Johnson, Philip E.2016-09-202016-09-202016-04-202016-09-20http://hdl.handle.net/10315/32325Isothermal Titration Calorimetry (ITC) studies were conducted wherein variances in the heat capacity of binding (Cp) among variants of the cocaine-binding aptamer were suggestive of two distinct binding mechanisms. Aptamer variants containing 6 base pairs in stem 1 are pre-folded and show little change in secondary structure with ligand binding. Aptamer variants with 3 base pairs in stem 1 are mostly unfolded and exhibit conformational changes that take place with ligand binding. ITC studies were extended to aptamer variants containing single nucleotide mutations and truncated stems. A relationship between nucleotide identity vs binding affinity was established while also noting that mutations within the aptamer core result in the switch of binding specificity from alkaloids to steroids. This altered specificity was most notable when one of two GA mismatched pairs in the aptamer core was converted to the Watson-Crick GC pair. Simultaneous mutation of both GA base pairs resulted in no detectable binding. Additional ITC and NMR spectroscopy studies demonstrated that the binding mechanism of steroid-binding aptamer constructs is nearly identical to that of cocaine-binding constructs. Conformational changes were noted for steroid-binding constructs with 4 base pairs in stem 1, as opposed to 3 base pairs as in cocaine-binding constructs. Combined ITC and NMR spectroscopy studies characterized the high affinity interaction of the cocaine-binding aptamer with quinine, showing a 30 40 fold increased affinity over cocaine. The binding mechanism with quinine was shown to be identical to that of cocaine, utilizing the same binding site while electrostatic interactions contributed only about 6% of the binding free energy. Fluorescence spectroscopy revealed that the aptamer is structurally stable at very high concentrations of urea. MN4 and MN19 both demonstrated high resistance to chemical denaturation with concentrations of urea reaching 6 M and 4.4 M, respectively. A final ITC study confirmed the bifunctionality of modified cocaine-binding aptamers. A titration of an equimolar mixture of deoxycholic acid (DCA) and cocaine had an enthalpy of (-32.5 0.2) kcal mol-1. This is comparable to the sum of enthalpies for independent titrations of cocaine and DCA at (-12 5) and (-11 2) kcal mol-1, respectively.enAuthor owns copyright, except where explicitly noted. Please contact the author directly with licensing requests.ChemistryElectronic Thesis or Dissertation2016-09-20CocaineAptamerThermodynamicsBiochemistryCocaine-bindingBiosensorNucleic acidChange in heat capacityNuclear magnetic resonanceIsothermal titration calorimetryFolding mechanismDNA3-way junctionBase pair mismatchSELEX