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Investigation of G-quadruplex and Small Molecule Interactions at the Single Molecule Level

Maleki, Parastoo
http://rave.ohiolink.edu/etdc/view?acc_num=kent1543944037378098

Abstract Details

2018, PHD, Kent State University, 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-mediated GQ unfolding both in steady state and in terms of unfolding rate. We also investigated how SMs influence folding rate and conformations of hGQ at physiological (150 mM KCl) and low (2 mM KCl) salt concentrations. We did not observe a significant change in the folding rate or conformations of hGQ at 150 mM KCl when the structure is folded in the presence or absence of SMs. However, SMs significantly increased the parallel conformation when hGQ was folded at 2 mM KCl. In the absence of SM, hGQ initially folds into the anti-parallel conformation (anti-parallel : parallel ratio of 6:1) but the conformation gradually shifts towards parallel conformation (ratio of 2:1 after 1 hour). Nevertheless, when folded in the presence of SM, the initial anti-parallel : parallel ratio becomes ~2.5:1.0, depending on SM. This ratio drifts towards parallel conformation at a much slower rate compared to the absence of SM and reaches 2:1 after 1 hour. These data suggest that SMs primarily influence the conformation during the initial folding and significantly reduce the rate of transitions between different conformations, presumably by stabilizing the already folded structures. With these and other exploratory work we performed, we have not only revealed significant mechanistic aspects of GQ, SM, and helicase interactions, but we also established protocols and guidelines to work with these challenging structures.
Hamza Balci , Dr. (Advisor)
John Portman, Dr. (Committee Member)
Bjorn Lussem, Dr. (Committee Member)
Gail Fraizer, Dr. (Committee Member)
Sanjaya Abeysirigunawardena, Dr. (Committee Member)
242 p.
Biophysics; Physics
G-quadruplex, single molecule FRET, Bloom Helicase, small molecule ligands, L1H1-7OTD, L2H2-6OTD, PDS, PhenDC3

Recommended Citations

Citations

  • Maleki, P. (2018). Investigation of G-quadruplex and Small Molecule Interactions at the Single Molecule Level [Doctoral dissertation, Kent State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=kent1543944037378098

    APA Style (7th edition)

  • Maleki, Parastoo. Investigation of G-quadruplex and Small Molecule Interactions at the Single Molecule Level. 2018. Kent State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=kent1543944037378098.

    MLA Style (8th edition)

  • Maleki, Parastoo. "Investigation of G-quadruplex and Small Molecule Interactions at the Single Molecule Level." Doctoral dissertation, Kent State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=kent1543944037378098

    Chicago Manual of Style (17th edition)

kent1543944037378098
344
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This open access ETD is published by Kent State University and OhioLINK.