Shoara, Aron AReinstein, OrenBorhani, Okty AbbasiMartin, Taylor RSlavkovic, SladjanaChurcher, Zachary RJohnson, Philip E2023-05-102023-05-102017-08-21Biochimie 145 (2018): 137-144https://doi.org/10.1016/j.biochi.2017.08.010http://hdl.handle.net/10315/41134We 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.enAttribution-NonCommercial-NoDerivatives 4.0 InternationalAptamer design; DNA melts; NMR spectroscopy; Fluorescence spectroscopy; Isothermal titration calorimetry; DNA-Small molecule interactionsArticle