: The gene recognition reagent according to item 16, wherein the γ-carbon is a chiral center.

[0079] Section 18: R2 [ka] 18. The gene recognition reagent according to item 17,

[0080] Item 19: The gene recognition reagent according to item 16, wherein R1, R3, R4, R5, and R6 are H.

[0081] Item 20: A gene recognition reagent described in any one of items 1 to 19, comprising a terminal group linked to a nucleic acid backbone or a nucleic acid analog backbone for self-assembly of two or more adjacent gene recognition reagents on a nucleic acid template.

[0082] Item 21: The gene recognition reagent according to Item 20, wherein the terminal group is a fused polycyclic aromatic moiety having 2 to 5 rings.

[0083] Item 22: The gene recognition reagent according to Item 20, wherein the terminal group is a sulfhydryl group or a thioester group.

[0084] Clause 23: A gene recognition reagent described in any one of clauses 1 to 22, wherein the multiple nucleobase moieties form a sequence comprising TTC, TTCTTC, TCT, TCTTCT, CTT, CTTCTT, CCG, CCGCCG, CGC, CGCCGC, GCC, GCCGCC, CGG, CGGCGG, GCG, GCGGCG, GGC, GGCGGC, CTG, CTGCTG, TGC, TGCTGC, GCT, GCTGCT, CAG, CAGCAG, AGC, AGCAGC, GCA, GCAGCA, CAGG, CAGGCAGG, AGGC, AGGCAGGC, GGCA, GGCAGGCA, GCAG, GCAGGCAG, AGAAT, GAATA, AATAG, ATAGA, TAGAA, GGCCCC, GCCCCG, CCCCGG, CCCGGC, CCGGCC, and CGGCCC, or consecutive repeats of any of the foregoing.

[0085] Item 24: A sequence in which a plurality of nucleobase segments are complementary to a target sequence of a nucleic acid Placed in 24. The gene recognition reagent according to any one of items 1 to 23, wherein

[0086] Item 25: The gene recognition reagent according to any one of items 1 to 24, which has 3 to 25 nucleic acid base moieties.

[0087] Section 26: Structure: [ka] (In the formula, X1 is =O, =S, =Se, or CH3; X2 is H, CH3, CN, NC, N3, C(O)OH, or C(O)NH2; X3 is O or S; X4 is H, C(O)CH3, or C(O)OCH3; and Y is N or CH; In (I), when X1 and X3 are O, X2 is not H or methyl. A compound comprising a nucleic acid backbone monomer or a nucleic acid analog backbone monomer linked to a nucleobase moiety having the formula:

[0088] Item 27: At least one nucleobase moiety is: [ka] 27. The compound according to claim 26, wherein

[0089] 28. At least one nucleobase moiety is: [ka] 27. The compound according to claim 26, wherein

[0090] Item 29: At least one nucleobase moiety is: [ka] 27. The compound according to claim 26, wherein

[0091] Item 30: At least one nucleobase moiety is: [ka] The compound described in No. 26, wherein

[0092] Item 31: The compound of any one of items 26 to 30, wherein the backbone monomer is DNA, RNA, peptide nucleic acid (PNA), phosphorothioate DNA (PS DNA), α,β-constrained nucleic acid (α,β-CNA), 2'-methoxyl RNA, 2'-fluoro RNA, locked nucleic acid (LNA), 2',4'-constrained ethyl nucleic acid ((S)-cEt), 2',4'-bridged nucleic acid NC(NH) (BNA-NC(NH)), 2',4'-bridged nucleic acid NC(N-methyl) (BNA-NC(N-Me)), 2'-(R)-(S)-5'-C-methyl DNA, or 2'-R-5'-E-vinylphosphonate nucleic acid (E-VP), wherein R is H, OH, F, OMe, or O(CH2)2OMe backbone monomer.

[0093] Item 32: The compound according to any one of items 26 to 31, wherein the backbone monomer is a peptide nucleic acid (PNA) backbone monomer.

