Quinine binding by the cocaine-binding aptamer. Thermodynamic and hydrodynamic analysis of high-affinity binding of an off-target ligand

Biochemistry. 2013 Dec 3;52(48):8652-62. doi: 10.1021/bi4010039. Epub 2013 Nov 14.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aptamers, Nucleotide / chemistry
  • Aptamers, Nucleotide / metabolism*
  • Base Sequence
  • Binding Sites
  • Binding, Competitive
  • Cocaine / chemistry
  • Cocaine / metabolism*
  • Hydrodynamics
  • Ligands
  • Molecular Sequence Data
  • Nucleic Acid Conformation
  • Osmolar Concentration
  • Quinine / chemistry
  • Quinine / metabolism*
  • Substrate Specificity
  • Thermodynamics

Substances

  • Aptamers, Nucleotide
  • Ligands
  • Quinine
  • Cocaine