CYP24A1-binding nucleic acid molecules and uses thereof

Abstract

To provide a novel nucleic acid molecule capable of specifically binding with CYP24A1.SOLUTION: A CYP24A1-binding nucleic acid molecule of the present invention includes DNA strand of the following (a). (a) DNA strand of the following (a1), (a2) or (a3): (a1) DNA strand consisting of any base sequence of sequence numbers 1 to 18; (a2) DNA strand consisting of a base sequence in which one or several bases are deleted, substituted, inserted and / or added in any base sequence of the (a1), and binds with CYP24A1; and (a3) DNA strand consisting of a base sequence having 80% or more identity with any base sequence of the (a1), and binds with CYP24A1.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a CYP24A1-binding nucleic acid molecule and uses thereof. [Background technology]

[0002] Low molecular weight compounds such as ketoconazole are known as inhibitors of CYP24A1, an enzyme that inactivates vitamin D (Non-Patent Document 1).

[0003] Vitamin D is known to be involved in bone and muscle weakness and cancer development associated with aging, and it is known that inhibiting the activity of CYP24A1, an enzyme that acts on and inactivates vitamin D, can treat prostate cancer, chronic kidney disease, rickets, and osteomalacia (Non-patent Documents 2, 3, 4). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] DS Loose, et al. “Ketoconazole blocks adrenal steroidogenesis by inhibiting cytochrome P450-dependent enzymes.” The Journal of Clinical Investigation, 1983;71(5):1495-1499. [Non-patent document 2] Aruna V Krishnan, et al. “The role of vitamin D in prostate cancer” Recent Results in Cancer Research, Vol. 164, 2003;164:205-21 [Non-patent document 3] Gary H.Posner, et al. “Vitamin D analogues targeting CYP24 in chronic kidney disease” The Journal of Steroid Biochemistry and Molecular Biology Volume 121, Issues 1-2, July 2010, Pages 13-19 [Non-patent document 4] Xiuying Bai, et al. “CYP24 inhibition as a therapeutic target in FGF23-mediated renal phosphate wasting disorders” The Journal of Clinical Investigation, 2016;126(2):667-680. Summary of the Invention [Problem to be solved by the invention]

[0005] However, CYP24A1 inhibitors based on low molecular weight compounds have low specificity and may inhibit not only CYP24A1 but also enzymes such as CYP27B1, which is involved in the metabolism of vitamin D to its active form in the body.

[0006] Therefore, an object of the present invention is to provide a nucleic acid molecule capable of specifically binding to CYP24A1. [Means for solving the problem]

[0007] The CYP24A1-binding nucleic acid molecule of the present invention (hereinafter also referred to as the "nucleic acid molecule of the present invention") comprises the following DNA strand (a): (a) a DNA strand of the following (a1), (a2), or (a3): (a1) a DNA strand consisting of any one of the nucleotide sequences of SEQ ID NOs: 1 to 18; (a2) a DNA strand that binds to CYP24A1, comprising a base sequence in which one or several bases are deleted, substituted, inserted, and / or added in any of the base sequences of (a1); (a3) A DNA strand that consists of a base sequence having 80% or more identity to any of the base sequences of (a1), and that binds to CYP24A1.

[0008] The CYP24A1 activity inhibitor of the present invention (hereinafter also referred to as "activity inhibitor") comprises the CYP24A1-binding nucleic acid molecule of the present invention.

[0009] The pharmaceutical of the present invention comprises the CYP24A1-binding nucleic acid molecule of the present invention and a pharmaceutically acceptable excipient.

[0010] The lung cancer therapeutic agent of the present invention comprises the CYP24A1-binding nucleic acid molecule of the present invention.

[0011] The prostate cancer therapeutic agent of the present invention comprises the CYP24A1-binding nucleic acid molecule of the present invention.

[0012] The therapeutic agent for chronic kidney disease of the present invention comprises the CYP24A1-binding nucleic acid molecule of the present invention.

[0013] The therapeutic agent for rickets of the present invention comprises the CYP24A1-binding nucleic acid molecule of the present invention.

[0014] The therapeutic agent for osteomalacia of the present invention comprises the CYP24A1-binding nucleic acid molecule of the present invention.

[0015] The CYP24A1 detection reagent of the present invention (hereinafter also referred to as "detection reagent") contains the CYP24A1-binding nucleic acid molecule of the present invention.

[0016] The CYP24A1 detection method of the present invention (hereinafter also referred to as the "detection method") comprises the steps of contacting a sample with a nucleic acid molecule and detecting CYP24A1 in the sample, wherein the nucleic acid molecule is the CYP24A1-binding nucleic acid molecule of the present invention, and in the detection step, CYP24A1 in the sample is bound to the nucleic acid molecule, and CYP24A1 in the sample is detected through this binding. [Effects of the Invention]

[0017] The CYP24A1-binding nucleic acid molecules of the present invention can specifically bind to CYP24A1. [Brief explanation of the drawings]

[0018] [Figure 1] FIG. 1 (FIGS. 1A-1, A-2, B, C, D, E-1, and E-2) is a schematic diagram showing the predicted secondary structure of the CYP24A1-binding nucleic acid molecule of the present invention. [Figure 2] FIG. 2 is a gel image showing the results of EMSA of the CYP24A1-binding nucleic acid molecule of the present invention in Example 1. [Figure 3] 3(A) and (B) are graphs showing the results of detecting the binding of the CYP24A1-binding nucleic acid molecule of the present invention to CYP24A1 in Example 1. [Figure 4] FIG. 4 is a graph showing the results of detecting the binding of the CYP24A1-binding nucleic acid molecule of the present invention to CYP24A1 and CYP27B1 in Example 1. [Figure 5] FIG. 5 is a graph showing the relative CYP24A1 activity in the presence of the CYP24A1-binding nucleic acid molecule of the present invention in Example 2. [Figure 6] 6(A) to 6(D) are graphs showing the relative activity of CYP24A1 in the presence of the CYP24A1-binding nucleic acid molecule of the present invention in Example 2. FIG. [Figure 7] FIG. 7 is a graph showing the relative activity of CYP27B1 in the presence of a CYP24A1-binding nucleic acid molecule of the present invention in a reference example. [Figure 8]FIG. 8(A) is a graph showing the relative value of the activity of CYP27A1 in the presence of the CYP24A1-binding nucleic acid molecule of the present invention in the reference example, and FIG. 8(B) is a graph showing the relative value of the activity of CYP11A1 in the presence of the CYP24A1-binding nucleic acid molecule of the present invention in the reference example. [Figure 9] FIGS. 9(A) and (B) are photographs showing the results of Example 3(1). [Figure 10] FIGS. 10(A) and (B) are photographs showing the results of Example 3(2). [Figure 11] FIGS. 11(A) and (B) are gel images showing the results of Example 4. [Figure 12] FIGS. 12(A) to (E) are graphs showing the results of HPLC of Example 5. [Figure 13] FIG. 13 is a graph showing the survival rate of A549 cells in Example 6.

BEST MODE FOR CARRYING OUT THE INVENTION

[0019] <CYP24A1-binding nucleic acid molecule> The CYP24A1-binding nucleic acid molecule of the present invention contains the DNA strand of the following (a). The nucleic acid molecule of the present invention contains the DNA strand of the following (a) and is characterized by being composed of DNA, and other constitutions and conditions are not particularly limited. The CYP24A1-binding nucleic acid molecule of the present invention is also referred to as a DNA aptamer, for example, as follows. (a) The DNA strand of the following (a1), (a2) or (a3): (a1) A DNA strand consisting of any of the base sequences of SEQ ID NOs: 1 to 18; (a2) A DNA strand consisting of a base sequence in which one or several bases are deleted, substituted, inserted and / or added in any of the base sequences of (a1) and binds to CYP24A1 (a3) A DNA strand consisting of a base sequence having 80% or more identity to any of the base sequences of (a1) and binds to CYP24A1.

[0020] In the present invention, CYP24A1 (Cytochrome P450 family 24 subfamily A member 1) refers to an enzyme belonging to the cytochrome P450 superfamily that is encoded by the CYP24A1 gene. CYP24A1 may be derived from, for example, humans or non-human animals. Examples of non-human animals include mice, rats, monkeys, rabbits, dogs, cats, horses, cows, and pigs. An example of the human-derived CYP24A1 is a protein consisting of the amino acid sequence (SEQ ID NO: 20) encoded by the nucleotide sequence (SEQ ID NO: 19) registered in GeneBank under Accession No. NM_000782.

[0021] Human CYP24A1 gene (SEQ ID NO: 19)

[0022] Human CYP24A1 (SEQ ID NO: 20) MSSPISKSRS LAAFLQQLRS PRQPPRLVTS TAYTSPQPRE VPVCPLTAGG ETQNAAALPG PTSWPLLGSL LQILWKGGLK KQHDTLVEYH KKYGKIFRMK LGSFESVHLG SPCLLEALYR TESAYPQRLE IKPWKAYRDY RKEGYGLLIL EGEDWQRVRS AFQKKLMKPG EVMKLDNKIN EVLADFMGRI DELCDERGHV EDLYSELNKW SFESICLVLY EKRFGLLQKN AGDEAVNFIM AIKTMMSTFG RMMVTPVELH KSLNTKVWQD HTLAWDTIFK SVKACIDNRL EKYSQQPSAD FLCDIYHQNR LSKKELYAAV TELQLAAVET TANSLMWILY NLSRNPQVQQ KLLKEIQSVL PENQVPRAED LRNMPYLKAC LKESMRLTPS VPFTTRTLDK ATVLGEYALP KGTVLMLNTQ VLGSSEDNFE DSSQFRPERW LQEKEKINPF AHLPFGVGKR MCIGRRLAEL QLHLALCWIV RKYDIQATDN EPVEMLHSGT LVPSRELPIA FCQR

[0023] The nucleic acid molecule of the present invention may be any molecule as long as it is capable of binding to CYP24A1, and the binding site in CYP24A1 is not particularly limited.

[0024] The nucleic acid molecule of the present invention has a detection limit of CYP24A1 concentration of, for example, 0.5 nM.

[0025] In the present invention, "binding" or "capable of binding" may mean that the nucleic acid molecule of the present invention actually binds to CYP24A1 or that it binds in a simulation using a molecular docking method or the like; however, the former is preferred. The binding between the nucleic acid molecule and CYP24A1 can be detected, for example, by analyzing protein-protein interactions, such as co-immunoprecipitation, pull-down assay, ELISA, or flow cytometry, which utilizes a mechanism similar to that of an antibody-antigen reaction. Specifically, the binding between the nucleic acid molecule and CYP24A1 can be detected by contacting a cell expressing CYP24A1 with a labeled nucleic acid molecule and then detecting the label in the cell. Alternatively, the binding between the nucleic acid molecule of the present invention and CYP24A1 may be detected, for example, by the method described in Example 1 below.

[0026] Specific examples of the CYP24A1-binding nucleic acid molecule of the present invention are shown below. The nucleic acid molecule of the present invention is a nucleic acid molecule comprising the following DNA strand (a). In the present invention, the nucleic acid molecule comprising the DNA strand (a) also means a nucleic acid molecule comprising at least one DNA strand selected from the group consisting of, for example, (a1) to (a4). (a) A DNA strand of the following (a1), (a2), or (a3): (a1) A DNA strand consisting of any of the base sequences of SEQ ID NOs: 1 to 18. (a2) A DNA strand that binds to CYP24A1 and that consists of a base sequence in which one or several bases are deleted, substituted, inserted and / or added in any of the base sequences of (a1). (a3) A DNA strand that consists of a base sequence having 80% or more identity to any of the base sequences of (a1), and that binds to CYP24A1. (a4) A DNA strand that binds to CYP24A1 and that has a base sequence complementary to a polynucleotide that hybridizes under stringent conditions to a DNA strand having any of the base sequences of (a1).

[0027] In the nucleic acid molecule of the present invention, the constituent units of the polynucleotide are, for example, nucleotide residues, including deoxyribonucleotide residues and ribonucleotide residues. As described below, the polynucleotide may be, for example, DNA consisting of deoxyribonucleotide residues, DNA containing deoxyribonucleotide residues and ribonucleotide residues, or may further contain non-nucleotide residues or a combination thereof.

[0028] The nucleic acid molecule of the present invention may be, for example, a molecule consisting of any one of the DNA strands (a1) to (a4) above, or a molecule containing any one of the DNA strands (a1) to (a4). In the latter case, the nucleic acid molecule of the present invention may contain two or more of the DNA strands (a1) to (a4) above, as described below. The two or more DNA strands may have the same sequence or different sequences. In the latter case, the nucleic acid molecule of the present invention may further include, for example, a linker, an additional sequence, and / or a primer binding sequence.

[0029] The DNA strand (a1) is a DNA strand consisting of any one of the base sequences of SEQ ID NOs: 1 to 18. The DNA strand (a1) is preferably a DNA strand consisting of any one of the base sequences of SEQ ID NOs: 5, 7, 12, and 18 (AptCYP24-#5 (SEQ ID NO: 5), AptCYP24-#7 (SEQ ID NO: 7), AptCYP24-#12 (SEQ ID NO: 12), AptCYP24-#18 (SEQ ID NO: 18)), for example, as shown in the Examples below, because of its high ability to inhibit CYP24A1 activity.

[0030] AptCYP24-#1 (SEQ ID NO: 1) 5′-CGCCTGGGACCAACATCATGTCATCGTCTA-3′ AptCYP24-#2 (SEQ ID NO: 2) 5′-CGCAAGCCACACTTTGTATGGACCAGTATC-3′ AptCYP24-#3 (SEQ ID NO: 3) 5′-CAGGGCCATAAGCTGACCCAATCTTGTCGG-3′ AptCYP24-#4 (SEQ ID NO: 4) 5′-AGACCAGTTATGCAAGCTCATCTGTTGT-3′ AptCYP24-#5 (SEQ ID NO: 5) 5′-CAGGACAGCACCCTGACCCAGTTCTGTATA-3′ AptCYP24-#6 (SEQ ID NO: 6) 5′-CAGGGTCAATGGTCCCCAATCTACACAGAT-3′ AptCYP24-#7 (SEQ ID NO: 7) 5′-CATGCAGGAAGTAACCCAATGTCGTTAAC-3′ AptCYP24-#8 (SEQ ID NO: 8) 5′-CAGCGTCACCATACCCTTTAACACCACGCA-3′ AptCYP24-#9 (SEQ ID NO: 9) 5′-ACCTATACACCGACGAACCCTACTTCCTGA-3′ AptCYP24-#10 (SEQ ID NO: 10) 5′-CTTTTACCCCCCCGCTGCCTTTATACTTGA-3′ AptCYP24-#11 (SEQ ID NO: 11) 5′-CAAGCCGAAGTTACACACGGACCAATATGT-3′ AptCYP24-#12 (SEQ ID NO: 12) 5′-GCCACTCTTCGTACTCCTCCCTCTATCGGA-3′ AptCYP24-#13 (SEQ ID NO: 13) 5′-TCCCCCCCATAACCAACATTTAGCATCATG-3′ AptCYP24-#14 (SEQ ID NO: 14) 5′-TAGGTAGCAAGCAAACATGTATTCCTTTTTC-3′ AptCYP24-#15 (SEQ ID NO: 15) 5′-GTAGCGCGACGTACAATATTTCTTTCCTCC-3′ AptCYP24-#16 (SEQ ID NO: 16) 5′-TGGACTTTTTCCGGTGGGAGACGAGGTTGC-3′ AptCYP24-#17 (SEQ ID NO: 17) 5′-ACCCAAGGACGTCACCGACTGCAGTGTTAT-3′ AptCYP24-#18 (SEQ ID NO: 18) 5′-ACCCTTAGCTCACCCTTCCCCGCCCGCCAC-3′

[0031] In (a2), "one or several" may be within a range in which the DNA strand of (a2) binds to CYP24A1, for example. The "one or several" may be, for example, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 or 2 in any of the base sequences of (a2). In the present invention, a numerical range of the number of bases, the number of sequences, etc., discloses, for example, all positive integers within that range. That is, for example, the description "1 to 5 bases" means the disclosure of all "1, 2, 3, 4, and 5 bases" (the same applies hereinafter).