[0094] Item 33: The compound according to item 32, wherein the backbone monomer is PEGylated with one or more PEG moieties of 2 to 50 (-O-CH2-CH2-) residues linked to the backbone monomer.

[0095] Item 34: The compound of item 32, wherein the backbone comprises one or more guanidine moieties linked to the backbone.

[0096] Item 35: The compound according to item 26, wherein the backbone monomer is a gamma peptide nucleic acid (γPNA) backbone monomer.

[0097] Item 36: The backbone monomer has the structure: [ka] (Wherein R1, R2, R3, R4, R5, and R6 are independently H; CH3, CH2OH, CH(CH3)OH, CH2SH, CH(CH3)CH3, CH2CH(CH3)CH3, CH(CH3)CH2CH3, CH2CH2SCH3, CH2CH3, CH2-C6H5, 1H-indol-3-ylmethyl, CH2-C6H4OH, CH2C(O)OH, CH2CH2C(O)OH, CH2C(O)NH2, CH2CH2C(O)NH2, 1H- imidazol-4-ylmethyl, CH2CH2CH2CH2NH2, or CH2CH2CH2NHC(NH)NH2; straight-chain or branched (C3-C8) alkyl, (C2-C8) alkenyl, (C2-C8) alkynyl, (C3-C8) aryl, (C3-C8) cycloalkyl, (C3-C8) aryl(C1-C6) alkylene, (C3-C8) cycloalkyl(C1-C6) alkylene, guanidine-containing groups, CH2-(OCH2-CH2) n -OH, CH2-(OCH2-CH2) n -NH2, CH2-(OCH2-CH2) n -SH, CH2-(OCH2-CH2) n-NHC(NH)NH2, CH2-(OCH2-CH2) n -morpholine, CH2-(OCH2-CH2) n -piperazine, [ka] wherein X is a linker, R1 and R2 together form a 1,3-propylene bond, R3 and R4 together form a 1,3-propylene bond, or R5 and R6 together form a 1,3-propylene bond, and each occurrence of R7 is independently a nucleobase. 27. The compound according to paragraph 26, wherein the PNA backbone has the formula:

[0098] Item 37: The compound according to item 36, wherein at least one of R1, R2, R3, R4, R5, and R6 is S1A, S1B, S1C, S1D, S1E, S1F, S1G, S1H, S1I, S1J, S1K, or S1L.

[0099] Item 38: The compound according to item 36 or 37, wherein the α-carbon, β-carbon, or γ-carbon is a chiral center.

[0100] Item 39: The compound according to item 38, wherein the γ-carbon is a chiral center.

[0101] Article 40: R2 [ka] 40. The compound according to claim 39, wherein

[0102] Item 41: The compound according to item 37, wherein R1, R3, R4, R5, and R6 are H.

[0103] Item 42: A kit comprising a compound according to any one of items 36 to 41 in a container.

[0104] Item 43: A kit according to item 42, comprising in separate containers monomer-linked adenine, guanine, cytosine and one or both of uracil and thymine.

[0105] Item 44: A kit comprising a gene recognition reagent according to any one of items 1 to 25 in a container.

[0106] Item 45: A kit according to any one of items 42 to 44, wherein the container is one or more compartments within a cartridge for use in an automated device.

[0107] Item 46: An array comprising the gene recognition reagent according to any one of items 1 to 19.

[0108] Item 47: A method for detecting a target sequence in a nucleic acid, comprising contacting a gene recognition reagent described in any one of items 1 to 25 with a sample containing nucleic acid, and detecting binding between the gene recognition reagent and the nucleic acid.

[0109] Item 48: A method for isolating and purifying a nucleic acid sample containing a target sequence, comprising contacting the nucleic acid sample with a gene recognition reagent described in any one of items 1 to 29, separating the nucleic acid sample from the gene recognition reagent, leaving behind the nucleic acid bound to the gene recognition reagent, and separating the gene recognition reagent from the nucleic acid bound to the gene recognition reagent.

[0110] Item 49: The method of item 48, wherein the gene recognition reagent is immobilized on a substrate, comprising contacting the substrate with nucleic acid, washing the substrate to remove unbound nucleic acid from the substrate, but leaving the nucleic acid bound to the substrate bound, and eluting the bound nucleic acid from the substrate.

