title: Monitoring of an ATP-binding aptamer and its conformational changes using an α-hemolysin nanopore.
abstract: An aptamer is a specific oligonucleotide sequence that spontaneously forms a secondary structure capable of selectively binding an analyte. An aptamer's conformation is the key to specific binding of a target molecule, even in the case of very closely related targets. Nanopores are a sensitive tool for the single-molecule analysis of DNA, peptides, and proteins transporting through the pore. Herein, a single α-hemolysin natural nanopore is utilized to sense the conformational changes of an adenosine 5'-triphosphate (ATP)-binding aptamer (ABA). The known DNA sequence of the ABA is used as a model to develop real-time monitoring of molecular conformational changes that occur by binding targets. The native, folded ABA structure has a nanopore unfolding time of 4.17 ms, compared with 0.29 ms for the ABA:ATP complex. A complementary 14-mer strand, which binds the ABA sequence in the key nucleic acids responsible for folding, forms linear duplex DNA, resulting in a nanopore transit time of 0.50 ms and a higher capture probability than that of the folded ABA oligomer. Competition assays between the ABA:ATP and ABA:reporter complexes are carried out, and the results suggest that the ABA:ATP complex is formed preferentially. The nanopore allows for the detection of an ABA in its folded, ATP-bound, and linear conformations.
authors.affiliation: Key Laboratory for Advanced Materials & Department of Chemistry, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, P. R. China.
authors.forename: Yi-Lun
authors.initials: YL
authors.lastname: Ying
authors.forename: Hai-Yan
authors.initials: HY
authors.lastname: Wang
authors.forename: Todd C
authors.initials: TC
authors.lastname: Sutherland
authors.forename: Yi-Tao
authors.initials: YT
authors.lastname: Long
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry*; D052157:Aptamers, Nucleotide / Q000737:chemistry*; D015374:Biosensing Techniques; D006460:Hemolysin Proteins / Q000737:chemistry*; D058608:Nanopores; D036103:Nanotechnology / Q000379:methods*; D011487:Protein Conformation
chemical_list: D052157:Aptamers, Nucleotide; D006460:Hemolysin Proteins; D000255:Adenosine Triphosphate