Nanopore Biosensors: Monitoring of an ATP‐Binding Aptamer and its Conformational Changes Using an α‐Hemolysin Nanopore (Small 1/2011)

Abstract

AbstractAn 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.