Method for preparing nucleic acid aptamer

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.

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.   
     
     
         2 . 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. 
     
     
         3 . 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. 
     
     
         4 . 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. 
     
     
         5 . 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. 
     
     
         6 . The production method according to  claim 1 , wherein the nucleic acid is a DNA. 
     
     
         7 . The production method according to  claim 1 , wherein the target substance is a peptide. 
     
     
         8 . The production method according to  claim 1 , wherein the connectors are biotin and avidin, streptavidin, or NeutrAvidin. 
     
     
         9 . The production method according to  claim 1 , wherein the solid-phase support is hydrophilic. 
     
     
         10 . 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. 
     
     
         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.   
     
     
         12 . 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. 
     
     
         13 . The nucleic acid molecule according to  claim 12 , wherein the nucleic acid molecule is a DNA. 
     
     
         14 . The nucleic acid molecule according to  claim 11 , wherein the target substance is a peptide. 
     
     
         15 . 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. 
     
     
         16 . The nucleic acid molecule according to  claim 15 , wherein the transcriptional regulator is NF-κB. 
     
     
         17 . 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. 
     
     
         18 . 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. 
     
     
         19 . 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. 
     
     
         20 . An inhibitor of target substance function comprising a nucleic acid molecule according to  claim 11  as an active ingredient. 
     
     
         21 . A pharmaceutical composition comprising an inhibitor of target substance function according to  claim 20 . 
     
     
         22 . 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. 
     
     
         23 . A method comprising detecting NF-κB p50 in a sample using a nucleic acid molecule according to  claim 16 . 
     
     
         24 . The method according to  claim 22 , wherein the detection is performed using surface plasmon resonance assay, quartz crystal microbalance assay, turbidimetry, colorimetry, or fluorometry. 
     
     
         25 . A kit for NF-κB p50 detection comprising at least one nucleic acid molecule according to  claim 16 .