[0032] In (a3), the "identity" may be within a range that allows the DNA strand of (a3) ​​to bind to CYP24A1. The identity may be, for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. The identity can be calculated using analysis software such as BLAST or BLAST2 with default parameters (the same applies hereinafter).

[0033] In (a4), the "hybridizable polynucleotide" is, for example, a polynucleotide that is completely or partially complementary to the DNA strand of (a1). The hybridization can be detected, for example, by various hybridization assays. The hybridization assay is not particularly limited, and for example, the method described in "Molecular Cloning: A Laboratory Manual 2nd Ed." (eds.) Sambrook et al. [Cold Spring Harbor Laboratory Press (1989)] can be used.

[0034] In (a4), "stringent conditions" may be, for example, low stringency conditions, moderate stringency conditions, or high stringency conditions. "Low stringency conditions" are, for example, 5x SSC, 5x Denhardt's solution, 0.5% SDS, 50% formamide, and 32°C. "Medium stringency conditions" are, for example, 5x SSC, 5x Denhardt's solution, 0.5% SDS, 50% formamide, and 42°C. "High stringency conditions" are, for example, 5x SSC, 5x Denhardt's solution, 0.5% SDS, 50% formamide, and 50°C. Those skilled in the art can set the level of stringency by appropriately selecting conditions such as temperature, salt concentration, probe concentration and length, ionic strength, and time. The "stringent conditions" may be, for example, those described in "Molecular Cloning: A Laboratory Manual 2nd Ed." edited by Sambrook et al. [Cold Spring Harbor Laboratory Press (1989)].

[0035] The nucleic acid molecule of the present invention may contain, for example, one DNA strand sequence of any one of (a1) to (a4) above, or may contain multiple DNA strand sequences of any one of (a1) to (a4) above. In the latter case, it is preferable that the multiple DNA strand sequences are linked to form a single-stranded DNA strand. The multiple DNA strand sequences may, for example, be directly linked to each other, or indirectly linked to each other via a linker. It is preferable that the DNA strand sequences are linked directly or indirectly at their respective ends. The multiple DNA strand sequences may, for example, be the same or different. It is preferable that the multiple DNA strand sequences are the same, for example. When the nucleic acid molecule contains multiple DNA strand sequences, the number of sequences is not particularly limited and is, for example, 2 or more, 2 to 18, 2 to 10, 2, or 3.

[0036] The linker is not particularly limited. The length of the linker is not particularly limited and may be, for example, 1 to 200 bases long, 1 to 20 bases long, 3 to 12 bases long, or 5 to 9 bases long. The constituent unit of the linker is, for example, a nucleotide residue, such as a deoxyribonucleotide residue or a ribonucleotide residue. The linker is not particularly limited and may be, for example, a polynucleotide such as DNA composed of deoxyribonucleotide residues or DNA containing ribonucleotide residues. Specific examples of the linker include polydeoxythymine (poly dT), polydeoxyadenine (poly dA), and poly dAdT, which is a repeating sequence of A and T, with poly dT and poly dAdT being preferred.

[0037] In the nucleic acid molecule of the present invention, the DNA strand is preferably a single-stranded DNA. The single-stranded DNA is preferably capable of forming, for example, a stem structure and a loop structure by self-annealing. The DNA strand is preferably capable of forming, for example, a stem-loop structure, an internal loop structure, and / or a bulge structure.

[0038] The nucleic acid molecule of the present invention may be, for example, double-stranded. In the case of double-stranded DNA, for example, one single-stranded DNA comprises any one of the DNA strands (a1) to (a4) above, and the other single-stranded DNA is not limited. The other single-stranded DNA can be, for example, a DNA strand comprising a base sequence complementary to any one of the DNA strands (a1) to (a4). When the nucleic acid molecule of the present invention is double-stranded, it is preferably dissociated into single-stranded DNA by, for example, denaturation prior to use. Furthermore, the dissociated single-stranded DNA of any one of the single-stranded DNAs (a1) to (a4) preferably forms a stem structure and a loop structure, for example, as described above.

[0039] In the present invention, "capable of forming a stem structure and a loop structure" includes, for example, actually forming a stem structure and a loop structure, as well as being able to form a stem structure and a loop structure under certain conditions even if the stem structure and the loop structure are not formed. "Capable of forming a stem structure and a loop structure" includes, for example, both cases confirmed experimentally and cases predicted by computer simulation or the like.

[0040] A constituent unit of the nucleic acid molecule of the present invention is, for example, a nucleotide residue. Examples of the nucleotide residue include deoxyribonucleotide residues and ribonucleotide residues. Examples of the nucleic acid molecule of the present invention include DNA composed only of deoxyribonucleotide residues and DNA containing one or several ribonucleotide residues. In the latter case, the term "one or several" is not particularly limited and may be, for example, 1 to 30, 1 to 15, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 or 2 in the DNA strand.

[0041] In the nucleotide residue, the base is not particularly limited. The base may be, for example, a natural base or a non-natural base, i.e., a modified base. The base may be, for example, a naturally occurring base or a synthetic base. The base may be, for example, a common base or a modified analog thereof.

[0042] Examples of the natural bases include purine bases such as adenine (a) and guanine (g), and pyrimidine bases such as cytosine (c), thymine (t), and uracil (u).

[0043] The modified base is not particularly limited, and examples thereof include bases obtained by modifying natural bases (non-artificial bases), and preferably has the same functions as the natural bases. The modified base may have inosine, xanthine, hypoxanthine, purine, isoguanine, isocytosine, 7-deazaadenine, or the like instead of deoxyribose or ribose. The modification site of the base is not particularly limited. When the base is a purine base, examples of the modification site of the purine base include the 7th and 8th positions of the purine skeleton. When the base is a pyrimidine base, examples of the modification site of the pyrimidine base include the 5th and 6th positions of the pyrimidine skeleton. When "=O" is bonded to the 4th carbon and a group other than "-CH3" or "-H" is bonded to the 5th carbon in the pyrimidine skeleton, the base can be referred to as a modified uracil or modified thymine.

[0044] The modifying group of the modified base is not particularly limited, and examples thereof include a methyl group, a fluoro group, an amino group, and a thio group.

[0045] The modified base is not particularly limited, and examples thereof include modified adenine obtained by modifying adenine, modified thymine obtained by modifying thymine, modified guanine obtained by modifying guanine, modified cytosine obtained by modifying cytosine, and modified uracil obtained by modifying uracil, with modified thymine, modified uracil, and modified cytosine being preferred.

[0046] Specific examples of the modified base include alkyl derivatives such as 2-aminoadenine and 6-methylated purine; alkyl derivatives such as 2-propylated purine; 5-halouracil and 5-halocytosine; 5-propynyluracil and 5-propynylcytosine; 6-azouracil, 6-azocytosine and 6-azothymine; 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-azocytosine, 5-azothymine, 5-azo ... Aminoallyluracil; 8-haloated, aminated, thiolated, thioalkylated, hydroxylated, and other 8-substituted purines; 5-trifluoromethylated and other 5-substituted pyrimidines; 7-methylguanine; 5-substituted pyrimidines; 6-azapyrimidines; N-2, N-6, and O-6 substituted purines (including 2-aminopropyladenine); 5-propynyluracil and 5-propynylcytosine; dihydrouracil; 3-deaza-5-azacytosine; 2-amino Nopurine;5-Alkyluracil;7-Alkylguanine;5-Alkylcytosine;7-Deazaadenine;N6,N6-Dimethyladenine;2,6-Diaminopurine;5-Amino-allyl-uracil;N3-Methyluracil;Substituted 1,2,4-triazoles;2-Pyridinone;5-Nitroindole;3-Nitropyrrole;5-Methoxyuracil;Uracil-5-oxyacetic acid;5-Methoxycarbonylmethyluracil;5-Methyl-2-thiouracil Examples include uracil; 5-methoxycarbonylmethyl-2-thiouracil; 5-methylaminomethyl-2-thiouracil; 3-(3-amino-3-carboxypropyl)uracil; 3-methylcytosine; 5-methylcytosine (5-Me-dC); N4-acetylcytosine; 2-thiocytosine; N6-methyladenine; N6-isopentyladenine; 2-methylthio-N6-isopentenyladenine; N-methylguanine; O-alkylated bases, etc.Further, examples of the purine base and pyrimidine base include those disclosed in U.S. Pat. No. 3,687,808, "Concise Encyclopedia of Polymer Science and Engineering," pp. 858-859, edited by Kroschwitz JI, John Wiley & Sons, 1990, and Englisch et al., Angewandte Chemie, International Edition, 1991, Vol. 30, p. 613.

[0047] The DNA strand may contain, for example, only one type of the modified base, or may contain two or more types of the modified base.

[0048] The nucleic acid molecule of the present invention may contain, for example, a modified nucleotide. The modified nucleotide may be a nucleotide having the modified base described above, a nucleotide having a modified sugar phosphate backbone in which the sugar phosphate backbone has been modified, or a nucleotide having the modified base and the modified sugar phosphate backbone.

[0049] In the sugar phosphate backbone, the deoxyribose residue and / or ribose residue may be modified, for example, to improve the stability of the nucleic acid molecule. The modification site in the sugar residue is not particularly limited, and examples include the 2'-position and the 4'-position of the sugar residue, and either or both may be modified. Examples of the modification group in the modified sugar include a methyl group, a fluoro group, an amino group, and a thio group. Specific examples of the deoxyribose residue include a modified 2'-carbon; specifically, the hydrogen bonded to the 2'-carbon can be substituted with a halogen such as fluoro. Furthermore, the deoxyribose residue can be substituted with a ribose residue by substituting the hydrogen at the 2'-carbon with a hydroxyl group. The ribose residue can be modified, for example, with a modified 2'-carbon; specifically, the hydroxyl group bonded to the 2'-carbon can be substituted with a hydrogen or a halogen such as fluoro. Furthermore, the ribose residue may be substituted by substituting the hydrogen atom of the hydroxyl group bonded to the 2'-carbon atom. Specific examples include 2'-O-methylribose residue, 2'-O-methoxyethylribose residue, 2'-O-dimethylaminoethylribose residue, and 2'-O-dimethylaminoethoxyethylribose residue. The ribose residue may be substituted with a deoxyribose residue by substituting the hydroxyl group of the 2'-carbon atom with a hydrogen atom. The ribose residue may be substituted with a stereoisomer, such as an arabinose residue. The modified nucleotide residue may also be a bicyclic sugar residue in which the cyclic structures of the sugar residues constituting the nucleotide residue are crosslinked. Specific examples of modified nucleotide residues containing a bicyclic sugar residue are not particularly limited, and include known artificial bicyclic nucleic acid monomer residues. Examples of the bicyclic artificial nucleic acid monomer residue include cEt (constrained ethyl bicyclic nucleic acid, manufactured by Ionis Pharmaceuticals), LNA (trademark, Locked Nucleic Acid), and ENA (registered trademark, 2'-O,4'-C-Ethylenebridged Nucleic Acid), and LNA is preferred.

[0050] When the base in the modified nucleotide residue is a pyrimidine base, for example, the 2'-position and / or 4'-position of the sugar residue is preferably modified. Specific examples of the modified nucleotide residue include 2'-methylated uracil nucleotide residues, 2'-methylated cytosine nucleotide residues, 2'-fluorolated uracil nucleotide residues, 2'-fluorolated cytosine nucleotide residues, 2'-aminolated uracil nucleotide residues, 2'-aminolated cytosine nucleotide residues, 2'-thiolated uracil nucleotide residues, and 2'-thiolated cytosine nucleotide residues, each of which is modified at the 2'-position of a deoxyribose residue or ribose residue.

[0051] The sugar phosphate backbone may be substituted with a non-sugar phosphate backbone having, for example, a non-deoxyribose residue, a non-ribose residue, and / or a non-phosphate residue. Examples of the non-sugar phosphate backbone include uncharged versions of the sugar phosphate backbone. Examples of the nucleotide substitute substituted with the non-sugar phosphate backbone include morpholino, cyclobutyl, pyrrolidine, PNA (peptide nucleic acid), etc.

[0052] In the sugar phosphate backbone, for example, the phosphate group can be modified. In the sugar phosphate backbone, the phosphate group closest to the sugar residue is called the α-phosphate group. The α-phosphate group is negatively charged, and the charge is uniformly distributed over the two oxygen atoms not bonded to the sugar residue. Of the four oxygen atoms in the α-phosphate group, the two oxygen atoms not bonded to the sugar residue in the phosphodiester bond between nucleotide residues are hereinafter also referred to as "non-linking oxygens." On the other hand, the two oxygen atoms bonded to the sugar residue in the phosphodiester bond between nucleotide residues are hereinafter referred to as "linking oxygens." The α-phosphate group is preferably modified, for example, to become uncharged or to have an asymmetric charge distribution at the non-linking oxygens.

[0053] The phosphate group may, for example, substitute for the non-bonding oxygen. The oxygen can be substituted with any of the following atoms: S (sulfur), Se (selenium), B (boron), C (carbon), H (hydrogen), N (nitrogen), and OR (R is an alkyl group or an aryl group), preferably with S. Preferably, both non-bonding oxygens are substituted, more preferably with S. Examples of the modified phosphate group include phosphorothioate, phosphorodithioate, phosphoroselenate, boranophosphate, boranophosphate ester, hydrogen phosphonate, phosphoramidate, alkyl or aryl phosphonate, and phosphotriester. Of these, phosphorothioate and phosphorodithioate, in which both non-bonding oxygens are substituted with S, are preferred.

[0054] The phosphate group may, for example, substitute for the bonded oxygen. The oxygen can be substituted with any of the atoms S (sulfur), C (carbon), and N (nitrogen), and examples of the modified phosphate group include bridged phosphoramidates substituted with N, bridged phosphorothioates substituted with S, and bridged methylene phosphonates substituted with C. The bonded oxygen is preferably substituted, for example, at least one of the 5'-terminal nucleotide residue and the 3'-terminal nucleotide residue of the nucleic acid molecule of the present invention. In the case of the 5'-side, substitution with C is preferred, and in the case of the 3'-side, substitution with N is preferred.

[0055] The phosphate group may be substituted with a non-phosphorus-containing linker, such as a siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo, and methyleneoxymethylimino, preferably a methylenecarbonylamino group or a methylenemethylimino group.

[0056] The nucleic acid molecule of the present invention may be modified, for example, at least one of the 3'-end and 5'-end nucleotide residues. The modification may be, for example, either the 3'-end or the 5'-end, or both. The modification may be, for example, as described above, and is preferably performed on the terminal phosphate group. The phosphate group may be modified in its entirety, or one or more atoms in the phosphate group may be modified. In the former case, for example, the entire phosphate group may be substituted or deleted.

[0057] The modification of the terminal nucleotide residue can be, for example, the addition of another molecule. Examples of the other molecule include functional molecules such as labeling substances and protecting groups, as described below. Examples of the protecting group include S (sulfur), Si (silicon), B (boron), and ester-containing groups. The functional molecule such as the labeling substance can be used, for example, for detecting the nucleic acid molecule of the present invention.

[0058] The other molecule may be attached to, for example, the phosphate group of the nucleotide residue, or to the phosphate group or the sugar residue via a spacer. The terminal atom of the spacer can be attached to or substituted for, for example, the bonded oxygen of the phosphate group or the O, N, S, or C of the sugar residue. The binding site of the sugar residue is preferably, for example, the 3'-C or 5'-C, or an atom bonded thereto. The spacer can also be attached to or substituted for, for example, the terminal atom of a nucleotide substitute such as a PNA.

[0059] The spacer is not particularly limited and may be, for example, -(CH2) n -, -(CH2) n N-, -(CH2) n O-, -(CH2) n S-, O(CH2CH2O) n The reagents may include CH2CH2OH, abasic sugars, amides, carboxyls, amines, oxyamines, oxyimines, thioethers, disulfides, thioureas, sulfonamides, and morpholinos, as well as biotin and fluorescein reagents, etc. In the above formula, n is a positive integer, and n=3 or 6 is preferred.

