PHD, Kent State University, 2022, College of Arts and Sciences / Department of Physics
Nucleic acids play a significant role in biological processes involving genetic information, such as replication, transcription, and translation. Aside from the well-known canonical double helix structure, DNA can adopt a variety of alternative non-canonical structures such as DNA hairpins, holiday junctions, cruciforms, triplexes, or G-quadruplexes (GQ). GQ structures may form in the guanine-rich (G-rich) regions of the genome, including telomeres, promoters, and other significant regulatory sites. Formation of GQs at telomeres protects chromosome ends against degradation and end-to-end joining. The cytosine-rich (C-rich) sequences that are complementary to GQ forming sequences can adopt an intercalated motif (i-motif) structure at acidic pH. In addition to their physiological functions, both the GQ and the i-motif have potential biotechnological applications.
In my dissertation, I will describe several projects on single molecule fluorescence and force spectroscopy studies of GQ and i-motif structures. In the first study, we developed a multiplexed, high throughput single-molecule force sensor that converts fluorescence signal into force information by hybridizing the ends of short looped double-stranded DNA (dsDNA) with a nucleic acid secondary (NAS) structure, such as a hairpin or a GQ. When looped, the dsDNA maintains a constant tension across the ends of the NAS. Initially, we completed proof-of-principle studies on this system using different lengths of dsDNA and hairpin structures under various salt conditions. Later, we applied this method to study of the interaction of an under-tension GQ structure with a destabilizing protein, Replication Protein A (RPA), and a stabilizing small molecule, an oxazole telomestatin derivative. The force applied to the GQ via the looped dsDNA was calibrated, with the help of a collaborator, against high-resolution optical tweezers measurements, which is only a few picoNewtons for the construct and assay conditions we used. (open full item for complete abstract)
Committee: Hamza Balci (Advisor); John Portman (Committee Member); Liang-Chy Chien (Committee Member); Soumitra Basu (Committee Member); Thorsten-Lars Schmidt (Committee Member)
Subjects: Biophysics; Physics