PHD, Kent State University, 2018, College of Arts and Sciences / Department of Physics
One of the targets for cancer therapy is telomeric G-quadruplex (GQ) structures, which are layered, G-rich DNA structures. GQ structures are known to inhibit telomerase, which is over expressed in ~90% of all cancers, and thereby prevent telomere extension. Therefore, stabilizing GQs with small molecule (SM) drugs has emerged as a potential cancer treatment therapy. GQ stabilizing SMs are typically characterized by their specificity to GQ and the additional thermal stability they impart to GQ. However, to date, there are no quantitative studies that probe the dynamics of GQ-SM interactions, and how these interactions influence the interaction of GQ with destabilizing proteins.
We performed single molecule measurements, primarily single molecule F¿rster resonance energy transfer (smFRET), to study binding kinetics, rotational freedom, and dwell time distributions of a Cy5-labeled oxazole telomestatin derivative (L1Cy5-7OTD) as it interacted with several different GQs. These measurements show that L1Cy5-7OTD dwells on more stable GQ for longer times and binds to such GQ with higher frequency. The dwell times illustrate a broad distribution, but are longer than a minute for a significant fraction of molecules (characteristic dwell time t=192±15 s and t=98±15 s for the more and less stable GQ, respectively). In addition, L1Cy5-7OTD may be able to bind to GQ in at least two different primary orientations and occasionally transition between these orientations. The dwell time in one of these orientations is significantly longer than that in the other one, suggesting different stabilities for different binding orientations.
In a different line of inquiry, we studied the impact of several prominent GQ-stabilizing SMs, including pyridostatin (PDS), Phen-DC3, and oxazole telomestatin derivatives (OTD), on helicase mediated GQ unfolding. Bloom helicase (BLM) and human telomeric GQ (hGQ) were used as model systems. The SMs we studied result in 2-3 fold reduction in the BLM-m (open full item for complete abstract)
Committee: Hamza Balci Dr. (Advisor); John Portman Dr. (Committee Member); Bjorn Lussem Dr. (Committee Member); Gail Fraizer Dr. (Committee Member); Sanjaya Abeysirigunawardena Dr. (Committee Member)
Subjects: Biophysics; Physics