title: Quinine binding by the cocaine-binding aptamer. Thermodynamic and hydrodynamic analysis of high-affinity binding of an off-target ligand.
abstract: The cocaine-binding aptamer is unusual in that it tightly binds molecules other than the ligand it was selected for. Here, we study the interaction of the cocaine-binding aptamer with one of these off-target ligands, quinine. Isothermal titration calorimetry was used to quantify the quinine-binding affinity and thermodynamics of a set of sequence variants of the cocaine-binding aptamer. We find that the affinity of the cocaine-binding aptamer for quinine is 30-40 times stronger than it is for cocaine. Competitive-binding studies demonstrate that both quinine and cocaine bind at the same site on the aptamer. The ligand-induced structural-switching binding mechanism of an aptamer variant that contains three base pairs in stem 1 is retained with quinine as a ligand. The short stem 1 aptamer is unfolded or loosely folded in the free form and becomes folded when bound to quinine. This folding is confirmed by NMR spectroscopy and by the short stem 1 construct having a more negative change in heat capacity of quinine binding than is seen when stem 1 has six base pairs. Small-angle X-ray scattering (SAXS) studies of the free aptamer and both the quinine- and the cocaine-bound forms show that, for the long stem 1 aptamers, the three forms display similar hydrodynamic properties, and the ab initio shape reconstruction structures are very similar. For the short stem 1 aptamer there is a greater variation among the SAXS-derived ab initio shape reconstruction structures, consistent with the changes expected with its structural-switching binding mechanism.
authors.affiliation: Department of Chemistry, York University , 4700 Keele Street, Toronto, Ontario, Canada M3J 1P3.
authors.forename: Oren
authors.initials: O
authors.lastname: Reinstein
authors.forename: Mina
authors.initials: M
authors.lastname: Yoo
authors.forename: Chris
authors.initials: C
authors.lastname: Han
authors.forename: Tsering
authors.initials: T
authors.lastname: Palmo
authors.forename: Simone A
authors.initials: SA
authors.lastname: Beckham
authors.forename: Matthew C J
authors.initials: MC
authors.lastname: Wilce
authors.forename: Philip E
authors.initials: PE
authors.lastname: Johnson
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D003042:Cocaine / Q000737:chemistry / Q000378:metabolism*; D057446:Hydrodynamics; D008024:Ligands; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009994:Osmolar Concentration; D011803:Quinine / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D052157:Aptamers, Nucleotide; D008024:Ligands; D011803:Quinine; D003042:Cocaine