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Biochemical Characterization of Fluorophore-Binding Nucleic Acids

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dc.contributor.authorTormann, Alexandra
dc.date.accessioned2022-09-27T17:07:54Z
dc.date.available2023-09-28T04:50:04Z
dc.date.issued2022-09-27
dc.date.submitted2022-09-14
dc.description.abstractAptamers, single stranded RNA or DNA that bind to target ligands with high affinity and specificity, have a wide range of applications, from therapeutics to biosensing, which makes biochemical characterization of the utmost importance. This research aimed to implement various analytical instrumentation methods for the biochemical characterization of DNA Mango aptamers sequences 2a, 4a, and 6b, to TO1-3PEG. In Chapter 3, fluorescence spectroscopy titrations were conducted. An equilibrium dissociation constant of 160.6 nM was calculated for the 4a aptamer sequence in sodium phosphate buffer. The increase in fluorescence enhancement was weaker than anticipated; therefore, further investigation is required. Isothermal titration calorimetry experiments were also conducted, summarized in Chapter 4. The binding affinity for the DNA Mango aptamer sequences was determined. It was concluded that no binding interactions between the 2a or 6b aptamer sequences and the target ligand were observed. The 4a sequence was found to bind weakly to the target ligand in both a sodium phosphate and a HEPES buffer. Native PAGE studies were also completed, outlined in Chapter 5. The native PAGE experiments indicated the presence of multimeric structures with structural heterogeneity in both the 4a and 6b sequences. A monomeric structure was also observed in the 4a aptamer. Chapter 6 investigated the binding affinity of two DNA Mango aptamer sequences, 4a and 6b, to the target ligand via Surface Plasmon Resonance. Numerous experimental conditions were explored, but binding interactions were not detected for the explored aptamers, therefore either eluding to a weak binding interaction, or the absence of binding altogether.en
dc.identifier.urihttp://hdl.handle.net/10012/18821
dc.language.isoenen
dc.pendingfalse
dc.publisherUniversity of Waterlooen
dc.subjectcharacterizationen
dc.subjectfluorophoreen
dc.subjectaptameren
dc.subjectbindingen
dc.subjectnucleic acidsen
dc.subjectmangoen
dc.subjectDNAen
dc.subjectRNAen
dc.subjectanalytical instrumentationen
dc.subjectITCen
dc.subjectSPRen
dc.titleBiochemical Characterization of Fluorophore-Binding Nucleic Acidsen
dc.typeMaster Thesisen
uws-etd.degreeMaster of Scienceen
uws-etd.degree.departmentChemistryen
uws-etd.degree.disciplineChemistryen
uws-etd.degree.grantorUniversity of Waterlooen
uws-etd.embargo.terms1 yearen
uws.contributor.advisorDieckmann, Thorsten
uws.contributor.affiliation1Faculty of Scienceen
uws.peerReviewStatusUnrevieweden
uws.published.cityWaterlooen
uws.published.countryCanadaen
uws.published.provinceOntarioen
uws.scholarLevelGraduateen
uws.typeOfResourceTexten

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