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- W3208265471 abstract "Author(s): Turner, Marc Benjamin | Advisor(s): Purse, Byron W | Abstract: Intrinsically fluorescent nucleoside analogues with minimally perturbing architecture offer a non-disruptive method for optically studying nucleic acid structure, dynamics, and metabolism. These nucleoside analogues have inherently fluorescent nucleobases, thus replacing the natural canonical bases of DNA, but retain Watson-Crick base-pairing. Minimally perturbing structures are those that induce few or mild adverse deviations from native, unmodified biomolecular conformations. Using fluorescent nucleobases eliminates any disruptive fluorophore conjugation chemistry often employed in conventional labeling strategies. Their minimally perturbing structure better preserves the native biomolecular topography and function of DNA. Since the discovery of 2-aminopurine 51 years ago, a multitude of nucleoside analogue scaffolds have been synthesized, including the tricyclic cytidine (tC) structure that the Purse Lab uses as a platform to develop novel derivatives with varied photophysical properties. Advancing these novel derivatives into meaningful biological applications requires characterization of their fluorescent, biophysical properties in DNA helices, as well as their compatibility with polymerase enzymes for metabolic labeling. This dissertation describes the biophysical characterization of a fluorescence turn-on probe in DNA-RNA hybrids, the polymerization kinetics exhibited by viral reverse transcriptase when incorporating the fluorescent nucleotides into nascent DNA strands, and preliminary cell culture studies with the fluorescent nucleotides. To better understand environmental parameters modulating the turn-on response in the derivative 8-diethylamino tC, spectroscopic experiments with the probe in DNA-RNA hybrids were performed. Shielding from solvent water molecules attenuated excited-state proton transfer and were identified to be a major factor modulating the increase in brightness by up to 37-fold compared to the free nucleoside in water. Michaelis-Menten kinetics for viral reverse transcriptase inserting the nucleotide analogues in growing DNA show efficiencies from 0.09 – 5 times that of natural dCTP across from G, with continued strand elongation. In a simplified model of the reverse transcription cycle, HIV-1 RT effectively recognized the unnatural analogues as the incoming nucleotide substrates and as the templating base during complementary DNA synthesis. Live cell culture work with bacterial and human cells has strongly suggested that tC nucleotides are retained in cells following delivery and that cytotoxicity is not a major concern. The findings of these studies suggest these tC-derivatives are appropriate biophysical tools for applications including sequence detection, DNA/RNA structural changes, and metabolic labeling." @default.
- W3208265471 created "2021-11-08" @default.
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- W3208265471 date "2020-01-01" @default.
- W3208265471 modified "2023-09-27" @default.
- W3208265471 title "Biophysical and Biochemical Applications of Fluorescent Tricyclic Cytidine for Nucleic Acid Labeling and DNA Structural Dynamics" @default.
- W3208265471 hasPublicationYear "2020" @default.
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