Cooperative binding by a bifunctional deoxycholic acid and cocaine-binding aptamer

A bifunctional cocaine and deoxycholic acid-binding aptamer was constructed from the individual ligand binding aptamers and the binding affinity and thermodynamics were measured using isothermal titration calorimetry. We show that the bifunctional aptamer binds its ligands with positive cooperativity, having a Hill coefficient of 1.2-1.5, whether the ligands are added individually or as an equimolar mixture. When the helix between the binding sites is decreased from eight to six base pairs the Hill coefficient increased indicating a greater degree of cooperativity. Reducing the distance between the binding sites to four base pairs resulted in the bifunctional aptamer displaying negative cooperativity as the two sites are now too close to function efficiently. A mechanism where molecular motion, or dynamics, at one ligand-binding site are affected by the presence of the ligand at the other is proposed to account for the observed cooperative binding.