Single-molecule investigation and nanopore-integrated biochip

Abstract

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT REQUEST OF AUTHOR.] Chlorella-encoded membrane protein Kcv was synthesized and purified in vitro. By co-synthesis and assembly of wild type with tagged version of Kcv, tetrameric stoichiometry was verified. The functionalities of purified wild type homo-tetramer single-channel of Kcv were investigated in artificial lipid bilayer, and the single-channel activities were consistent with the heterologously expressed Kcv. Thrombin-binding aptamer (TBA) folds into the G-quadruplex in the presence of cation. Trapping the TBA in the nanocavity of the alpha-hemolysin resulted in electrical characteristic changes to the pore conductance that revealed important molecular processes including spontaneous unfolding of the quartet structure and translocation of unfolded TBA through the pore. This guest-nanocavity supramolecule was used to detect regulation by cations of the folding/unfolding kinetics of the G-quadruplex formed by TBA. A portable, durable, and single protein channel-integrated chip device was developed for investigation of ion channel activities and single-molecule bio-sensing over long observation times.