[0111] Item 50: A composition comprising the gene-recognizing reagent or compound according to any one of items 1 to 49 and a pharmaceutically acceptable excipient.

[0112] The present invention has been described with reference to certain exemplary embodiments, dispersible compositions, and uses thereof. However, those skilled in the art will recognize that various substitutions, modifications, or combinations of any of the exemplary embodiments may be made without departing from the spirit and scope of the present invention. Accordingly, the present invention is not limited by the description of the exemplary embodiments, but rather by the appended claims of the parent application.

Claims

1. comprising a plurality of nucleobase moieties attached to a nucleic acid backbone or nucleic acid analog backbone, wherein at least one nucleobase moiety is: 【Chemistry 1】 (In the formula, X ３ is O or S; and Y is N) and a gene recognition reagent, wherein the nucleic acid backbone or nucleic acid analog backbone is selected from one of DNA, RNA, peptide nucleic acid (PNA), phosphorothioate DNA (PS DNA), α,β-constrained nucleic acid (α,β-CNA), 2'-methoxyl RNA, 2'-fluoro RNA, locked nucleic acid (LNA), 2',4'-constrained ethyl nucleic acid ((S)-cEt), 2',4'-bridged nucleic acid NC(N-H) (BNA-NC(N-H)), 2',4'-bridged nucleic acid NC(N-methyl) (BNA-NC(N-Me)), 2'-(R)-(S)-5'-C-methyl DNA, or 2'-R-5'-E-vinylphosphonate nucleic acid (E-VP), where R is H, OH, F, OMe, or O(CH 2 ) 2 OMe.

2. A gene recognition reagent as described in claim 1, wherein the backbone is a nucleic acid analog backbone, and the nucleic acid analog backbone is a peptide nucleic acid (PNA) backbone.

3. A gene recognition reagent as described in claim 2, wherein the PNA backbone comprises one or more guanidine moieties linked to the PNA backbone.

4. A gene recognition reagent as described in claim 1, wherein the backbone is a nucleic acid analog backbone, and the nucleic acid analog backbone is a gamma-peptide nucleic acid (gamma-PNA) backbone.

5. the backbone is a nucleic acid analog backbone, the nucleic acid analog backbone is a PNA backbone; The PNA backbone and the plurality of nucleobase moieties together comprise: 【Chemistry 2】 (wherein n′ is 2 or more, and R １ , R ２ , R ３ , R ４ , R ５ , and R ６ are independently H, CH ３ , C.H. ２ OH, CH(CH ３ ) OH, CH ２ SH, CH (CH ３ ) CH ３ , C.H. ２ CH (CH ３ ) CH ３ , CH(CH ３ ) CH ２ CH ３ , C.H. ２ CH ２ SCH ３ , C.H. ２ CH ３ , C.H. ２ -C ６ H ５ , C.H. ２ -C ６ H ４ OH, 1H-indol-3-ylmethyl, CH ２ C(O)OH, CH ２ CH ２ C(O)OH, CH ２ C(O)NH ２ , C.H. ２ CH ２ C(O)NH ２ , 1H-imidazol-4-ylmethyl, CH ２ CH ２ CH ２ CH ２ NH ２ , C.H. ２ CH ２ CH ２ NHC (NH) NH ２ , linear or branched chain (C ３ -C ８ ) alkyl, (C ２ -C ８ ) alkenyl, (C ２ -C ８ ) alkynyl, (C ３ -C ８ ) aryl, (C ３ -C ８ ) cycloalkyl, (C ３ -C ８ ) aryl (C １ -C ６ ) alkylene, (C ３ -C ８ ) cycloalkyl (C １ -C ６ ) alkylene, guanidine-containing group, CH ２ -(OCH ２ -CH ２ ) ｎ -OH, CH ２ -(OCH ２ -CH ２ ) ｎ -NH ２ , C.H. ２ -(OCH ２ -CH ２ ) ｎ -SH, CH ２ -(OCH ２ -CH ２ ) ｎ -NHC(NH)NH ２ , C.H. ２ -(OCH ２ -CH ２ ) ｎ -morpholine, CH ２ -(OCH ２ -CH ２ ) ｎ -piperazine, where n is 1 to 50; 【Transformation 3】 wherein X is a linker: 【Chemistry 4】 ) and R １ and R ２ together form a 1,3-propylene bond, and R ３ and R ４ together form a 1,3-propylene bond, or R ５ and R ６ together form a 1,3-propylene bond, and R ７ are independently nucleobases of said plurality of nucleobase moieties) The gene recognition reagent according to claim 1 , wherein