[0060] In addition to these, the molecule to be added to the end may be, for example, a dye, an intercalating agent (e.g., acridine), a crosslinking agent (e.g., psoralen, mitomycin C), an anticancer drug, a porphyrin (TPPC4, texaphyrin, sapphyrin), a polycyclic aromatic hydrocarbon (e.g., phenazine, dihydrophenazine), an artificial endonuclease (e.g., EDTA), a lipophilic carrier (e.g., cholesterol, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, a heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide, RGD peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), antibodies, transport / absorption enhancers (e.g., aspirin, vitamin A, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bis-imidazole, histamine, imidazole clusters, acridine-imidazole conjugates, tetraazamacrocyclic Eu 3+ complexes), and sugars (e.g., N-acetylgalactosamine, galactose, mannose).

[0061] The 5' end of the nucleic acid molecule of the present invention may be modified, for example, with a phosphate group or a phosphate group analog. The phosphate group can be, for example, 5' monophosphate ((HO)2(O)PO-5'), 5' diphosphate ((HO)2(O)POP(HO)(O)-O-5'), 5' triphosphate ((HO)2(O)PO-(HO)(O)POP(HO)(O)-O-5'), 5'-guanosine cap (7-methylated or unmethylated, 7m-GO-5'-(HO)(O)PO-(HO)(O)POP(HO)(O)-O-5'), 5'-adenosine cap (Appp), any modified or unmodified nucleotide cap structure (NO-5'-(HO)(O)PO-(HO)(O)POP(HO)(O)-O-5'), 5' monothiophosphate (phosphorothioate: (HO)2(S)PO-5'), 5' monodithiophosphate (phosphorodi ... Examples of the thiolates include thioates ((HO)(HS)(S)PO-5'), 5'-phosphorothiolic acid ((HO)2(O)PS-5'), sulfur-substituted monophosphates, diphosphates, and triphosphates (e.g., 5'-α-thiotriphosphate, 5'-γ-thiotriphosphate, etc.), 5'-phosphoramidates ((HO)2(O)P-NH-5', (HO)(NH2)(O)PO-5'), 5'-alkylphosphonic acids (e.g., RP(OH)(O)-O-5', (OH)2(O)P-5'-CH2, where R is alkyl (e.g., methyl, ethyl, isopropyl, propyl, etc.)), and 5'-alkyl ether phosphonic acids (e.g., RP(OH)(O)-O-5', where R is alkyl ether (e.g., methoxymethyl, ethoxymethyl, etc.)).

[0062] The number of modified nucleotides in the DNA strand is not particularly limited and may be, for example, 1 to 30, 1 to 20, 1 to 10, 1 to 5, 1 to 4, 1 to 3, or 1 or 2. The number of modified nucleotides in the entire length of the nucleic acid molecule containing the DNA strand is also not particularly limited and may be, for example, 1 to 30, 1 to 20, 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1, or 2.

[0063] The nucleic acid molecule of the present invention may contain, for example, one or several artificial nucleic acid monomer residues. The term "one or several" is not particularly limited and may be, for example, 1 to 30, 1 to 20, 1 to 10, 1 to 5, 1 to 4, 1 to 3, or 1 or 2 in the DNA strand. Examples of the artificial nucleic acid monomer residues include PNA (peptide nucleic acid), LNA (locked nucleic acid), and ENA (2'-O,4'-C-ethylene bridged nucleic acid). The nucleic acid in the monomer residue is, for example, the same as described above.

[0064] The nucleic acid molecule of the present invention is preferably, for example, nuclease-resistant. To be nuclease-resistant, the nucleic acid molecule of the present invention preferably has, for example, the modified nucleotide residue and / or the artificial nucleic acid monomer residue. To be nuclease-resistant, the nucleic acid molecule of the present invention may have, for example, several tens of kDa of PEG (polyethylene glycol) or deoxythymidine bound to the 5'-end or 3'-end, or, as described above, the 5'-end may be modified with a phosphate group. The modified phosphate group is preferably, for example, a phosphorothioate group.

[0065] The nucleic acid molecule of the present invention may further have, for example, an additional sequence. The additional sequence is preferably bound to at least one of the 5'-end and 3'-end of the nucleic acid molecule, more preferably to the 3'-end. The additional sequence is not particularly limited. The length of the additional sequence is not particularly limited, and may be, for example, 1 to 200 bases, 1 to 50 bases, 1 to 25 bases, or 1 to 12 bases. The constituent units of the additional sequence are, for example, nucleotide residues, such as deoxyribonucleotide residues and ribonucleotide residues. The additional sequence is not particularly limited, and may be, for example, a polynucleotide such as DNA composed of deoxyribonucleotide residues or DNA containing ribonucleotide residues. Specific examples of the additional sequence include poly(dT) and poly(dA).

[0066] The nucleic acid molecule of the present invention may further have, for example, a primer-binding sequence. The primer-binding sequence is a region to which a primer used in producing (synthesizing) the nucleic acid molecule of the present invention, for example, by the method described in the Examples below, can hybridize. The primer-binding sequence is preferably bound to at least one of the 5'-end and 3'-end of the nucleic acid molecule, and more preferably to both the 5'-end and 3'-end. The constituent units of the primer-binding sequence are, for example, nucleotide residues, such as deoxyribonucleotide residues and ribonucleotide residues, with deoxyribonucleotide residues being preferred. The length of the primer-binding sequence is not particularly limited and may be, for example, 1 to 40 bases, 1 to 30 bases, 1 to 20 bases, or 20 bases. When the nucleic acid molecule of the present invention has the primer binding sequence, it is preferable that, for example, the 5' end of each of the sequences is bound to the 3' end of a polynucleotide consisting of the base sequence represented by SEQ ID NO: 21 (5'-AGCAGCACAG AGGTCAGATG), and the 3' end of each of the sequences is bound to the 5' end of a polynucleotide consisting of the base sequence represented by SEQ ID NO: 22 (CCTATCGCTG CTACCGTGAA-3').

[0067] The nucleic acid molecule of the present invention may be used by immobilizing it on a carrier, for example. The nucleic acid molecule of the present invention may be immobilized, for example, at either the 5' end or the 3' end. When immobilizing the nucleic acid molecule of the present invention, the nucleic acid molecule may be immobilized directly or indirectly on the carrier, for example. In the latter case, it is preferable to immobilize it via the additional sequence, for example.

[0068] The nucleic acid molecule of the present invention may further have, for example, a labeling substance. The labeling substance is preferably bound to at least one of the 5' end and the 3' end of the nucleic acid molecule, more preferably the 5' end.

[0069] The labeling substance is not particularly limited, and examples thereof include fluorescent substances, dyes, isotopes, enzymes, tags, etc. Examples of the labeling substance include fluorophores such as pyrene, TAMRA, fluorescein, Cy3 dye, Cy5 dye, FAM dye, rhodamine dye, Texas Red dye, JOE, MAX, HEX, and TYE. Examples of the dye include Alexa dyes such as Alexa488. Examples of the enzyme include luciferase, alkaline phosphatase, peroxidase, β-galactosidase, and glucuronidase. Examples of the isotope include stable isotopes and radioactive isotopes, and stable isotopes are preferred. Stable isotopes are easy to handle, for example, because they pose little risk of exposure and do not require dedicated facilities, and they can also reduce costs. Furthermore, stable isotopes do not change the physical properties of the labeled compound, and they also have excellent properties as a tracer. The stable isotope is not particularly limited, and examples thereof include 2 H, 13 C. 15 N, 17 O. 18 O. 33 S, 34 S and 36 Examples of the tag include vitamins such as biotin.

[0070] The nucleic acid molecule of the present invention may further include, for example, a carrier. In this case, the nucleic acid molecule of the present invention may be contained in the carrier or may exist separately. In the former case, the nucleic acid molecule of the present invention may be immobilized or encapsulated in the carrier. In the latter case, the nucleic acid molecule of the present invention may also be referred to as, for example, a kit containing the nucleic acid molecule. The size of the carrier is not particularly limited and is, for example, 100 nm to 600 nm.

[0071] The carrier may be, for example, a structure capable of holding the nucleic acid molecule of the present invention, such as a micelle, liposome, emulsion, or niosome. The components of the carrier may be appropriately selected depending on the type of carrier. Examples of the components include polymers such as amphipathic polymers; surfactants; and lipids such as cationic lipids, non-cationic lipids, cholesterol, and polyethylene glycol (PEG)-modified lipids.

[0072] Examples of the cationic lipid include N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 5-carboxyspermylglycine dioctadecylamide (DOGS), 2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminium (DOSPA), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-distearoyl-3-methylammonium-propane (DOTAP), and 1,2-distearoyl-3-methylammonium-propane (DOTAP). N-dimethyl-3-aminopropane (DSDMA), 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA), 1,2-linoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA), N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1,2-dimyristyloxyprop-3-yl) -N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), 3-dimethylamino-2-(cholest-5-ene-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienooxy)propane (CLinDMA), 2-[5'-(cholest-5-ene-3-beta-oxy)-3'-oxapentoxy]-3-dimethyl-1-(cis,cis-9',1-2'-octadecadienooxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA) , 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 2,3-dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP), 1,2-N,N'-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1,2-dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-DMA), 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-K-XTC2-DMA), 2-(2,2-di((9Z,12Z)-octadeca-9,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethanamine (DLin-KC2-DMA), etc.

[0073] Examples of the non-cationic lipid include neutral, zwitterionic, and anionic lipids. Examples of the non-cationic lipid include distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylethanolamine (P ... Examples include 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, and 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE).

[0074] Examples of the cholesterol include N,N-dimethyl-N-ethylcarboxamidocholesterol (DC-Choi) and 1,4-bis(3-N-oleylamino-propyl)piperazine.

[0075] Examples of the polymer include polyacrylate, polyalkyl cyanoacrylate, polylactide, polylactide-polyglycolide copolymer, polycaprolactone, dextran, albumin, gelatin, alginate, collagen, chitosan, linear cyclodextrin copolymer, linear oxidized cyclodextrin copolymer, cyclodextrin, protamine, PEGylated protamine, poly-L-lysine (PLL), PEGylated PLL, and polyethyleneimine (PEI).

[0076] The method for immobilizing the nucleic acid molecule of the present invention on the carrier can be carried out, for example, in accordance with the method for producing liposomes.

[0077] The method for producing the nucleic acid molecule of the present invention is not particularly limited, and the molecule can be synthesized by, for example, a nucleic acid synthesis method using chemical synthesis, a genetic engineering technique, or a known method. The nucleic acid molecule of the present invention can also be obtained, for example, by the so-called SELEX method. In this case, the target is preferably CYP24A1. The nucleic acid molecule of the present invention is also preferably obtained by a competitive non-SELEX method called the SELCOS method (Systematic Evolution of Ligands by Competitive Selection), as described in Reference 1 below. In this case, it is preferable to use CYP24A1 as the target and CYP27B1 as the competitive control target. CYP27B1 is an enzyme involved in the metabolism of vitamin D to its active form in vivo. Specific procedures for the SELCOS method can be found, for example, in the Examples section below. By producing the nucleic acid molecule of the present invention using the SELCOS method, it is possible to obtain, for example, a nucleic acid molecule that more specifically binds to CYP24A1. Reference 1: Ankita Kushwaha et al. “Competitive non-SELEX for the selective and rapid enrichment of DNA aptamers and its use in electrochemical aptasensor”, SCIENTIFIC REPORT, 2019 Apr 30, doi: 10.1038 / s41598-019-43187-6

[0078] As described above, the nucleic acid molecule of the present invention exhibits binding to CYP24A1. Therefore, the use of the nucleic acid molecule of the present invention is not particularly limited as long as it utilizes its binding to CYP24A1. The nucleic acid molecule of the present invention can be used in various methods, for example, in place of an antibody against CYP24A1.

[0079] <Pharmaceuticals> As described above, the pharmaceutical of the present invention comprises the CYP24A1-binding nucleic acid molecule of the present invention and a pharmaceutically acceptable excipient. The pharmaceutical of the present invention is characterized by comprising the CYP24A1-binding nucleic acid molecule of the present invention, and other configurations and conditions are not particularly limited. Because the pharmaceutical of the present invention comprises the nucleic acid molecule of the present invention, it can regulate CYP24A1 activity. It is also known that CYP24A1 acts on and inactivates the vitamin D metabolites 1,25(OH)2D3 and 25(OH)D3. Therefore, the pharmaceutical of the present invention can be used as a pharmaceutical for diseases caused by vitamin D deficiency. It has also been reported that CYP24A1 is overexpressed in cancer cells. Therefore, the pharmaceutical of the present invention is expected to function as a pharmaceutical for cancer. The above description of the nucleic acid molecule of the present invention can be applied to the pharmaceutical of the present invention.

[0080] Examples of diseases caused by vitamin D deficiency include chronic kidney disease, rickets, and osteomalacia.

[0081] Examples of the cancer include lung cancer and prostate cancer.

[0082] The method of use (administration conditions) of the pharmaceutical of the present invention is not particularly limited, and for example, the nucleic acid molecule may be administered to the subject.

[0083] The administration target may be, for example, a cell, a tissue, or an organ. The administration target may be, for example, a human or a non-human animal other than a human. The non-human animal may be, for example, a non-human mammal such as a mouse, a rat, a rabbit, a sheep, a cow, a horse, a dog, a pig, or a monkey. The administration may be, for example, in vivo but in In vitro That's fine too.

[0084] The cells are not particularly limited and include, for example, cells derived from humans and mice, and specific examples include proximal tubule epithelial cells, small intestinal epithelial cells, large intestinal epithelial cells, epidermal keratinocytes, etc. The cells exclude, for example, cells in human fertilized eggs, human embryos, and cells within human individuals.

[0085] The administration method is not particularly limited and can be appropriately determined depending on, for example, the subject of administration. When the subject of administration is cells isolated from a living body, examples of the administration method include a method using a transfection reagent, an electroporation method, and a nanobubble method. When the subject of administration is a living body, examples of the administration method include parenteral administration and oral administration. Examples of the parenteral administration include topical administration, subcutaneous administration, and intravenous administration. The administration conditions of the medicament of the present invention, such as the number of administrations and the dosage, are not particularly limited.

[0086] The form of the pharmaceutical of the present invention is not particularly limited, and examples thereof include injections, intravenous drip infusions, eye drops, eye ointments, skin ointments, patches, inhalants, liquids, aerosols, pump sprays, and oral preparations.

[0087] In the pharmaceutical composition of the present invention, the dosage of the nucleic acid molecule is not particularly limited. Also, the administration conditions of the nucleic acid molecule are not particularly limited. As a specific example, when the pharmaceutical composition of the present invention is systemically administered to a human by subcutaneous injection, intravenous injection, or the like, the dosage (total) per administration is, for example, 100 to 500 mg. The frequency of administration of the pharmaceutical composition of the present invention is, for example, once every two to eight weeks. In the pharmaceutical composition of the present invention, it is preferable that the nucleic acid molecule is contained at a concentration that can achieve the exemplified administration conditions.

[0088] Examples of the pharmaceutically acceptable additives include complexing agents, carriers, binding substances to target cells, condensing agents, fusing agents, excipients, bases, stabilizers, preservatives, and the like.

[0089] The additive may, for example, form a complex with the nucleic acid molecule. In this case, the additive may also be referred to as a complexing agent, for example. In the composition of the present invention, by forming a complex of the nucleic acid molecule, for example, the nucleic acid molecule can be efficiently delivered. The binding between the nucleic acid molecule and the complexing agent is not particularly limited, and examples thereof include non-covalent bonds. Examples of the complex include an inclusion complex. The complexing agent may, for example, refer to the description of the aforementioned carrier.

[0090] The dosage of the additive is not particularly limited as long as it does not interfere with the function of the nucleic acid molecule. The type of the additive is not particularly limited, and can be appropriately selected, for example, according to the type of the administration subject.

