The Cocaine-Binding Aptamer: A Thermodynamic and Structural Overview of Specific and Non-Specific Binding Interactions

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dc.contributor.advisorJohnson, Philip E.
dc.creatorReinstein, Oren
dc.date.accessioned2016-09-20T18:54:38Z
dc.date.available2016-09-20T18:54:38Z
dc.date.copyright2016-04-20
dc.date.issued2016-09-20
dc.date.updated2016-09-20T18:54:38Z
dc.degree.disciplineChemistry
dc.degree.levelDoctoral
dc.degree.namePhD - Doctor of Philosophy
dc.description.abstractIsothermal 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.
dc.identifier.urihttp://hdl.handle.net/10315/32325
dc.language.isoen
dc.rightsAuthor owns copyright, except where explicitly noted. Please contact the author directly with licensing requests.
dc.subjectChemistry
dc.subject.keywordsCocaine
dc.subject.keywordsAptamer
dc.subject.keywordsThermodynamics
dc.subject.keywordsBiochemistry
dc.subject.keywordsCocaine-binding
dc.subject.keywordsBiosensor
dc.subject.keywordsNucleic acid
dc.subject.keywordsChange in heat capacity
dc.subject.keywordsNuclear magnetic resonance
dc.subject.keywordsIsothermal titration calorimetry
dc.subject.keywordsFolding mechanism
dc.subject.keywordsDNA
dc.subject.keywords3-way junction
dc.subject.keywordsBase pair mismatch
dc.subject.keywordsSELEX
dc.titleThe Cocaine-Binding Aptamer: A Thermodynamic and Structural Overview of Specific and Non-Specific Binding Interactions
dc.typeElectronic Thesis or Dissertation

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