6. R １ , R ２ , R ３ , R ４ , R ５ , and R ６ 6. The gene recognition reagent according to claim 5, wherein at least one of the following is S1A, S1B, S1C, S1D, S1E, S1F, S1G, S1H, S1I, S1J, S1K, or S1L.

7. 7. The gene recognition reagent according to claim 5, wherein the α-carbon, β-carbon, or γ-carbon is the chiral center.

8. 8. The gene recognition reagent according to claim 7, wherein the γ-carbon is a chiral center.

9. R ２ but: 【Transformation 5】 The gene recognition reagent according to claim 8,

10. The plurality of nucleobase moieties are TTC, TTCTTC, TCT, TCTTCT, CTT, CTTCTT, CCG, CCGCCG, CGC, CGCCGC, GCC, GCCGCC, CGG, CGGCGG, GCG , GCGGCG, GGC, GGCGGC, CTG, CTGCTG, TGC, TGCTGC, GCT, GCTGCT, CAG, CAGCAG, AGC, AGCAGC, GCA, GCAGCA, CAGG, C The gene recognition reagent according to any one of claims 1 to 8, which forms a sequence comprising AGGCAGG, AGGC, AGGCAGGC, GGCA, GGCAGGCA, GCAG, GCAGGCAG, AGAAT, GAATA, AATAG, ATAGA, TAGAA, GGCCCC, GCCCCG, CCCCGG, CCCGGC, CCGGCC, or CGGCCC, or consecutive repeats of any of the foregoing.

11. A gene recognition reagent described in any one of claims 1 to 10, wherein the multiple nucleic acid base portions are arranged in a sequence complementary to a target sequence of a nucleic acid.

12. The gene recognition reagent according to any one of claims 1 to 11, which has 3 to 25 nucleic acid base moieties.

13. comprising nucleic acid backbone monomers or nucleic acid analog backbone monomers, The backbone monomer has the structure: 【Transformation 6】 (In the formula, X ３ is O or S; and Y is N) and linked to a nucleobase moiety having the formula: The compound wherein the nucleic acid backbone monomer or nucleic acid analog backbone monomer is DNA, RNA, peptide nucleic acid (PNA), phosphorothioate DNA (PS DNA), α,β-constrained nucleic acid (α,β-CNA), 2'-methoxyl RNA, 2'-fluoro RNA, locked nucleic acid (LNA), 2',4'-constrained ethyl nucleic acid ((S)-cEt), 2',4'-bridged nucleic acid NC(N-H) (BNA-NC(N-H)), 2',4'-bridged nucleic acid NC(N-methyl) (BNA-NC(N-Me)), 2'-(R)-(S)-5'-C-methyl DNA, or 2'-R-5'-E-vinylphosphonate nucleic acid (E-VP), where R is H, OH, F, OMe, or O(CH 2 ) 2 OMe.

14. The compound of claim 13, wherein the backbone monomer is a nucleic acid analog backbone monomer, and the nucleic acid analog backbone monomer is a peptide nucleic acid (PNA) backbone monomer.

15. The compound of claim 14, wherein the PNA backbone monomer comprises one or more guanidine moieties linked to the PNA backbone monomer.

16. The compound of claim 13, wherein the backbone monomer is a nucleic acid analog backbone monomer, and the nucleic acid analog backbone monomer is a gamma-peptide nucleic acid (gamma-PNA) backbone monomer.