[0091] The pharmaceutical composition of the present invention is expected to be suitably used, for example, for the treatment of diseases caused by vitamin D deficiency or the cancer. In the present invention, the term "treatment" includes, for example, the meanings of prevention, improvement, and improvement of prognosis, and any of them is acceptable.

[0092] <CYP24A1 activity inhibitor> As described above, the CYP24A1 activity inhibitor of the present invention comprises the CYP24A1-binding nucleic acid molecule of the present invention. The activity inhibitor of the present invention is characterized by comprising the CYP24A1-binding nucleic acid molecule of the present invention, and other configurations and conditions are not particularly limited. Since the activity inhibitor of the present invention comprises the nucleic acid molecule of the present invention, it can inhibit CYP24A1 activity. Furthermore, CYP24A1 functions in the inactivation of vitamin D. Therefore, the activity inhibitor of the present invention is expected to function as a vitamin D inactivation inhibitor. The explanations of the nucleic acid molecule and pharmaceutical of the present invention can be applied to the activity inhibitor of the present invention.

[0093] In the present invention, the "inhibition of activity" means that when a target nucleic acid molecule is introduced into CYP24A1, if the activity of CYP24A1 is significantly reduced compared to CYP24A1 that has not been introduced with the nucleic acid molecule or that has been introduced with a control nucleic acid molecule, the nucleic acid molecule can be evaluated as inhibiting the activity of CYP24A1, for example.

[0094] The method for using the activity inhibitor of the present invention can be described in the above explanation of the medicament of the present invention.

[0095] <Lung cancer treatment drug> As described above, the lung cancer therapeutic agent of the present invention comprises the CYP24A1-binding nucleic acid molecule of the present invention. The therapeutic agent of the present invention is characterized by comprising the CYP24A1-binding nucleic acid molecule of the present invention, and other configurations and conditions are not particularly limited. The lung cancer therapeutic agent of the present invention preferably comprises, for example, 1,25-dihydroxyvitamin D3 (also known as calcitriol). Because the therapeutic agent of the present invention comprises the nucleic acid molecule of the present invention, it can inhibit CYP24A1 activity. As shown in the Examples below, the nucleic acid molecule of the present invention can inhibit the proliferation of lung cancer cells, and, for example, in the presence of 1,25-dihydroxyvitamin D3, it can further inhibit the proliferation of lung cancer cells. The lung cancer therapeutic agent of the present invention comprises the CYP24A1 of the present invention and can inhibit CYP24A1 activity, thereby treating lung cancer. The explanations of the nucleic acid molecule and pharmaceutical of the present invention can be applied to the therapeutic agent of the present invention.

[0096] The method of using the therapeutic agent of the present invention can be based on the explanation of the pharmaceutical agent of the present invention described above.

[0097] <Prostate cancer treatment drug> As described above, the prostate cancer therapeutic agent of the present invention comprises the CYP24A1-binding nucleic acid molecule of the present invention. The therapeutic agent of the present invention is characterized by comprising the CYP24A1-binding nucleic acid molecule of the present invention, and other configurations and conditions are not particularly limited. Because the therapeutic agent of the present invention comprises the nucleic acid molecule of the present invention, it can inhibit CYP24A1 activity. As described in Reference 2 below, it is known that prostate cancer can be treated by inhibiting CYP24A1 activity. The prostate cancer therapeutic agent of the present invention comprises the CYP24A1 of the present invention and can inhibit CYP24A1 activity, thereby enabling prostate cancer treatment. The explanations of the nucleic acid molecule and pharmaceutical of the present invention can be used for the therapeutic agent of the present invention. Reference 2 Aruna V Krishnan, et al. “The role of vitamin D in prostate cancer” Recent Results in Cancer Research, Vol. 164, 2003;164:205-21

[0098] The method of using the therapeutic agent of the present invention can be based on the explanation of the pharmaceutical agent of the present invention described above.

[0099] <Chronic kidney disease treatment drug> As described above, the therapeutic agent for chronic kidney disease of the present invention comprises the CYP24A1-binding nucleic acid molecule of the present invention. The therapeutic agent of the present invention is characterized by comprising the CYP24A1-binding nucleic acid molecule of the present invention, and other configurations and conditions are not particularly limited. Since the therapeutic agent of the present invention comprises the nucleic acid molecule of the present invention, it can inhibit CYP24A1 activity. As described in References 3 and 4 below, it is known that chronic kidney disease can be treated by inhibiting CYP24A1 activity. The therapeutic agent for chronic kidney disease of the present invention comprises the CYP24A1 of the present invention and can inhibit CYP24A1 activity, thereby treating chronic kidney disease. The explanations of the nucleic acid molecule and pharmaceutical of the present invention can be applied to the therapeutic agent of the present invention. Reference 3 Gary H.Posner, et al. “Vitamin D analogues targeting CYP24 in chronic kidney disease” The Journal of Steroid Biochemistry and Molecular Biology Volume 121, Issues 1-2, July 2010, Pages 13-19 Reference 4 Xiuying Bai, et al. “CYP24 inhibition as a therapeutic target in FGF23-mediated renal phosphate wasting disorders” The Journal of Clinical Investigation, 2016;126(2):667-680.

[0100] The method of using the therapeutic agent of the present invention can be based on the explanation of the pharmaceutical agent of the present invention described above.

[0101] <Rickets treatment drug> As described above, the therapeutic drug for rickets of the present invention comprises the CYP24A1-binding nucleic acid molecule of the present invention. The therapeutic drug of the present invention is characterized by comprising the CYP24A1-binding nucleic acid molecule of the present invention, and other configurations and conditions are not particularly limited. Since the therapeutic drug of the present invention comprises the nucleic acid molecule of the present invention, it can inhibit CYP24A1 activity. As described in Reference 4 below, it is known that rickets can be treated by inhibiting CYP24A1 activity. The therapeutic drug for rickets of the present invention comprises the CYP24A1 of the present invention and can inhibit CYP24A1 activity, thereby treating rickets. The explanations of the nucleic acid molecule and medicament of the present invention can be used for the therapeutic drug of the present invention. Reference 4 Xiuying Bai, et al. “CYP24 inhibition as a therapeutic target in FGF23-mediated renal phosphate wasting disorders” The Journal of Clinical Investigation, 2016;126(2):667-680.

[0102] The method of using the therapeutic agent of the present invention can be based on the explanation of the pharmaceutical agent of the present invention described above.

[0103] <Osteomalacia treatment drug> As described above, the osteomalacia therapeutic agent of the present invention comprises the CYP24A1-binding nucleic acid molecule of the present invention. The therapeutic agent of the present invention is characterized by comprising the CYP24A1-binding nucleic acid molecule of the present invention, and other configurations and conditions are not particularly limited. Since the therapeutic agent of the present invention comprises the nucleic acid molecule of the present invention, it can inhibit CYP24A1 activity. As described in Reference 4 below, it is known that osteomalacia can be treated by inhibiting CYP24A1 activity. Since the osteomalacia therapeutic agent of the present invention comprises the CYP24A1 of the present invention and can inhibit CYP24A1 activity, it can treat rickets. The explanations of the nucleic acid molecule and medicament of the present invention can be used for the therapeutic agent of the present invention. Reference 4 Xiuying Bai, et al. “CYP24 inhibition as a therapeutic target in FGF23-mediated renal phosphate wasting disorders” The Journal of Clinical Investigation, 2016;126(2):667-680.

[0104] The method of using the therapeutic agent of the present invention can be based on the explanation of the pharmaceutical agent of the present invention described above.

[0105] <Detection reagents> As described above, the CYP24A1 detection reagent of the present invention comprises the CYP24A1-binding nucleic acid molecule of the present invention. The detection reagent of the present invention is characterized by comprising the CYP24A1-binding nucleic acid molecule of the present invention, and other configurations and conditions are not particularly limited. The nucleic acid molecule of the present invention binds to CYP24A1. Therefore, the detection reagent of the present invention can detect CYP24A1. For the detection reagent of the present invention, for example, the explanation of the nucleic acid molecule of the present invention can be used.

[0106] According to the present invention, since the nucleic acid molecule of the present invention binds to CYP24A1, it is possible to detect CYP24A1 in a sample, for example, by detecting the binding between CYP24A1 and the nucleic acid molecule. Specifically, for example, the presence or absence of CYP24A1 in a sample or the amount of CYP24A1 can be analyzed. Therefore, the detection reagent of the present invention can also be referred to as, for example, an analytical reagent. It can be said that the detection reagent of the present invention enables, for example, qualitative or quantitative analysis.

[0107] In the present invention, the sample is not particularly limited. Examples of the sample include the aforementioned cell and tissue extracts. The sample may be a sample containing CYP24A1, a sample not containing CYP24A1, or a sample whose presence or absence of CYP24A1 is unknown.

[0108] The sample may be, for example, a liquid sample or a solid sample. A liquid sample is preferred because it is easy to contact with the nucleic acid molecule and is easy to handle. In the case of a solid sample, for example, a mixture, extract, or dissolution solution may be prepared using a solvent and used. The solvent is not particularly limited, and examples thereof include water, physiological saline, and buffer solutions.

[0109] The detection step includes, for example, a contacting step of contacting the sample with the nucleic acid molecule to bind CYP24A1 in the sample to the nucleic acid molecule, and a binding detection step of detecting the binding between CYP24A1 and the nucleic acid molecule. The detection step also includes, for example, an analysis step of analyzing the presence or amount of CYP24A1 in the sample based on the results of the binding detection step.

[0110] In the contacting step, the method for contacting the sample with the nucleic acid molecule is not particularly limited. The sample is preferably contacted with the nucleic acid molecule in, for example, a liquid. The liquid is not particularly limited, and examples thereof include water, physiological saline, and a buffer solution.

[0111] In the contacting step, the conditions for contacting the sample with the nucleic acid molecule are not particularly limited. The contact temperature is, for example, 4 to 37° C. or 18 to 25° C., and the contact time is, for example, 10 to 120 minutes or 30 to 60 minutes.

[0112] In the contacting step, the nucleic acid molecule may be, for example, an immobilized nucleic acid molecule immobilized on a carrier, or an unimmobilized free nucleic acid molecule. In the latter case, the nucleic acid molecule is contacted with the sample, for example, in a container. The nucleic acid molecule may be, for example, an immobilized nucleic acid molecule because of its excellent handleability. The carrier is not particularly limited, and examples thereof include a substrate, beads, a container, etc., and examples of the container include a microplate, a tube, etc.

[0113] As described above, the binding detection step is a step of detecting the binding between CYP24A1 and the nucleic acid molecule in the sample. By detecting the presence or absence of the binding between the two, for example, the presence or absence of CYP24A1 in the sample can be analyzed (qualitatively determined), and by detecting the degree of binding between the two (amount of binding), for example, the amount of CYP24A1 in the sample can be analyzed (quantitatively determined).

[0114] If the binding between CYP24A1 and the nucleic acid molecule cannot be detected, it can be determined that CYP24A1 is not present in the sample, and if the binding is detected, it can be determined that CYP24A1 is present in the sample.

[0115] The method for analyzing the binding between CYP24A1 and the nucleic acid molecule is not particularly limited. For example, a conventionally known method for detecting binding between substances can be used, and specific examples include the above-mentioned detection methods. The binding may also be, for example, the detection of a complex between CYP24A1 and the nucleic acid molecule. In this case, the nucleic acid molecule is preferably labeled with a labeling substance. When the nucleic acid molecule is labeled, the binding between CYP24A1 and the nucleic acid molecule can be analyzed, for example, by detecting the label.

[0116] <Detection kit> The detection kit of the present invention comprises the CYP24A1-binding nucleic acid molecule of the present invention. The detection kit of the present invention is characterized by comprising the nucleic acid molecule of the present invention, and other configurations and conditions are not particularly limited. The detection kit of the present invention can be used to detect, for example, CYP24A1. The explanations for the nucleic acid molecule and detection reagent of the present invention can be applied to the detection kit of the present invention.

[0117] The detection kit of the present invention may contain, in addition to the nucleic acid molecule of the present invention, other components such as the carrier, buffer solution, and instruction manual.

[0118] <Treatment method> The treatment method of the present invention comprises an administration step of administering a CYP24A1-binding nucleic acid molecule of the present invention to a subject. The treatment method of the present invention is characterized by administering a CYP24A1-binding nucleic acid molecule of the present invention to a subject, and other steps and conditions are not particularly limited. The treatment method of the present invention can suppress CYP24A1 activity in the subject by administering the nucleic acid molecule of the present invention. The treatment method of the present invention can be applied to the treatment method of the present invention as described above with reference to the nucleic acid molecule, pharmaceutical agent, and activity regulator of the present invention.

[0119] When the subject of administration is, for example, a patient with a disease caused by vitamin D deficiency, the treatment method of the present invention can also be referred to as, for example, a method for treating the disease.

[0120] The administration conditions for the treatment method of the present invention can be determined by reference to the above explanation of the pharmaceutical agent of the present invention.

[0121] <Activity suppression method> The method for inhibiting CYP24A1 activity of the present invention (hereinafter also referred to as the "activity inhibition method") uses the CYP24A1-binding nucleic acid molecule of the present invention. The activity inhibition method of the present invention is characterized by using the CYP24A1-binding nucleic acid molecule of the present invention, and other steps and conditions are not particularly limited. Since the activity inhibition method of the present invention uses the nucleic acid molecule of the present invention, it can inhibit CYP24A1 activity. Furthermore, CYP24A1 functions in the inactivation of vitamin D. Therefore, the activity inhibition method of the present invention is expected to function as a method for inhibiting vitamin D inactivation. The explanations of the nucleic acid molecule, pharmaceutical, and activity inhibitor of the present invention can be applied to the activity inhibition method of the present invention.

[0122] The administration conditions for the activity suppression method of the present invention can be determined by referring to the explanation of the medicament of the present invention described above.

[0123] <Lung cancer treatment methods> As described above, the method for treating lung cancer of the present invention includes an administration step of administering a lung cancer therapeutic agent to a subject, wherein the lung cancer therapeutic agent is the lung cancer therapeutic agent of the present invention. The method for treating lung cancer of the present invention is characterized by administering the lung cancer therapeutic agent of the present invention to a subject, i.e., administering the CYP24A1-binding nucleic acid molecule of the present invention, and other steps and conditions are not particularly limited. The method for treating lung cancer of the present invention administers the lung cancer therapeutic agent of the present invention to a subject, thereby treating lung cancer in the subject. The explanations of the CYP24A1-binding nucleic acid molecule, pharmaceutical, and lung cancer therapeutic agent of the present invention can be used for the method for treating lung cancer of the present invention.

[0124] In the lung cancer treatment method of the present invention, the administration step may, for example, in In vitro or in vivo For the subjects and conditions for administration of the lung cancer therapeutic agent of the present invention, the explanation of the subjects and conditions for administration of the pharmaceutical agent of the present invention can be cited, for example.

[0125] <Prostate cancer treatment methods> As described above, the method for treating prostate cancer of the present invention includes an administration step of administering a prostate cancer therapeutic agent to a subject, where the prostate cancer therapeutic agent is the prostate cancer therapeutic agent of the present invention. The method for treating prostate cancer of the present invention is characterized by administering the prostate cancer therapeutic agent of the present invention to a subject, i.e., administering the CYP24A1-binding nucleic acid molecule of the present invention, and other steps and conditions are not particularly limited. The method for treating prostate cancer of the present invention administers the prostate cancer therapeutic agent of the present invention to a subject, thereby treating prostate cancer in the subject. The explanations of the CYP24A1-binding nucleic acid molecule, pharmaceutical, and prostate cancer therapeutic agent of the present invention can be used for the method for treating prostate cancer of the present invention.

[0126] In the prostate cancer treatment method of the present invention, the administration step may, for example, in In vitro or in vivo For the subjects and conditions for administration of the prostate cancer therapeutic agent of the present invention, the explanation of the subjects and conditions for administration of the pharmaceutical agent of the present invention can be cited, for example.

