# US 2014/0194320 A1 · Method for preparing nucleic acid aptamer > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2014/0194320 A1 - Document ID: US20140194320A1 - Status: Published application - Kind: A1 - Application number: 14/237,645 - Filed: 2012-08-08 - Published: 2014-07-10 - Earliest claimed: 2011-08-12 - Assignees: Tagcyx Bitoecnologies; Riken - Inventors: Ichiro Hirao; Michiko Hirao; Shigeyuki Yokoyama - Web page: https://patentgrep.com/patent/US20140194320A1 ## CPC - G01N 33/543: with an insoluble carrier for immobilising immunochemicals - A61K 31/7105: Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links - A61P 37/02: Immunomodulators - A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00 - C12N 15/1048: {SELEX} - C12N 15/115: Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith {; Nucleic acids binding to non-nucleic acids, e.g. aptamers} - C12Q 1/6834: Enzymatic or biochemical coupling of nucleic acids to a solid phase - G01N 33/5308: {for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites} - C12N 2310/16: Aptamers - C12N 2320/13: in a process of directed evolution, e.g. SELEX, acquiring a new function - C12N 2320/30: Special therapeutic applications - C12N 2330/30: chemically synthesised - G01N 2333/47: Assays involving proteins of known structure or function as defined in the subgroups - G01N 2333/4703: Regulators; Modulating activity - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES ## Abstract An object of the present invention is to develop and provide a method for efficiently and conveniently producing a nucleic acid aptamer, particularly, a DNA aptamer, having high specificity for and high binding activity against a target substance. The present invention provides a method for producing a nucleic acid aptamer, comprising: a complex formation step of mixing a single-stranded nucleic acid library with a target substance in a solution to form a complex of a single-stranded nucleic acid and the target substance; an immobilization step of mixing the solution after the preceding step with a solid-phase support to immobilize the complex onto the solid-phase support via connector(s) adsorbed on the target substance and/or the solid-phase support; a recovery step of recovering the complex immobilized on the solid-phase support from the solution; an amplification step of recovering the single-stranded nucleic acid from the complex, followed by amplification by a nucleic acid amplification method; and a single-stranded nucleic acid preparation step of converting the double-stranded nucleic acids obtained in the amplification step into single strands and then forming an intramolecular conformation. ## Claims ### Claim 1 A method for producing a nucleic acid aptamer, comprising: a complex formation step of mixing a single-stranded nucleic acid library with a target substance in a solution to form a complex of a single-stranded nucleic acid and the target substance; an immobilization step of mixing the solution after the complex formation step with a solid-phase support to immobilize the complex onto the solid-phase support via connector(s) adsorbed on the target substance and/or the solid-phase support; a recovery step of recovering the complex immobilized on the solid-phase support from the solution; an amplification step of recovering the single-stranded nucleic acid from the complex and then amplifying the single-stranded nucleic acid by a nucleic acid amplification method; and a single-stranded nucleic acid preparation step of converting the double-stranded nucleic acids obtained in the amplification step into single strands and then forming an intramolecular conformation. ### Claim 2 (depends on 1) The production method according to claim 1, further comprising a repetitive step of repeating several times the round from the complex formation step to the single-stranded nucleic acid preparation step using the single-stranded nucleic acids obtained in the single-stranded nucleic acid preparation step as a new single-stranded nucleic acid library. ### Claim 3 (depends on 2) The production method according to claim 2, wherein the repetitive step involves repeating 2 to 15 times the round from the complex formation step to the single-stranded nucleic acid preparation step. ### Claim 4 (depends on 2) The production method according to claim 2, further comprising a selection step of selecting a single-stranded nucleic acid molecule from among the single-stranded nucleic acids obtained after the repetitive step, wherein the single-stranded nucleic acid molecule comprises in its secondary structure one or more double-stranded regions each consisting of a pair of consecutive 5 to 20 bases base-paired each other and at least one of the double-stranded regions comprises 1 to 10 base pairs consisting of non-Watson-Crick base pairs. ### Claim 5 (depends on 1) The production method according to claim 1, wherein in the complex formation step, the solution comprises a competitive substance that competes with the single-stranded nucleic acid for binding with the target substance. ### Claim 6 (depends on 1) The production method according to claim 1, wherein the nucleic acid is a DNA. ### Claim 7 (depends on 1) The production method according to claim 1, wherein the target substance is a peptide. ### Claim 8 (depends on 1) The production method according to claim 1, wherein the connectors are biotin and avidin, streptavidin, or NeutrAvidin. ### Claim 9 (depends on 1) The production method according to claim 1, wherein the solid-phase support is hydrophilic. ### Claim 10 (depends on 1) The production method according to claim 1, wherein the solution or a buffer used in the complex formation step and/or the recovery step comprises a surfactant. ### Claim 11 A nucleic acid molecule binding to a target substance, wherein the nucleic acid molecule comprises one or more double-stranded regions each consisting of a pair of consecutive 5 to 20 bases base-paired each other, and at least one of the double-stranded regions comprises 1 to 10 base pairs consisting of non-Watson-Crick base pairs. ### Claim 12 (depends on 11) The nucleic acid molecule according to claim 11, wherein the nucleic acid molecule consists of a single-stranded nucleic acid or a double-stranded nucleic acid. ### Claim 13 (depends on 12) The nucleic acid molecule according to claim 12, wherein the nucleic acid molecule is a DNA. ### Claim 14 (depends on 11) The nucleic acid molecule according to claim 11, wherein the target substance is a peptide. ### Claim 15 (depends on 14) The nucleic acid molecule according to claim 14, wherein the peptide is a transcriptional regulator, a signaling factor, a protein ligand, or a receptor protein. ### Claim 16 (depends on 15) The nucleic acid molecule according to claim 15, wherein the transcriptional regulator is NF-κB. ### Claim 17 (depends on 16) The nucleic acid molecule according to claim 16, wherein the NF-κB is p50, and the nucleic acid molecule comprises a double-stranded region consisting of the nucleotide sequences represented by SEQ ID NOs: 1 and 2. ### Claim 18 (depends on 17) The nucleic acid molecule according to claim 17, wherein the nucleic acid molecule comprises a double-stranded region consisting of the nucleotide sequences represented by SEQ ID NOs: 3 and 4, SEQ ID NOs: 5 and 6, or SEQ ID NOs: 7 and 8. ### Claim 19 (depends on 18) The nucleic acid molecule according to claim 18, wherein the nucleic acid molecule comprises the nucleotide sequence represented by any of SEQ ID NOs: 9 to 21. ### Claim 20 (depends on 11) An inhibitor of target substance function comprising a nucleic acid molecule according to claim 11 as an active ingredient. ### Claim 21 (depends on 20) A pharmaceutical composition comprising an inhibitor of target substance function according to claim 20. ### Claim 22 (depends on 11) A method comprising using a nucleic acid molecule according to claim 11 to detect a target substance to which the nucleic acid molecule binds, in a sample. ### Claim 23 (depends on 16) A method comprising detecting NF-κB p50 in a sample using a nucleic acid molecule according to claim 16. ### Claim 24 (depends on 22) The method according to claim 22, wherein the detection is performed using surface plasmon resonance assay, quartz crystal microbalance assay, turbidimetry, colorimetry, or fluorometry. ### Claim 25 (depends on 16) A kit for NF-κB p50 detection comprising at least one nucleic acid molecule according to claim 16.