17. The backbone monomer of claim 17, wherein the backbone monomer is a nucleic acid analog backbone monomer, the nucleic acid analog backbone monomer having the structure: 【Transformation 7】 (In the formula, R １ , R ２ , R ３ , R ４ , R ５ , and R ６ are independently H, CH ３ , C.H. ２ OH, CH(CH ３ ) OH, CH ２ SH, CH (CH ３ ) CH ３ , C.H. ２ CH (CH ３ ) CH ３ , CH(CH ３ ) CH ２ CH ３ , C.H. ２ CH ２ SCH ３ , C.H. ２ CH ３ , C.H. ２ -C ６ H ５ , 1H-indol-3-ylmethyl, CH ２ -C ６ H ４ OH, CH ２ C(O)OH, CH ２ CH ２ C(O)OH, CH ２ C(O)NH ２ , C.H. ２ CH ２ C(O)NH ２ , 1H-imidazol-4-ylmethyl, CH ２ CH ２ CH ２ CH ２ NH ２ , C.H. ２ CH ２ CH ２ NHC (NH) NH ２ , linear or branched chain (C ３ -C ８ ) alkyl, (C ２ -C ８ ) alkenyl, (C ２ -C ８ ) alkynyl, (C ３ -C ８ ) aryl, (C ３ -C ８ ) cycloalkyl, (C ３ -C ８ ) aryl (C １ -C ６ ) alkylene, (C ３ -C ８ ) cycloalkyl (C １ -C ６ ) alkylene, guanidine-containing group, CH ２ -(OCH ２ -CH ２ ) ｎ -OH, CH ２ -(OCH ２ -CH ２ ) ｎ -NH ２ , C.H. ２ -(OCH ２ -CH ２ ) ｎ -SH, CH ２ -(OCH ２ -CH ２ ) ｎ -NHC(NH)NH ２ , C.H. ２ -(OCH ２ -CH ２ ) ｎ -morpholine, CH ２ -(OCH ２ -CH ２ ) ｎ -piperazine, where n is 1 to 50; 【Transformation 8】 wherein X is a linker: 【Chemistry 9】 ) and R １ and R ２ together form a 1,3-propylene bond, and R ３ and R ４ together form a 1,3-propylene bond, or R ５ and R ６ together form a 1,3-propylene bond) 14. The compound of claim 13, which is a PNA backbone monomer having the formula:

18. R １ , R ２ , R ３ , R ４ , R ５ , and R ６ 18. The compound of claim 17, wherein at least one of: S1A, S1B, S1C, S1D, S1E, S1F, S1G, S1H, S1I, S1J, S1K, or S1L.

19. 19. The compound of claim 17 or 18, wherein the α-carbon, β-carbon, or γ-carbon is a chiral center.

20. 20. The compound of claim 19, wherein the γ-carbon is a chiral center.

21. R ２ but 【Chemistry 10】 21. The compound of claim 20, wherein:

22. A method for in vitro detection of a target sequence in a nucleic acid, comprising contacting a gene recognition reagent according to any one of claims 1 to 12 with a sample comprising a nucleic acid, and detecting binding between the gene recognition reagent and the nucleic acid.

23. A method for in vitro isolation and purification of nucleic acids containing a target sequence, comprising contacting a nucleic acid sample with the gene recognition reagent of any one of claims 1 to 12, separating the nucleic acid sample from the gene recognition reagent, leaving the nucleic acid bound to the gene recognition reagent bound to the gene recognition reagent, and separating the gene recognition reagent from the nucleic acid bound to the gene recognition reagent.

24. The gene recognition reagent is immobilized on a substrate, 24. The method of claim 23, comprising contacting nucleic acid with the substrate, washing the substrate to remove unbound nucleic acid from the substrate, but leaving bound nucleic acid on the substrate bound, and eluting the bound nucleic acid from the substrate.

25. A composition comprising the gene recognition reagent according to any one of claims 1 to 12 or the compound according to any one of claims 13 to 21, and a pharmaceutically acceptable excipient.

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