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Biochemical Characterization of Fluorophore-Binding Nucleic Acids

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Tormann_Alexandra.pdf (2.84 MB)

Date

2022-09-27

Authors

Tormann, Alexandra

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University of Waterloo

Abstract

Aptamers, 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.

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Keywords

characterization, fluorophore, aptamer, binding, nucleic acids, mango, DNA, RNA, analytical instrumentation, ITC, SPR

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http://hdl.handle.net/10012/18821

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Chemistry