[0127] <Chronic kidney disease treatment method> As described above, the method for treating chronic kidney disease of the present invention includes an administration step of administering a therapeutic agent for chronic kidney disease to a subject, wherein the therapeutic agent for chronic kidney disease is the therapeutic agent for chronic kidney disease of the present invention. The method for treating chronic kidney disease of the present invention is characterized by administering the therapeutic agent for chronic kidney disease of the present invention to a subject, i.e., administering the CYP24A1-binding nucleic acid molecule of the present invention, and other steps and conditions are not particularly limited. The method for treating chronic kidney disease of the present invention administers the therapeutic agent for chronic kidney disease of the present invention to a subject, thereby treating chronic kidney disease in the subject. The explanations for the CYP24A1-binding nucleic acid molecule, pharmaceutical, and therapeutic agent for chronic kidney disease of the present invention can be used for the method for treating chronic kidney disease of the present invention.

[0128] In the method for treating chronic kidney disease of the present invention, the administration step may, for example, in In vitro or in vivo For the subjects and conditions for administration of the therapeutic agent for chronic kidney disease of the present invention, the explanation of the subjects and conditions for administration of the pharmaceutical agent of the present invention can be cited, for example.

[0129] <Treatment method for rickets> As described above, the method for treating rickets of the present invention includes an administration step of administering a therapeutic drug for rickets to a subject, and the therapeutic drug for rickets is the therapeutic drug for rickets of the present invention. The method for treating rickets of the present invention is characterized by administering the therapeutic drug for rickets of the present invention, i.e., administering the CYP24A1-binding nucleic acid molecule of the present invention, to a subject, and other steps and conditions are not particularly limited. The method for treating rickets of the present invention administers the therapeutic drug for rickets of the present invention to a subject, and therefore can treat rickets in the subject. The explanations for the CYP24A1-binding nucleic acid molecule, pharmaceutical, and therapeutic drug for rickets of the present invention can be used for the method for treating rickets of the present invention.

[0130] In the method for treating rickets of the present invention, the administration step may, for example, in In vitro or in vivoFor the subjects and conditions for administering the therapeutic drug for rickets of the present invention, the explanation of the subjects and conditions for administering the pharmaceutical of the present invention can be cited, for example.

[0131] <Osteomalacia treatment method> As described above, the method for treating osteomalacia of the present invention includes an administration step of administering a therapeutic agent for osteomalacia to a subject, wherein the therapeutic agent for osteomalacia is the therapeutic agent for osteomalacia of the present invention. The method for treating osteomalacia of the present invention is characterized by administering the therapeutic agent for osteomalacia of the present invention, i.e., administering the CYP24A1-binding nucleic acid molecule of the present invention, to a subject, and other steps and conditions are not particularly limited. The method for treating osteomalacia of the present invention involves administering the therapeutic agent for osteomalacia of the present invention to a subject, thereby treating osteomalacia in the subject. The explanations for the CYP24A1-binding nucleic acid molecule, pharmaceutical, and therapeutic agent for osteomalacia of the present invention can be used for the method for treating osteomalacia of the present invention.

[0132] In the osteomalacia treatment method of the present invention, the administration step may, for example, in In vitro or in vivo For the subjects and conditions for administering the osteomalacia therapeutic agent of the present invention, the explanation of the subjects and conditions for administering the pharmaceutical agent of the present invention can be cited, for example.

[0133] <Detection method> The CYP24A1 detection method of the present invention (hereinafter also referred to as the "detection method") comprises the step of detecting CYP24A1 in a sample by contacting the sample with the CYP24A1-binding nucleic acid molecule of the present invention and allowing CYP24A1 in the sample to bind to the nucleic acid molecule. The detection method of the present invention is characterized by using the nucleic acid molecule of the present invention, and other steps and conditions are not particularly limited. The nucleic acid molecule of the present invention binds to CYP24A1. Therefore, the detection method of the present invention can detect CYP24A1. For the detection method of the present invention, the explanations of the nucleic acid molecule, detection reagent, and detection kit of the present invention can be used, for example.

[0134] The detection method of the present invention may use the detection kit of the present invention as the nucleic acid molecule.

[0135] <Use of Nucleic Acid Molecules> The present invention relates to a nucleic acid molecule of the present invention or use thereof for regulating CYP24A1 activity, a nucleic acid molecule of the present invention or use thereof for detecting CYP24A1, a nucleic acid molecule of the present invention or use thereof for treating lung cancer, a nucleic acid molecule of the present invention or use thereof for treating prostate cancer, a nucleic acid molecule of the present invention or use thereof for treating chronic kidney disease, a nucleic acid molecule of the present invention or use thereof for treating rickets, and a nucleic acid molecule of the present invention or use thereof for treating osteomalacia. The explanations for the nucleic acid molecule, pharmaceutical, activity inhibitor, therapeutic agent for lung cancer, therapeutic agent for prostate cancer, therapeutic agent for chronic kidney disease, therapeutic agent for rickets, therapeutic agent for osteomalacia, detection reagent, detection kit, treatment method, activity inhibition method, method for treating lung cancer, method for treating prostate cancer, method for treating chronic kidney disease, method for treating rickets, method for treating osteomalacia, and detection method of the present invention can be cited in the present invention. [Example]

[0136] Next, examples of the present invention will be described. However, the present invention is not limited to the following examples. Commercially available reagents were used according to their protocols unless otherwise specified.

[0137] [Example 1] It was confirmed that the nucleic acid molecule of the present invention can be obtained by the SELCOS method described in Reference 1 above.

[0138] (1) Preparation of DNA pool A pool of polynucleotides consisting of the base sequence of SEQ ID NO: 23 below was prepared as a single-stranded nucleic acid pool for obtaining CYP24A1-binding nucleic acid molecules. In SEQ ID NO: 23 below, the 20 bases at the 5' and 3' ends are primer sequences, and the central 30 bases are a random sequence. In SEQ ID NO: 23 below, N is A, C, G, or T. A DNA library was then prepared by PCR using the following forward primer (SEQ ID NO: 24) and biotinylated reverse primer (SEQ ID NO: 25). The DNA library contained approximately 10 18 There is a diversity of types. Nucleic Acid Pool (SEQ ID NO: 23) 5'-AGCAGCACAGAGGTCAGATGNNNNNNNNNNNNNNNNNNNNNNNNNNNNCCTATGCGTGCTACCGTGAA-3' Forward primer (SEQ ID NO: 24) 5'-AGCAGCACAGAGGTCAGATG-3' Biotinylated reverse primer (SEQ ID NO: 25) 5'-TTCACGGTAGCAGCGATAGG-3'

[0139] (2) Preparation of immobilized targets First, E. coli expressing human-derived CYP24A1 was prepared with reference to Reference 5 below, and human-derived CYP24A1 was isolated from the E. coli. Next, E. coli expressing mouse-derived CYP27B1 was prepared with reference to Reference 6 below, and mouse-derived CYP27B1 was isolated from the E. coli. It is known that there are no species differences between mice and humans in CYP27B1, and it can be assumed that the results for mouse-derived CYP27B1 will be similar to those for human-derived CYP27B1. Then, the human-derived CYP24A1 was immobilized on Ni-NTA magnetic beads (50 μm, QIAGEN) and the mouse-derived CYP27B1 was immobilized on Ni-NTA agarose resin beads (45-165 μm, QIAGEN) according to the protocols for each bead, to prepare immobilized CYP24A1 and immobilized CYP27B1, respectively. The immobilized CYP24A1 is a target protein for the binding nucleic acid molecule of the present invention, and the immobilized CYP27B1 is a competing target protein for the binding nucleic acid molecule of the present invention. Reference 5: Kusudo et al., “Metabolism of A-ring diastereomers of 1α,25-dihydroxyvitamin D3by CYP24A1”, BBRC 321, 774, 2004 Reference 6: Uchida et al., “Purification and characterization of mouse CYP27B1 overproduced by an Escherichia coli system coexpressing molecular chaperonins GroEL / ES”, BBRC 323, 505, 2004

[0140] (3) Selection Next, using the immobilized target obtained in (2) above and the nucleic acid pool in (1) above, the SELCOS method was carried out in the following steps with reference to Reference 1 below. Reference 1: Ankita Kushwaha et al. “Competitive non-SELEX for the selective and rapid enrichment of DNA aptamers and its use in electrochemical aptasensor”, SCIENTIFIC REPORT, 2019 Apr 30, doi: 10.1038 / s41598-019-43187-6

[0141] (1st step) First, the approximately 10 obtained in (1) above 18 The nucleic acid pools were heated at 90°C for 5 minutes. The mixture was then cooled to 4°C and allowed to stand for 5 minutes, followed by incubation at 25°C for 15 minutes. 500 μl of the nucleic acid pool containing 10 pmol of nucleic acid molecules was mixed with 20 μl of the Ni-NTA magnetic bead-immobilized CYP24A1 and 20 μl of the Ni-NTA agarose resin bead-immobilized CYP27B1 in the presence of a binding buffer and incubated for 60 minutes at 25°C to allow binding of the candidate nucleic acid molecules in the nucleic acid pool to CYP24A1 or CYP27B1. The binding buffer consisted of 20 mmol / L Tris-HCl buffer (pH 7.4; containing 0.01% Tween® 20), 100 mmol / L NaCl, 5 mmol / L KCl, 2 mmol / L MgCl, and 1 mmol / L CaCl.

[0142] (Second step) Next, the sample was washed with a washing buffer (20 mmol / L Tris-HCl buffer (pH 7.4, 0.05% Tween® 20), 100 mmol / L NaCl, 5 mmol / L KCl, 2 mmol / L MgCl, 1 mmol / L CaCl), and the sample solution was centrifuged at 1,000 g for 10 seconds. The supernatant was carefully removed to remove nucleic acid molecules that were not bound to CYP24A1 or CYP27B1 or that had weak binding. This washing was performed twice. The first and second steps were then repeated five times. Each time a round was repeated, 50 pmol, 250 pmol, 1000 pmol, and 1000 pmol of the nucleic acid pool were added. Furthermore, each time a round was repeated, the incubation time and the NaCl concentration of the washing buffer were changed to 30 minutes (200 mmol / L NaCl), 15 minutes (400 mmol / L NaCl), 15 minutes (800 mmol / L NaCl), and 15 minutes (2000 mmol / L NaCl), respectively.

[0143] (Third Step) The washed supernatant was then separated using a magnetic stand to separate the Ni-NTA magnetic bead-immobilized CYP24A1, and then centrifuged at 1000 g for 10 seconds to separate the Ni-NTA agarose resin bead-immobilized CYP27B1. The supernatant was then removed. The separated Ni-NTA magnetic bead-immobilized CYP24A1 and Ni-NTA agarose resin bead-immobilized CYP27B1 were heat-treated (heated at 90°C for 5 minutes, and the supernatant was immediately removed) to recover the nucleic acid molecules bound to each bead, which were used as a candidate nucleic acid pool for CYP24A1-binding nucleic acid molecules and a candidate nucleic acid pool for CYP27B1-binding nucleic acid molecules.

[0144] (Step 4) The candidate nucleic acid pool for CYP24A1-binding nucleic acid molecules obtained in step 3 was incubated with the Ni-NTA agarose resin beads in the presence of the binding buffer at 25°C for 15 to 20 minutes, and then the Ni-NTA agarose resin beads were removed in the same manner as in step 3 to remove nonspecific candidates.The candidate nucleic acid pool for CYP24A1-binding nucleic acid molecules obtained was then further incubated with the Ni-NTA agarose resin bead-immobilized CYP27B1 at 25°C for 10 to 15 minutes, and then the Ni-NTA agarose resin bead-immobilized CYP27B1 was removed in the same manner as in step 3 to remove nonspecific candidates that bind to CYP27B1, thereby obtaining a CYP24A1-binding nucleic acid pool. Similarly, the candidate nucleic acid pool for CYP27B1-binding nucleic acid molecules was incubated with the Ni-NTA agarose resin beads in the presence of the binding buffer at 25°C for 15 to 20 minutes, and then the Ni-NTA agarose resin beads were removed in the same manner as in step 3 to remove nonspecific candidates. The resulting candidate nucleic acid pool for CYP27B1-binding nucleic acid molecules was further incubated with the Ni-NTA magnetic bead-immobilized CYP24A1 at 25°C for 10 minutes, and then the Ni-NTA magnetic bead-immobilized CYP24A1 was removed in the same manner as in step 3 to remove nonspecific candidates that bind to CYP24A1, thereby obtaining a CYP27B1-binding nucleic acid pool.

[0145] (5th step) The CYP24A1-binding nucleic acid pool and the CYP27B1-binding nucleic acid pool obtained in the fourth step were amplified by PCR using the forward primer (SEQ ID NO: 24) and the biotinylated reverse primer (SEQ ID NO: 25). The PCR conditions were 20 cycles of 98°C for 2 minutes, 98°C for 10 seconds, 59°C for 5 seconds, 72°C for 10 seconds, and 72°C for 4 minutes. Amplification was confirmed by gel electrophoresis using an 8% polyacrylamide gel containing 8 mol / L urea at 60°C. Forward primer (SEQ ID NO: 24) 5'-AGCAGCACAGAGGTCAGATG-3' Biotinylated reverse primer (SEQ ID NO: 25) 5'-TTCACGGTAGCAGCGATAGG-3'

[0146] (4) Sequence analysis The CYP24A1-binding nucleic acid pool and the CYP27B1-binding nucleic acid pool obtained in (3) above were each subjected to sequence analysis using Illumina NGS, and 10 5 Approximately several types of sequences were obtained. As a result, the CYP24A1-binding nucleic acid molecules shown below as SEQ ID NOS: 1 to 18 were obtained. Note that the primer binding sequences shown as SEQ ID NOS: 21 and 22 were bound to the 5' and 3' ends of each sequence, respectively, but are not shown here. AptCYP24-#1 (SEQ ID NO: 1) 5′-CGCCTGGGACCAACATCATGTCATCGTCTA-3′ AptCYP24-#2 (SEQ ID NO: 2) 5′-CGCAAGCCACACTTTGTATGGACCAGTATC-3′ AptCYP24-#3 (SEQ ID NO: 3) 5′-CAGGGCCATAAGCTGACCCAATCTTGTCGG-3′ AptCYP24-#4 (SEQ ID NO: 4) 5′-AGACCAGTTATGCAAGCTCATCTGTTGT-3′ AptCYP24-#5 (SEQ ID NO: 5) 5′-CAGGACAGCACCCTGACCCAGTTCTGTATA-3′ AptCYP24-#6 (SEQ ID NO: 6) 5′-CAGGGTCAATGGTCCCCAATCTACACAGAT-3′ AptCYP24-#7 (SEQ ID NO: 7) 5′-CATGCAGGAAGTAACCCAATGTCGTTAAC-3′ AptCYP24-#8 (SEQ ID NO: 8) 5′-CAGCGTCACCATACCCTTTAACACCACGCA-3′ AptCYP24-#9 (SEQ ID NO: 9) 5′-ACCTATACACCGACGAACCCTACTTCCTGA-3′ AptCYP24-#10 (SEQ ID NO: 10) 5′-CTTTTACCCCCCCGCTGCCTTTATACTTGA-3′ AptCYP24-#11 (SEQ ID NO: 11) 5′-CAAGCCGAAGTTACACACGGACCAATATGT-3′ AptCYP24-#12 (SEQ ID NO: 12) 5′-GCCACTCTTCGTACTCCTCCCTCTATCGGA-3′ AptCYP24-#13 (SEQ ID NO: 13) 5′-TCCCCCCCATAACCAACATTTAGCATCATG-3′ AptCYP24-#14 (SEQ ID NO: 14) 5′-TAGGTAGCAAGCAAACATGTATTCCTTTTTC-3′ AptCYP24-#15 (SEQ ID NO: 15) 5′-GTAGCGCGACGTACAATATTTCTTTCCTCC-3′ AptCYP24-#16 (SEQ ID NO: 16) 5′-TGGACTTTTTCCGGTGGGAGACGAGGTTGC-3′ AptCYP24-#17 (SEQ ID NO: 17) 5′-ACCCAAGGACGTCACCGACTGCAGTGTTAT-3′ AptCYP24-#18 (SEQ ID NO: 18) 5′-ACCCTTAGCTCACCCTTCCCCGCCCGCCAC-3′ 5' end primer binding sequence (SEQ ID NO: 21) 5´-AGCAGCACAG AGGTCAGATG 3' end primer binding sequence (SEQ ID NO: 22) CCTATCGCTG CTACCGTGAA-3´

[0147] The 18 sequences of the binding nucleic acid molecules were filtered according to the number of repeats and then clustered based on their homology. ClustalW was used for filtering and clustering. As a result, the 18 sequences of the binding nucleic acid molecules could be classified into six types of clusters, as shown in Table 1 below, based on the predicted secondary structure and stem-loop position.

