Development of a thermal-stable structure-switching cocaine-binding aptamer

We have developed a new cocaine-binding aptamer variant that has a significantly higher melt tem- perature when bound to a ligand than the currently used sequence. Retained in this new construct is the ligand-induced structure-switching binding mechanism that is important in biosensing applications of the cocaine-binding aptamer. Isothermal titration calorimetry methods show that the binding affinity of this new sequence is slightly tighter than the existing cocaine-binding aptamer. The improved thermal performance, a Tm increase of 4 C for the cocaine-bound aptamer and 9 C for the quinine-bound aptamer, was achieved by optimizing the DNA sequence in stem 2 of the aptamer to have the highest stability based on the nearest neighbor thermodynamic parameters and confirmed by UV and fluores- cence spectroscopy. The sequences in stem 1 and stem 3 were unchanged in order to retain the structure switching and ligand binding functions. The more favorable thermal stability characteristics of the OR3 aptamer should make it a useful construct for sensing applications employing the cocaine-binding aptamer system.