Johnson, Philip E.Shoara, Amin Aron2022-12-142022-12-142022-08-082022-12-14http://hdl.handle.net/10315/40726Since its first report in 1990, aptamers have been utilized in biosensor modeling technologies. One of the most important advantages of using aptamers is the structural flexibility and thermal stability of nucleic acids. These structural merits enable aptamers to be linked on solid surfaces, attached to chemical labels, or extended to build nanostructures for advanced therapeutic and diagnostic modeling purposes. The cocaine-binding aptamer was originally selected through a systematic evolution of ligands by exponential enrichment (SELEX) method to select cocaine molecules from cocaine metabolites in biological solutions. However, the aptamer showed binding to quinine and other antimalaria drugs tighter than its original ligand, cocaine. Work presented in this dissertation demonstrate how the cocaine-binding aptamer can be exploited as a model system for the structural analysis of aptamers using biophysical techniques including fluorometry methods. The results discussed in this study demonstrate how intrinsic fluorescence of ligands was exploited for aptamer-ligand binding and thermal stability analyses. Furthermore, photoisomerization of stilbene coupled with ligand-induced binding mechanism of the cocaine-binding aptamer were employed for the development of the Photochrome Aptamer Switch Assay. This research aims to gain insight into how aptamers interact with their ligands by utilizing the fluorescence properties of the ligands. Investigating the binding mechanisms of aptamers is essential in sensing technology since biosensors yield greater analytical sensitivity upon ligand-induced structural changes.Author owns copyright, except where explicitly noted. Please contact the author directly with licensing requests.BiochemistryChemistryMolecular biologyElectronic Thesis or Dissertation2022-12-14AptamerDNARNAProteinFluorescenceSpectroscopyLigandStructureDynamicsIntrinsicMethodAnalysisBiophysicalX-ray crystallographyIsothermal titration calorimetryNuclear magnetic resonanceDifferential scanning calorimetryCocaineQuinineAntimalariaAntimalarialArtemisininElectrochemicalSensorBiosensorDetectionLevamisoleStilbeneSITSPhotoisomerizationPhotochrome aptamer switch assayPHASACocaine bindingOchratoxin AOTAOTXQuenchingEnhancementDifferential scanning fluorimetryFluorometryUltravioletVisibleLightThermal stabilityThermal melt denaturationIsothermFunctionMN4MN19Ligand induced binding folding mechanismDopamineSerotoninStructure modelingAptachainAptameshFluorescence polarizationProtocolPhotomultiplierCLODCLOQThresholdOptimizationQuantum yieldAffinityKaKdDissociation constantMerocyanineFPhOBtzDyeThree way junction3WJTWJThermal shift assayConjugationFluorescence decay kineticsFluorescence anisotropyMethod validationDrugSensitivityCalibrationCurvePlotNon linear regressionSpike-recoveryAccuracyCircular dichroismExcitationEmissionin vitroStern Volmer constantStokes shiftSELEXEnthalpyGibbs free energyEntropyWavelengthRefractive indexExtinction coefficientFluorescence lifetime