[0148] [Table 1]

[0149] The secondary structures of 11 representative sequences from the six clusters, i.e., AptCYP24-#1 (SEQ ID NO: 1), AptCYP24-#2 (SEQ ID NO: 2), AptCYP24-#3 (SEQ ID NO: 3), AptCYP24-#5 (SEQ ID NO: 5), AptCYP24-#7 (SEQ ID NO: 7), AptCYP24-#11 (SEQ ID NO: 11), AptCYP24-#12 (SEQ ID NO: 12), AptCYP24-#15 (SEQ ID NO: 15), AptCYP24-#16 (SEQ ID NO: 16), AptCYP24-#17 (SEQ ID NO: 17), and AptCYP24-#18 (SEQ ID NO: 18), were analyzed using Mfold. The predicted secondary structures of each binding nucleic acid molecule are shown in Figure 1 (Figures 1A-1, A-2, B, C, D, E-1, and E-2). The left diagram of Figure 1A-1 shows the predicted secondary structure of AptCYP24-#1 (SEQ ID NO: 1), and the right diagram shows the predicted secondary structure of AptCYP24-#2 (SEQ ID NO: 2). The left diagram of Figure 1A-2 shows the predicted secondary structure of AptCYP24-#3 (SEQ ID NO: 3), and the right diagram shows the predicted secondary structure of AptCYP24-#5 (SEQ ID NO: 5). Figure 1B shows the predicted secondary structure of AptCYP24-#7 (SEQ ID NO: 7). The left diagram of Figure 1C shows the predicted secondary structure of AptCYP24-#11 (SEQ ID NO: 11), and the right diagram shows the predicted secondary structure of AptCYP24-#12 (SEQ ID NO: 12). Figure 1D shows the predicted secondary structure of AptCYP24-#15 (SEQ ID NO: 15). The left diagram in Figure 1E-1 shows the predicted secondary structure of AptCYP24-#16 (SEQ ID NO: 16), the right diagram shows the predicted secondary structure of AptCYP24-#17 (SEQ ID NO: 17), and Figure 1E-2 shows the predicted secondary structure of AptCYP24-#18 (SEQ ID NO: 18).

[0150] (5) Confirmation of binding to CYP24A1 Furthermore, among the obtained CYP24A1-binding nucleic acid molecules, AptCYP24-#7 (SEQ ID NO: 7) was used in a gel shift assay (electrophoretic mobility shift assay (EMSA) targeting CYP24A1 to confirm that the CYP24A1-binding nucleic acid molecule of the present invention binds to CYP24A. Specifically, 80 nmol / L of AptCYP24-#7 (SEQ ID NO: 7) was mixed with predetermined concentrations of CYP24A1 (0.1 μmol / L, 0.5 μmol / L, 1 μmol / L, and 5 μmol / L) and incubated at room temperature for 30 minutes. Next, 10 μl of each sample was mixed with 4 μl of EMSA sample buffer (0 mmol / L Tris / 10% (v / v) bromophenol blue (pH 6.8)), loaded onto a 12% PAGE gel, and run at 100 V for 1 hour. The control was AptCYP24-#7 (SEQ ID NO: 7) alone without CYP24A1. The gel was then stained with SYBR™ Gold Nucleic Acid Gel Stain, photographed using a UV Transilluminator Imaging System, and analyzed using Imagej.

[0151] The results are shown in Figure 2. Figure 2 is a gel image showing the results of EMSA. In the gel image of Figure 2, each lane, from left to right, shows the results for a DNA marker (DNA ladder) and CYP24A1 concentrations of 0, 0.1 μmol / L, 0.5 μmol / L, 1 μmol / L, and 5 μmol / L. In Figure 2, the 70b band indicated by the arrow represents the free aptamer (AptCYP24-#7 (SEQ ID NO: 7)). As shown in Figure 2, the amount of the 70b band representing the free aptamer (AptCYP24-#7 (SEQ ID NO: 7)) decreases as the CYP24A1 concentration increases, indicating that AptCYP24-#7 (SEQ ID NO: 7) binds to CYP24A1. This demonstrates that the CYP24A1-binding nucleic acid molecule of the present invention binds to CYP24A1.

[0152] (6) Confirmation of specific binding to CYP24A1 Of the CYP24A1-binding nucleic acid molecules obtained, AptCYP24-#7 (sequence number 7) was subjected to a competitive electrochemical assay called Apta-DEPSOR using the three-electrode detection system DEPSOR (Disposable Electrochemical Printed Sensor), with reference to Reference 1, to electrically detect specific binding to CYP24A1.

[0153] (6-1) Preparation of DEP chip First, a disposable three-electrode screen-printed (DEP) chip (BioDevice Technology, Inc.) was prepared. This three-electrode system for electrochemical analysis included a carbon-based working electrode (3 mm diameter), a reference electrode, and an Ag / AgCl reference electrode. A 0.5, 5, 50, or 500 nmol / L CYP24A1 or CYP27B1 solution was dropped onto the working electrode of the DEP chip and incubated at 4°C for 1 hour. After incubation, the working electrode was rinsed three times with 100 mmol / L Tris-HCl buffer (pH 7.4) to remove excess CYP24A1 or CYP27B1, and the chip was air-dried. To suppress nonspecific adsorption, 3.5 μL of blocking buffer (100 mmol / L Tris-HCl solution containing 1% BSA) was then added to the chip and incubated overnight at 4°C. The chip was then rinsed three times with 100 mmol / L Tris-HCl buffer (pH 7.4) and dried, thereby preparing a CYP24A1-immobilized DEP chip and a CYP27B1-immobilized DEP chip.

[0154] (6-2) Preparation of aptamer-modified gold nanoparticles AptCYP24-#7 (SEQ ID NO: 7) was coated onto gold nanoparticles (40 nm, BBI Solutions, UK) and immobilized with a CYP24A1-binding nucleic acid molecule to prepare aptamer-modified gold nanoparticles. Specifically, the thiol-modified aptamer (AptCYP24-#7 (SEQ ID NO: 7)) was dissolved in nuclease-free water and deprotected with TCEP for two hours in the dark. The gold nanoparticles (AuNP, 40 nm) were then centrifuged (80 × 100 g) for 10 minutes at 4°C, and 45 μl of the supernatant was discarded. The remaining 5 μl was added to the deprotected aptamer solution (30 μl, 10 μg / ml) and sonicated for 3 seconds at 45 kHz. The mixture was then vortexed for 10 seconds and placed on a rotating wheel for 3 minutes. 260 μl of distilled water was then added to the mixture. Then, 60 μl of NaCl (1 mol / L) was added dropwise. The mixture was then heated at 90°C for 2 minutes and then left at room temperature for 15 minutes to allow the aptamer molecules bound to the gold nanoparticles to fold. The aptamer-bound gold nanoparticles were then added with 40 μl of 10% BSA, incubated at room temperature for 5 minutes, vortexed for 10 seconds, and placed on a rotating wheel for 2 hours for blocking. The mixture was then washed with 50 μl of the wash buffer, centrifuged, and the supernatant discarded to remove any aptamer that was not bound to the gold nanoparticles. The resulting aptamer-modified gold nanoparticles were suspended in 50 μl of the binding buffer and stored at 4°C until use.

[0155] (6-3) Detection of binding to CYP24A1 by DEPSOR 2 μL of the aptamer-modified gold nanoparticles prepared in (6-2) above was dropped onto the surface of the CYP24A1-immobilized DEP chip prepared in (6-1) above. The CYP24A1-immobilized DEP chip was then incubated at room temperature (approximately 25°C) for 15 minutes. The DEP chip after the reaction was then washed three times with a washing buffer (20 mmol / L Tris-HCl buffer (pH 7.4), 0.05% Tween® 20), 100 mmol / L NaCl, 5 mmol / L KCl, 2 mmol / L MgCl2, and 1 mmol / L CaCl2) and connected to an electrochemical analyzer (BioSeeds, Inc.) to measure the current.

[0156] The results are shown in Figure 3. Figure 3(A) is a graph showing the differential pulse voltammetry (DPV) curves for the response between 0.5, 5, 50, or 500 nmol / L CYP24A1-immobilized DEP chips and aptamer-modified gold nanoparticles, and Figure 3(B) is a graph showing the peak signal current values. In Figure 3(B), the vertical axis represents the current value (μA), and the horizontal axis represents the concentration of CYP24A1 immobilized on the DEP chip. In the DEPSOR analysis, the electrochemical signal increases depending on the amount of gold nanoparticles captured by CYP24A1 immobilized on the electrode surface. As shown in Figure 3, the signal increases in proportion to the CYP24A1 concentration, indicating an increase in the amount of aptamer-modified gold nanoparticles bound. This demonstrates that the CYP24A1-binding nucleic acid molecule of the present invention binds to CYP24A1 and can detect CYP24A1 at extremely low concentrations of 0.5 nmol / L.

[0157] (6-4) Confirmation of specific binding to CYP24A1 by DEPSOR Next, the concentration of AptCYP24-#7 (sequence number 7) immobilized on the aptamer-modified gold nanoparticles was changed to 10, 100, or 1000 nmol / l, and the binding of AptCYP24-#7 (sequence number 7) to CYP24A1 or CYP27B1 was detected in the same manner, except that a CYP24A1-immobilized DEP chip with 500 nmol / l CYP24A1 immobilized or a CYP27B1-immobilized DEP chip with 500 nmol / l CYP27B1 immobilized was used.

[0158] The results are shown in Figure 4. Figure 4 is a graph showing the peak signal current values ​​obtained by DEPSOR analysis. In Figure 4, the vertical axis represents the current value (μA), and the horizontal axis represents the concentration of AptCYP24-#7 (SEQ ID NO: 7) immobilized on gold nanoparticles. As shown in Figure 4, AptCYP24-#7 (SEQ ID NO: 7) bound to CYP24A1 but did not bind to CYP27B1. In other words, it was found that the CYP24A1-binding nucleic acid molecule of the present invention specifically binds to CYP24A1.

[0159] From the above, it was found that the CYP24A1-binding nucleic acid molecule of the present invention can be obtained by the SELCOS method, which uses CYP24A1 as the target and CYP27B1 as the competing target, and that the obtained CYP24A1-binding nucleic acid molecule specifically binds to CYP24A1.

[0160] [Example 2] It was confirmed that the nucleic acid molecules of the present invention exhibit the ability to inhibit CYP24A1.

[0161] Of the CYP24A1-binding nucleic acid molecules obtained in Example 1, AptCYP24-#1 (sequence number 1), AptCYP24-#2 (sequence number 2), AptCYP24-#3 (sequence number 3), AptCYP24-#5 (sequence number 5), AptCYP24-#7 (sequence number 7), AptCYP24-#11 (sequence number 11), AptCYP24-#12 (sequence number 12), AptCYP24-#15 (sequence number 15), AptCYP24-#16 (sequence number 16), AptCYP24-#17 (sequence number 17), and AptCYP24-#18 (sequence number 18) were used to confirm their ability to inhibit CYP24A1 activity.

[0162] The ability of each CYP24A1-binding nucleic acid molecule to inhibit CYP24A1 activity was evaluated with reference to Reference 5 below. Specifically, each aptamer was incubated at 95°C for 5 minutes, then placed on ice for 3 minutes and then left at room temperature (approximately 25°C, hereinafter the same) for 15 minutes. Next, each aptamer was added to 89.5 μL of reaction mixture (5 nmol / L CYP24A1, 0.5 μmol / L adrenodoxin, 0.05 μmol / L adrenodoxin reductase, and 1 mmol / L EDTA in 100 mmol / L Tris-HCl buffer (pH 7.5)) to a concentration of 200 nmol / L, and the mixture was left at room temperature for 15 minutes. Next, 0.5 μL of 1000 μmol / L 1α,25-dihydroxyvitamin D3 (substrate) (final concentration: 5 μmol / L) and 10 μL of 10 mmol / L NADPH (final concentration: 1 mmol / L) were added to 89.5 μL of the reaction mixture and allowed to react for 10 minutes at 37°C. After the reaction, a chloroform / methanol (3:1) solution was added to the mixture, the supernatant was removed, and the organic phase was dried. The dried product was dissolved in acetonitrile and analyzed by HPLC (Waters). The peak areas of the substrate 1α,25-dihydroxyvitamin D3 and the metabolite 1α,24,25-dihydroxyvitamin D3 formed after CYP24A1 action on the substrate were calculated, and the CYP24A1 activity in the presence of each aptamer was calculated. As controls, the following were used: no aptamer was added (Control 1, CYP24A1 only); the initial DNA library prepared in Example 1(1) was added instead of each aptamer (Control 2, Initial random library); and a poly T-rich sequence (TTTTTTTTTTTAACATT, SEQ ID NO: 26) was added instead of each aptamer (Control 3, Poly T-rich sequence). Reference 5: Kusudo et al., “Metabolism of A-ring diastereomers of 1α,25-dihydroxyvitamin D3by CYP24A1”, BBRC 321, 774, 2004

[0163] The results are shown in Figure 5. Figure 5 is a graph showing relative CYP24A1 activity values. In Figure 5, the vertical axis shows the relative CYP24A1 activity value, with the CYP24A1 activity value of Control 1 (CYP24A1 only) taken as 100%, and the horizontal axis shows the type of sample. As shown in Figure 5, AptCYP24-#1 (SEQ ID NO: 1) had a 72.5 ± 7.3% cytotoxicity, AptCYP24-#2 (SEQ ID NO: 2) had a 51.4 ± 2.7% cytotoxicity, AptCYP24-#3 (SEQ ID NO: 3) had a 52.8 ± 14.2% cytotoxicity, AptCYP24-#5 (SEQ ID NO: 5) had a 47.9 ± 11.2% cytotoxicity, AptCYP24-#7 (SEQ ID NO: 7) had a 39.1 ± 2.8% cytotoxicity, AptCYP24-#11 (SEQ ID NO: 11) had a 64.2 ± 8.2% cytotoxicity, and AptCYP24-#12 (SEQ ID NO: 13) had a 64.2 ± 8.2% cytotoxicity. AptCYP24-#12) exhibited 42.1±5.7%, AptCYP24-#15 (SEQ ID NO: 15) 58.2±9.5%, AptCYP24-#16 (SEQ ID NO: 16) 78.8±4.8%, AptCYP24-#17 (SEQ ID NO: 17) 54.2±12.4%, and AptCYP24-#18 (SEQ ID NO: 18) 42.9±2.9% inhibitory activity, demonstrating that all CYP24A1-binding nucleic acid molecules of the present invention bind to CYP24A1 and exhibit CYP24A1 inhibitory activity. Among them, AptCYP24-#5 (SEQ ID NO: 5), AptCYP24-#7 (SEQ ID NO: 7), AptCYP24-#12 (SEQ ID NO: 12), and AptCYP24-#18 (SEQ ID NO: 18) were found to exhibit particularly high CYP24A1 inhibitory activity.

[0164] Next, the CYP24A1 inhibitory activity of four aptamers, AptCYP24-#5 (SEQ ID NO: 5), AptCYP24-#7 (SEQ ID NO: 7), AptCYP24-#12 (SEQ ID NO: 12), and AptCYP24-#18 (SEQ ID NO: 18), which had particularly high CYP24A1 inhibitory activity, was confirmed in the same manner except that the aptamer concentration was changed to 10 nmol / L, 50 nmol / L, 100 nmol / L, 125 nmol / L, 150 nmol / L, 175 nmol / L, and 200 nmol / L. Control 1 alone was used as a control.

