title: Nanopore force spectroscopy of aptamer-ligand complexes.
abstract: The stability of aptamer-ligand complexes is probed in nanopore-based dynamic force spectroscopy experiments. Specifically, the ATP-binding aptamer is investigated using a backward translocation technique, in which the molecules are initially pulled through an α-hemolysin nanopore from the cis to the trans side of a lipid bilayer membrane, allowed to refold and interact with their target, and then translocated back in the trans-cis direction. From these experiments, the distribution of bound and unbound complexes is determined, which in turn allows determination of the dissociation constant Kd ≈ 0.1 mM of the aptamer and of voltage-dependent unfolding rates. The experiments also reveal differences in binding of the aptamer to AMP, ADP, or ATP ligands. Investigation of an aptamer variant with a stabilized ATP-binding site indicates fast conformational switching of the original aptamer before ATP binding. Nanopore force spectroscopy is also used to study binding of the thrombin-binding aptamer to its target. To detect aptamer-target interactions in this case, the stability of the ligand-free aptamer-containing G-quadruplexes-is tuned via the potassium content of the buffer. Although the presence of thrombin was detected, limitations of the method for aptamers with strong secondary structures and complexes with nanomolar Kd were identified.
authors.affiliation: Lehrstuhl für Bioelektronik, Physik Department, Technische Universität München, Garching, Germany.
authors.forename: Vera
authors.initials: V
authors.lastname: Arnaut
authors.forename: Martin
authors.initials: M
authors.lastname: Langecker
authors.forename: Friedrich C
authors.initials: FC
authors.lastname: Simmel
mesh_terms: D000227:Adenine Nucleotides / Q000378:metabolism; D052157:Aptamers, Nucleotide / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002021:Buffers; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D006460:Hemolysin Proteins / Q000737:chemistry / Q000378:metabolism; D008024:Ligands; D058608:Nanopores; D013057:Spectrum Analysis; D013917:Thrombin / Q000378:metabolism
chemical_list: D000227:Adenine Nucleotides; D052157:Aptamers, Nucleotide; D002021:Buffers; D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins; C036507:Hlya protein, E coli; D008024:Ligands; D013917:Thrombin