Comparison of the free and ligand-bound imino hydrogen exchange rates for the cocaine-binding aptamer

Abstract

Using NMR magnetization transfer experiments, the hydrogen exchange rate constants (k _ex ) of the DNA imino protons in the cocaine-binding aptamer have been determined for the free, cocaine-bound, and quinine-bound states. The secondary structure of the cocaine-binding aptamer is composed of three stems built around a three-way junction. In the free aptamer the slowest exchanging imino protons are located in the middle of the stems. The highest k _ex values were found for a nucleotide in the GAA loop of stem 3 and for nucleotides at the end of the stems that form the three-way junction structure and in the tandem GA mismatch. Upon ligand binding, the k _ex values of nucleotides at the ligand binding site are reduced, indicating that these base pairs become more stable or less solvent accessible in the bound state. The imino proton k _ex values of nucleotides located away from the binding site are only minimally affected by ligand binding.

Keywords: Aptamer function; DNA-small molecule interactions; Hydrogen exchange rate constant; Ligand binding.