[0165] The results are shown in Figure 6. Figure 6 is a graph showing relative CYP24A1 activity values, with (A) showing the results for AptCYP24-#7 (SEQ ID NO: 7), (B) showing the results for AptCYP24-#18 (SEQ ID NO: 18), (C) showing the results for AptCYP24-#12 (SEQ ID NO: 12), and (D) showing the results for AptCYP24-#5 (SEQ ID NO: 5). In Figure 6, the vertical axis shows the relative CYP24A1 activity value, with the CYP24A1 activity value of CYP24A1 alone (Control 1) taken as 100%, and the horizontal axis shows the concentration of each aptamer. As shown in Figure 6, AptCYP24-#5 (SEQ ID NO: 5), AptCYP24-#7 (SEQ ID NO: 7), AptCYP24-#12 (SEQ ID NO: 12), and AptCYP24-#18 (SEQ ID NO: 18) exhibited high CYP24A1 inhibitory activity at all concentrations. In particular, AptCYP24-#12 (SEQ ID NO: 12) and AptCYP24-#5 (SEQ ID NO: 5) exhibited high CYP24A1 inhibitory activity even at an extremely low concentration of 10 nmol / L. Furthermore, the concentration of the aptamer at which the relative activity of CYP24A1 was 50% (IC 50 The ATP values ​​were 123±13 nmol / L for AptCYP24-#7 (SEQ ID NO: 7), 72.7±37.7 nmol / L for AptCYP24-#18 (SEQ ID NO: 18), 62.8±12.2 nmol / L for AptCYP24-#12 (SEQ ID NO: 12), and 94.2±32.8 nmol / L for AptCYP24-#5 (SEQ ID NO: 5), indicating that all aptamers exhibit strong CYP24A1 inhibitory activity at extremely low concentrations.

[0166] [Reference example] It was confirmed that the nucleic acid molecules of the present invention do not inhibit the activity of enzymes other than CYP24A1.

[0167] The ability of each CYP24A1-binding nucleic acid molecule to inhibit CYP27B1 activity was evaluated with reference to Reference 6 below. Specifically, the same procedures as in Example 2 were carried out, except that a reaction solution (100 mmol / L Tris-HCl buffer (pH 7.5) containing 20 nmol / L CYP27B1, 2 μmol / L adrenodoxin, 0.2 μmol / L adrenodoxin reductase, 1 mmol / L EDTA, 5 μmol / L 25-dihydroxyvitamin D3 (substrate), and 1 mmol / L NADPH) was used instead of the reaction solution used in Example 2. Note that a control without the aptamer (CYP27B1 alone, No Aptamer) was used. Reference 6: Uchida et al., “Purification and characterization of mouse CYP27B1 overproduced by an Escherichia coli system coexpressing molecular chaperonins GroEL / ES”, BBRC 323, 505, 2004

[0168] The results are shown in Figure 7. Figure 7 is a graph showing relative CYP27B1 activity values. In Figure 7, the vertical axis shows the relative CYP27B1 activity value, with the CYP27B1 activity value of CYP27B1 alone (control) taken as 100%, and the horizontal axis shows the type of sample. As shown in Figure 7, none of the CYP24A1-binding nucleic acid molecules of the present invention inhibited CYP27B1 activity.

[0169] CYP27B1 is an enzyme that functions in vivo to metabolize 25(OH)D3, the body's stored form of vitamin D, into 1,25(OH)2D3. The CYP24A1-binding nucleic acid molecule of the present invention inhibits the function of CYP24A1 but not the function of CYP27B1, suggesting that when administered to a living body, it may be possible to increase the concentration of 1,25(OH)2D3, the active form of vitamin D, in the living body.

[0170] The ability of the CYP24A1-binding nucleic acid molecule of the present invention to inhibit CYP27A1 activity was confirmed in the same manner, except that CYP27A1, a P450 molecular species present in the same mitochondrial fraction as CYP27B1, was used instead of CYP27B1.The ability of the CYP24A1-binding nucleic acid molecule of the present invention to inhibit CYP27A1 activity was confirmed in the same manner, except that CYP11A1, a P450 molecular species present in the same mitochondrial fraction as CYP27B1, was used instead of CYP27B1, and the activity of CYP11A1 was measured with reference to Reference 7 below. Specifically, CYP11A1 activity was measured using a reaction mixture containing 20 mmol / L phosphate buffer, 0.3% Tween® 20, 0.2 nmol / L CYP11A1, 2 μmol / L ADX, 0.2 μmol / L ADR, 1 mmol / L NADPH, and 100 μmol / L cholesterol. The mixture was incubated at 37°C for 30 minutes and then incubated at 98°C for 10 minutes to terminate the reaction. Sodium cholate (final concentration: 1%) and cholesterol oxidase (final concentration: 8 units / ml) were then added to the reaction mixture, followed by incubation at 37°C for 20 minutes. Hexane was then added to extract metabolites, which were then analyzed by HPLC to measure CYP11A1 activity.

[0171] The results are shown in Figures 8(A) and (B). Figure 8(A) is a graph showing the relative activity of CYP27A1, and Figure 8(B) is a graph showing the relative activity of CYP11A1. In Figure 8(A), the vertical axis shows the relative activity of CYP27A1, where the activity of CYP27A1 alone (control, no aptamer) is taken as 100%, and the horizontal axis shows the type of sample. In Figure 8(B), the vertical axis shows the relative activity of CYP11A1, where the activity of CYP11A1 alone (control, no aptamer) is taken as 100%, and the horizontal axis shows the type of sample. As shown in Figures 8(A) and (B), it was found that the CYP24A1-binding nucleic acid molecules of the present invention do not inhibit the activity of CYP27A1 or CYP11A1.

[0172] These findings demonstrate that the CYP24A1-binding nucleic acid molecule of the present invention is capable of specifically inhibiting the activity of CYP24A1.

[0173] [Example 3] It was confirmed that the nucleic acid molecule of the present invention is taken up by cancer cells.

[0174] (1) Cellular uptake of CYP24A1-binding nucleic acid molecules Of the CYP24A1-binding nucleic acid molecules obtained in Example 1, AptCYP24-#7 (SEQ ID NO: 7) was labeled at its 5' end with Cy3 (registered trademark) to prepare a Cy3-labeled CYP24A1-binding nucleic acid molecule for observation under a fixed cell confocal microscope (product name: Zeiss LSM 700, Zeiss, Germany). Next, human alveolar basal epithelial adenocarcinoma cells (A549 cells, obtained from the JCRB Cell Bank, National Institutes of Biomedical Innovation, Health and Nutrition) were plated in a non-coated glass-bottom dish (9.5 mm x 4 wells) at a density of 1.5 x 10 4 The cells were seeded at 1000 nmol / L cells / whell. 24 hours after cell seeding, 500 nmol / L Cy3-labeled CYP24A1-binding nucleic acid molecules were added to the dish and incubated at 37°C for 2 hours. To remove excess fluorescent dye, the cells were washed three times with 1x PBS, and membrane staining was performed using a wheat germ agglutinin (WGA) conjugate (Biotium, USA) in 1x PBS at 37°C for 10 minutes. After staining, the cells were washed twice with 1x PBS, fixed with HCHO (4%), and intracellular aptamer signals were observed using a confocal microscope (Example 3-1). As a reference example, A549 cells were pretreated in DMEM with 100 μmol / L dynasore (Sigma-Aldrich, Japan), an endocytosis inhibitor, for 30 minutes. Similarly, aptamer treatment and membrane staining were performed, and the cells were observed using a confocal microscope (Reference Example 3).

[0175] The results are shown in Figure 9. Figure 9(A) is a photograph showing the results of Example 3-1, and Figure 9(B) is a photograph showing the results of Reference Example 3. In Figures 9(A) and (B), the photographs on the left are photographs of cells whose membranes were stained with WGA (WGA-membrane), the photographs in the middle show the fluorescence of the Cy3 label in the Cy3-labeled CYP24A1-binding nucleic acid molecule (Cy3-AptCYP24-#7), and the photographs on the right are merged photographs (merged view). In each of Figure 9, the stained areas are indicated by a border, and the areas where the staining of the Cy3-labeled CYP24A1-binding nucleic acid molecule (Cy3-AptCYP24-#7) is particularly localized are indicated by an open arrow. As shown in Figure 9(A), fluorescence indicating the Cy3-labeled CYP24A1-binding nucleic acid molecule was present in the cell membrane of A549 cells, confirming that the CYP24A1-binding nucleic acid molecule of the present invention was taken up into A549 cells. Furthermore, as shown in Figure 9(B) of Reference Example 3, fluorescence indicating the CYP24A1-binding nucleic acid molecule of the present invention was not observed in A549 cells pretreated with an endocytosis inhibitor, suggesting that the CYP24A1-binding nucleic acid molecule of the present invention was taken up into cells by endocytosis. However, the present invention is in no way limited to this assumption.

[0176] (2) Confirmation of the intracellular localization of CYP24A1-binding nucleic acid molecules Mitochondria are known to be the primary site in cells where CYP24A1 inactivates vitamin D metabolites, and therefore, the nucleic acid molecules of the present invention were confirmed to be co-localized in mitochondria.

[0177] First, A549 cells were incubated with the Cy3-labeled CYP24A1-binding nucleic acid molecule in the same manner as above, except that the Cy3-labeled CYP24A1-binding nucleic acid molecule was used at a concentration of 100 nmol / L or 400 nmol / L. To remove excess fluorescent dye, the cells were washed twice with 1x PBS and fixed with 4% paraformaldehyde for 15 minutes. The fixed cells were then treated with 100 nmol / L MitoGreen (PromoCell) and Hoechst® 33342 (ThermoFisher) diluted 1:2000 in PBS buffer for 30 minutes to stain the nuclei and mitochondria. The cells were then observed under the confocal microscope.

[0178] The results are shown in Figure 10. Figure 10 is a photograph showing the staining results, with Figure 10(A) showing the results when the CYP24A1-binding nucleic acid molecule was used at a concentration of 400 nmol / L, and Figure 10(B) showing the results when the CYP24A1-binding nucleic acid molecule was used at a concentration of 100 nmol / L. Figures 10(A) and (B) show, from left to right, the results of nuclear staining (Nucleic (Hoescht3342)), mitochondrial staining (Mitochondria (MitoGreen)), a Cy3 fluorescent signal (Cy3-AptCYP24-#7) showing the localization of the Cy3-labeled CYP24A1-binding nucleic acid molecule, and a merged photograph (Merge) of these. The photograph below the merged photograph is an enlarged view of the cells in the merged photograph. In addition, in Figure 10, the stained areas of the nucleus and mitochondria are indicated by a frame, and the area where the staining of the Cy3-labeled CYP24A1-binding nucleic acid molecule (Cy3-AptCYP24-#7) is particularly localized is indicated by an open arrow. As shown in Figure 10, in A549 cells, the CYP24A1-binding nucleic acid molecule was found to be localized in mitochondria. Therefore, it is presumed that the CYP24A1-binding nucleic acid molecule of the present invention co-localizes with CYP24A1 in mitochondria and interacts with CYP24A1 in cells. However, the present invention is in no way limited to this presumption.

[0179] [Example 4] It was confirmed that modification of the nucleic acid molecule of the present invention improves nuclease resistance.

[0180] To evaluate the stability of the nucleic acid molecules of the present invention in serum, AptCYP24-#7 (SEQ ID NO: 7), one of the CYP24A1-binding nucleic acid molecules obtained in Example 1, was modified with phosphorothioate to prepare a phosphorothioate-modified CYP24A1-binding nucleic acid molecule. 1 μmol / L of the phosphorothioate-modified CYP24A1-binding nucleic acid molecule was then incubated in PBS containing 10% fetal bovine serum (FBS) for 2, 4, 6, 24, 48, or 72 hours. The incubation temperature was 37°C. Similarly, an unmodified CYP24A1-binding nucleic acid molecule was also treated using 1 μmol / L of AptCYP24-#7 (SEQ ID NO: 7), which was not modified with phosphorothioate, except that the incubation time was 2, 4, 8, or 24 hours. After the predetermined time had elapsed, the remaining reaction product was collected, immediately heated at 95°C for 5 minutes, and then stored at -80°C. The remaining reaction mixture was then loaded onto a 2% agarose gel in 0.5x TBE buffer and run for 15 minutes at 120 V. The gel was then stained with SYBR™ Gold Nucleic Acid Gel Stain, photographed with a UV Transilluminator Imaging System, and analyzed with Imagej.

[0181] The results are shown in Figure 11. Figure 11(A) is a gel image showing the results for unmodified CYP24A1-binding nucleic acid molecules, and Figure 11(B) is a gel image showing the results for phosphorothioate-modified CYP24A1-binding nucleic acid molecules. In Figure 11, the numbers above each well indicate the incubation time, with 0 h representing the control (untreated) result. In Figure 11, the numbers below each well indicate the residual aptamer percentage (residual products (%)) when the control (0 h) is set to 100%. As shown in Figure 11(A), the unmodified CYP24A1-binding nucleic acid molecule remained undegraded even after 24 hours of incubation in serum. Furthermore, as shown in Figure 11(B), the residual percentage of the phosphorothioate-modified CYP24A1-binding nucleic acid molecule after 24 hours in serum was more than twice that of the unmodified CYP24A1-binding nucleic acid molecule, and remained undegraded even after 72 hours. Therefore, it was found that the CYP24A1-binding nucleic acid molecules of the present invention can be used in vivo, and that phosphorothioate-modified CYP24A1-binding nucleic acid molecules have improved nuclease resistance and are therefore more suitable for use in vivo.

[0182] [Example 5] It has been confirmed that the nucleic acid molecule of the present invention has the ability to inhibit endogenous CYP24A1.

[0183] First, 150 μl of DMEM containing 10% FBS was dispensed per well into a 48-well plate, and 3.38 × 10 3Cells were seeded at 1000 μl / well. Next, FBS-free DMEM containing 400 nmol / L of the CYP24A1-binding nucleic acid molecule (AptCYP24-#7 (SEQ ID NO: 7)) was added to the wells after 24 hours of culture to treat the A549 cells with the aptamer. Two hours later, 1 μmol / L of 1,25-dihydroxyvitamin D3 (hereinafter also referred to as "1,25-D3") was added to the wells. Eighteen hours after the addition of the CYP24A1-binding nucleic acid molecule, 150 μl of medium was removed, and a 3:1 chloroform / methanol solution was added and mixed. The supernatant was removed, and the organic phase was evaporated to dryness. The dried product was dissolved in acetonitrile, and CYP24A1 activity was analyzed by HPLC (Waters) (Example 5-1). Furthermore, A549 cells were treated in the same manner, except that a medium containing 400 nmol / L ketoconazole (Fujifilm Wako Pure Chemical Industries, Ltd.) was used instead of the medium containing the CYP24A1-binding nucleic acid molecule, and CYP24A1 activity was analyzed by HPLC (Comparative Example 5). To confirm the combined effect of CYP24A1-binding nucleic acid molecules and ketoconazole, A549 cells were treated in the same manner, except that a medium containing 100 nmol / L CYP24A1-binding nucleic acid molecules and 100 nmol / L ketoconazole was used instead of the medium containing 400 nmol / L CYP24A1-binding nucleic acid molecule, and CYP24A1 activity was analyzed by HPLC (Example 5-2). The controls were a fraction immediately after the addition of 1,25-D3 (Control 1) and a fraction 18 hours after the addition of 1,25-D3 without the addition of the CYP24A1-binding nucleic acid molecule (Control 2).

[0184] The HPLC results are shown in Figure 12. Figures 12(A) to 12(E) are graphs showing the HPLC results, where Figure 12(A) shows the results for Control 1 (1,25-D3 only (0 h)), Figure 12(B) shows the results for Control 2 (1,25-D3 only (18 h)), Figure 12(C) shows the results for Example 5-1 (1,25-D3 only (18 h) + AptCYP24-#7 (400 nM)), Figure 12(D) shows the results for Comparative Example 5 (1,25-D3 only (18 h) + Ketoconazole (400 nM)), and Figure 12(E) shows the results for Example 5-2 (1,25-D3 only (18 h) + Ketoconazole (400 nM) + AptCYP24-#7 (400 nM)). In each diagram in Figure 12, the peaks indicated by arrows are the major peaks of 1,25-D3 metabolites. As shown in Figure 12, the major peaks of 1,25-D3 metabolites were significantly reduced in Examples 5-1 and 5-2 compared to Controls 1 and 2. In other words, it was found that the CYP24A1-binding nucleic acid molecules of the present invention have the ability to inhibit the activity of endogenous CYP24A1 in A549 cells, and that this inhibitory ability is comparable to that of ketoconazole, a known CYP24A1 inhibitor.

[0185] Next, for Examples 5-1 and 5-2, the peak areas of the substrate 1α,25-dihydroxyvitamin D3 and the metabolite 1α,24,25-dihydroxyvitamin D3 formed after CYP24A1 acts on the substrate were calculated, and the CYP24A1 activity in the presence of each aptamer was calculated. The results are shown in Table 2 below. Table 2 below shows the relative CYP24A1 activity values, with the CYP24A1 activity value of the control (1,25-D3 only) set at 100%. As shown in Table 2 below, the CYP24A1-binding nucleic acid molecule of the present invention exhibited 41.5±3.6% of the endogenous CYP24A1 inhibitory activity, demonstrating significant inhibitory activity compared to the control. Furthermore, as shown in Example 5-2, when the CYP24A1 of the present invention was used in combination with ketoconazole, it exhibited an endogenous CYP24A1 inhibitory activity of 22.1±4.5%, demonstrating that the nucleic acid molecule of the present invention exhibits a synergistic effect when used in combination with a known CYP24A1 inhibitor. [Table 2]

[0186] It has been reported that CYP24A1 is abnormally expressed in human lung cancer cells, and that high expression of CYP24A1 suppresses antiproliferative 1,25-D3 signaling in lung cancer cells, including A549 cells. Because the CYP24A1-binding nucleic acid molecule of the present invention has excellent inhibitory activity against endogenous CYP24A1, it was suggested that the CYP24A1-binding nucleic acid molecule of the present invention could be used as a therapeutic agent for lung cancer.

[0187] [Example 6] It was confirmed that the nucleic acid molecule of the present invention suppresses the proliferation of cancer cells.

[0188] 3 x 10 A549 cells in a 96 well plate 3Cells were seeded at a density of 100 cells / well. After 24 hours of culture, A549 cells were treated with an aptamer by adding FBS-free DMEM containing 500 nmol / L of the CYP24A1-binding nucleic acid molecule of the present invention (AptCYP24-#7 (SEQ ID NO: 7)). Two hours later, 100 nmol / L of 1,25-dihydroxyvitamin D3 (hereinafter also referred to as "1,25-D3") was added. Furthermore, the medium containing 500 nmol / L of the CYP24A1-binding nucleic acid molecule (AptCYP24-#7 (SEQ ID NO: 7)) was replaced every 24 hours after the aptamer treatment, allowing for continuous aptamer treatment. After 72 hours of culture, cell viability was assessed using Cell Counting Kit-8 (CK-8 kit, Dojindo Laboratories, Inc.) (Example 6). Absorbance was measured at 450 nm using a microplate reader (Spark™ 10 M, TECAN). As a control, treatment was carried out in the same manner except that a medium not containing the binding nucleic acid molecule of the present invention was used (cells only, control 6). As comparative examples, treatment was carried out in the same manner except that a medium containing the initial random sequence (SEQ ID NO: 23) of Example 1(1) (Comparative Example 6-1) or a medium containing 400 nmol / L ketoconazole (Comparative Example 6-2) was used instead of the medium containing the binding nucleic acid molecule of the present invention. All values ​​were normalized to the control (cells only).

[0189] The results are shown in Figure 13. Figure 13 is a graph showing the viability of A549 cells after culture. The vertical axis indicates the relative absorbance, with the absorbance of Control 6 (Vehicle) set at 1, and the horizontal axis indicates the type of sample. In Figure 13, the value for each sample is the mean ± SD of triplicate measurements, and ** indicates P<0.01. As shown in Figure 13, Example 6 (1,25-D3 + Apt-7), which contained the binding nucleic acid molecule of the present invention, significantly inhibited cancer cell proliferation compared to Comparative Example 6-1 (1,25-D3 only) and also inhibited cancer cell proliferation to an equal or greater extent than Comparative Example 6-2 (1,25-D3 + KCZ). This demonstrates that the CYP24A1-binding nucleic acid molecule of the present invention can inhibit cancer cell proliferation and can be used as a cancer therapeutic agent.

[0190] Although the present invention has been described above with reference to the embodiments and examples, the present invention is not limited to the above embodiments and examples. Various modifications that can be understood by those skilled in the art can be made to the configuration and details of the present invention within the scope of the present invention.

[0191] This application claims priority based on Japanese Patent Application No. 2021-082904, filed on May 17, 2021, the disclosure of which is incorporated herein in its entirety.

[0192] <Additional Notes> Some or all of the above-described embodiments and examples can be described as, but are not limited to, the following supplementary notes. (Appendix 1) A CYP24A1-binding nucleic acid molecule comprising a DNA strand of the following (a): (a) a DNA strand of the following (a1), (a2), or (a3): (a1) a DNA strand consisting of any one of the nucleotide sequences of SEQ ID NOs: 1 to 18; (a2) a DNA strand that binds to CYP24A1, comprising a base sequence in which one or several bases are deleted, substituted, inserted, and / or added in any of the base sequences of (a1); (a3) A DNA strand that consists of a base sequence having 80% or more identity to any of the base sequences of (a1), and that binds to CYP24A1. (Appendix 2) 10. The nucleic acid molecule of claim 1, further comprising a carrier. (Appendix 3) 3. The nucleic acid molecule of claim 1, wherein the carrier is at least one selected from the group consisting of a micelle, a liposome, an emulsion, and a niosome. (Appendix 4) An agent for inhibiting CYP24A1 activity, comprising a CYP24A1-binding nucleic acid molecule described in any one of Appendices 1 to 3. (Appendix 5) A pharmaceutical comprising a CYP24A1-binding nucleic acid molecule according to any one of claims 1 to 3 and a pharmaceutically acceptable excipient. (Appendix 6) A therapeutic agent for lung cancer, comprising a CYP24A1-binding nucleic acid molecule described in any one of Appendices 1 to 3. (Appendix 7) Further, a therapeutic agent for lung cancer as described in Appendix 6, which contains 1,25-dihydroxyvitamin D3. (Appendix 8) A therapeutic agent for treating prostate cancer, comprising a CYP24A1-binding nucleic acid molecule described in any one of Appendices 1 to 3. (Appendix 9) A therapeutic agent for chronic kidney disease, comprising a CYP24A1-binding nucleic acid molecule described in any one of Appendices 1 to 3. (Appendix 10) A therapeutic agent for rickets, comprising a CYP24A1-binding nucleic acid molecule described in any one of Appendices 1 to 3. (Appendix 11) A therapeutic agent for osteomalacia, comprising a CYP24A1-binding nucleic acid molecule described in any one of Appendices 1 to 3. (Appendix 12) A reagent for detecting CYP24A1, comprising a CYP24A1-binding nucleic acid molecule described in any one of appendices 1 to 3. (Appendix 13) contacting a sample with a nucleic acid molecule and detecting CYP24A1 in the sample; the nucleic acid molecule is a CYP24A1-binding nucleic acid molecule according to any one of appendices 1 to 3; A method for detecting CYP24A1, wherein in the detection step, CYP24A1 in the sample is allowed to bind to the nucleic acid molecule, and CYP24A1 in the sample is detected based on the binding. [Industrial Applicability]

[0193] The CYP24A1-binding nucleic acid molecule of the present invention is capable of binding to CYP24A1. The CYP24A1-binding nucleic acid molecule of the present invention can regulate the activity of CYP24A1. CYP24A1 also functions in the inactivation of vitamin D. Therefore, the CYP24A1-binding nucleic acid molecule of the present invention is expected to function as a new pharmaceutical for diseases caused by vitamin D deficiency. Furthermore, the CYP24A1-binding nucleic acid molecule of the present invention can be used to detect CYP24A1. Therefore, the CYP24A1-binding nucleic acid molecule of the present invention can be said to be an extremely useful tool, for example, in the pharmaceutical field and for detecting CYP24A1.

Claims

1. A CYP24A1-binding nucleic acid molecule comprising a DNA strand of the following (a): (a) a DNA strand of the following (a1), (a2), or (a3): (a1) a DNA strand consisting of a nucleotide sequence in which, from the 5'-end, a nucleotide sequence consisting of SEQ ID NO: 21, a nucleotide sequence consisting of any one of SEQ ID NOs: 1 to 18, and a nucleotide sequence consisting of SEQ ID NO: 22 are arranged consecutively; (a2) A DNA strand that binds to CYP24A1, wherein the DNA strand is a base sequence in which 1 to 6 bases are deleted, substituted, inserted, and / or added in any of the base sequences of (a1); (a3) A DNA strand that consists of a base sequence having 90% or more identity to any of the base sequences of (a1), and that binds to CYP24A1.

2. The nucleic acid molecule of claim 1 , further comprising a carrier.

3. The nucleic acid molecule according to claim 1 or 2, wherein the carrier is at least one selected from the group consisting of a micelle, a liposome, an emulsion, and a niosome.

4. An agent for inhibiting CYP24A1 activity, comprising a CYP24A1-binding nucleic acid molecule of the following (a): (a) a DNA strand of the following (a1), (a2), or (a3): (a1) a DNA strand consisting of a base sequence in which, from the 5'-end, a base sequence consisting of SEQ ID NO: 21, a base sequence consisting of any one of SEQ ID NOs: 1 to 3, 5, 7, 11, 12, and 15 to 18, and a base sequence consisting of SEQ ID NO: 22 are consecutively arranged; (a2) A DNA strand that binds to CYP24A1, wherein the DNA strand is a base sequence in which 1 to 6 bases are deleted, substituted, inserted, and / or added in any of the base sequences of (a1); (a3) A DNA strand that consists of a base sequence having 90% or more identity to any of the base sequences of (a1), and that binds to CYP24A1.

5. A pharmaceutical comprising the following CYP24A1-binding nucleic acid molecule (a) and a pharmaceutically acceptable excipient: (a) a DNA strand of the following (a1), (a2), or (a3): (a1) a DNA strand consisting of a base sequence in which, from the 5'-end, a base sequence consisting of SEQ ID NO: 21, a base sequence consisting of any one of SEQ ID NOs: 1 to 3, 5, 7, 11, 12, and 15 to 18, and a base sequence consisting of SEQ ID NO: 22 are consecutively arranged; (a2) A DNA strand that binds to CYP24A1, wherein the DNA strand is a base sequence in which 1 to 6 bases are deleted, substituted, inserted, and / or added in any of the base sequences of (a1); (a3) A DNA strand that consists of a base sequence having 90% or more identity to any of the base sequences of (a1), and that binds to CYP24A1.

6. A therapeutic agent for lung cancer, comprising a CYP24A1-binding nucleic acid molecule represented by the following formula (a): (a) a DNA strand of the following (a1), (a2), or (a3): (a1) a DNA strand consisting of a base sequence in which, from the 5'-end, a base sequence consisting of SEQ ID NO: 21, a base sequence consisting of any one of SEQ ID NOs: 1 to 3, 5, 7, 11, 12, and 15 to 18, and a base sequence consisting of SEQ ID NO: 22 are consecutively arranged; (a2) A DNA strand that binds to CYP24A1, wherein the DNA strand is a base sequence in which 1 to 6 bases are deleted, substituted, inserted, and / or added in any of the base sequences of (a1); (a3) A DNA strand that consists of a base sequence having 90% or more identity to any of the base sequences of (a1), and that binds to CYP24A1.

7. The lung cancer therapeutic agent according to claim 6, further comprising 1,25-dihydroxyvitamin D3.

8. A therapeutic agent for prostate cancer, comprising a CYP24A1-binding nucleic acid molecule represented by the following formula (a): (a) a DNA strand of the following (a1), (a2), or (a3): (a1) a DNA strand consisting of a base sequence in which, from the 5'-end, a base sequence consisting of SEQ ID NO: 21, a base sequence consisting of any one of SEQ ID NOs: 1 to 3, 5, 7, 11, 12, and 15 to 18, and a base sequence consisting of SEQ ID NO: 22 are consecutively arranged; (a2) A DNA strand that binds to CYP24A1, wherein the DNA strand is a base sequence in which 1 to 6 bases are deleted, substituted, inserted, and / or added in any of the base sequences of (a1); (a3) A DNA strand that consists of a base sequence having 90% or more identity to any of the base sequences of (a1), and that binds to CYP24A1.

9. A therapeutic agent for chronic kidney disease, comprising a CYP24A1-binding nucleic acid molecule represented by the following formula (a): (a) a DNA strand of the following (a1), (a2), or (a3): (a1) a DNA strand consisting of a base sequence in which, from the 5'-end, a base sequence consisting of SEQ ID NO: 21, a base sequence consisting of any one of SEQ ID NOs: 1 to 3, 5, 7, 11, 12, and 15 to 18, and a base sequence consisting of SEQ ID NO: 22 are consecutively arranged; (a2) A DNA strand that binds to CYP24A1, wherein the DNA strand is a base sequence in which 1 to 6 bases are deleted, substituted, inserted, and / or added in any of the base sequences of (a1); (a3) A DNA strand that consists of a base sequence having 90% or more identity to any of the base sequences of (a1), and that binds to CYP24A1.

10. A therapeutic agent for rickets, comprising a CYP24A1-binding nucleic acid molecule represented by the following formula (a): (a) a DNA strand of the following (a1), (a2), or (a3): (a1) a DNA strand consisting of a base sequence in which, from the 5'-end, a base sequence consisting of SEQ ID NO: 21, a base sequence consisting of any one of SEQ ID NOs: 1 to 3, 5, 7, 11, 12, and 15 to 18, and a base sequence consisting of SEQ ID NO: 22 are consecutively arranged; (a2) A DNA strand that binds to CYP24A1, wherein the DNA strand is a base sequence in which 1 to 6 bases are deleted, substituted, inserted, and / or added in any of the base sequences of (a1); (a3) A DNA strand that consists of a base sequence having 90% or more identity to any of the base sequences of (a1), and that binds to CYP24A1.

11. A therapeutic agent for osteomalacia, comprising a CYP24A1-binding nucleic acid molecule represented by the following formula (a): (a) a DNA strand of the following (a1), (a2), or (a3): (a1) a DNA strand consisting of a base sequence in which, from the 5'-end, a base sequence consisting of SEQ ID NO: 21, a base sequence consisting of any one of SEQ ID NOs: 1 to 3, 5, 7, 11, 12, and 15 to 18, and a base sequence consisting of SEQ ID NO: 22 are consecutively arranged; (a2) A DNA strand that binds to CYP24A1, wherein the DNA strand is a base sequence in which 1 to 6 bases are deleted, substituted, inserted, and / or added in any of the base sequences of (a1); (a3) A DNA strand that consists of a base sequence having 90% or more identity to any of the base sequences of (a1), and that binds to CYP24A1.

12. A reagent for detecting CYP24A1, comprising the CYP24A1-binding nucleic acid molecule of any one of claims 1 to 3.

13. contacting a sample with a nucleic acid molecule and detecting CYP24A1 in the sample; The nucleic acid molecule is a CYP24A1-binding nucleic acid molecule according to any one of claims 1 to 3, A method for detecting CYP24A1, wherein in the detection step, CYP24A1 in the sample is allowed to bind to the nucleic acid molecule, and CYP24A1 in the sample is detected through the binding.

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