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Mechanochemistry, Transition Dynamics and Ligand-Induced Stabilization of Human Telomeric G-Quadruplexes at Single-Molecule Level

Koirala, Deepak P
http://rave.ohiolink.edu/etdc/view?acc_num=kent1397919270

Abstract Details

2014, PHD, Kent State University, College of Arts and Sciences / Department of Chemistry.
DNA secondary structures such as G-quadruplexes have attracted extensive research attention in recent years because of their vital biological roles and tremendous applications in therapeutics, biosensing and nanobiotechnology. Human telomeric G-quadruplex structures are of particular interest because human telomeres are closely associated with the genetic integrity, cell proliferation, aging and cancer. Fundamental understanding of the transition dynamics and the equilibrium population distribution of the G-quadruplexes, their intermediates and other alternative structures in the human telomeric DNA sequences and their interactions with small-molecule ligands or proteins are, therefore, crucial. This dissertation has uncovered the previously unknown folding dynamics and population distribution of human telomeric G-quadruplex, intermediate and misfolded G-quadruplex structures as well as their interaction with small-molecule ligands. Intramolecular folding in three and four tandem guanine repeats of human telomeric DNA have been investigated using Optical-Tweezers, Molecular Dynamics Simulation, Circular Dichroism and Dimethyl Sulphate footprinting. An unprecedented, mechanically and thermodynamically stable, folded species has been observed in the sequence with three tandem guanine repeats which is consistent with a G-triplex conformation. Similar species has also been found to coexist with a prevalent G-quadruplex structure in a DNA sequence with four tandem guanine repeats. Such observations suggest a complex folding pattern of the human telomeric DNA in which G-triplex could be an intermediate structure to the G-quadruplex. Due to complex folding dynamics and stunning structural polymorphism, population equilibrium in human telomeric sequence is expected to be intricate and beyond the resolution of ensemble-average techniques, such as CD, NMR, or X-ray crystallography. By combining a force-jump method at the single-molecular level and a statistical population deconvolution at the sub-nanometre resolution, a complex population network with previously unknown transition dynamics in human telomeric sequences that contain four to eight TTA GGG repeats has been revealed. The kinetics data firmly establish that G-triplexes are intermediates to G-quadruplexes while long-loop G-quadruplexes are misfolded population minorities whose formation and disassembly are faster than G-triplexes or regular G-quadruplexes. The existence of misfolded DNA supports the emerging view that structural and kinetic complexities of DNA can rival those of RNA or proteins. While G-quadruplexes are the most prevalent species in all the sequences studied, the abundance of a misfolded G-quadruplex in a particular telomeric sequence decreases with an increase in the loop length or the number of long-loops in the structure. These population patterns support the prediction that in the full-length 3' overhang of human telomeres, G-quadruplexes with shortest TTA loops would be the most dominant species, which justifies the modelling role of regular G-quadruplexes in the investigation of telomeric structures. Since ligand-induced stabilization of telomeric DNA G-quadruplex has potential in cancer treatment through the inhibition of telomerase - an enzyme over expressed in many cancer cells, understanding the kinetic, thermodynamic, and mechanical properties of small molecule binding to these structures is important. However, classical ensemble assays are unable to measure these parameters simultaneously in a single platform. The interactions between human telomeric G-quadruplex structure and small-molecule ligands have been investigated using optical-tweezers method. With a force jump approach, it has been observed that pyridostatin promote the folding of telomeric G-quadruplexes. The increased mechanical stability of pyridostatin bound G-quadruplex permitted the determination of the dissociation constant (Kd) of 490 ± 80 nM. The free energy change of binding obtained from a Hess-like process provided an identical Kd for pyridostatin and a Kd of 42 ± 3 µM for a weaker ligand RR110. Because of its unique abilities, this single-molecule platform is anticipated to provide detailed insights into the mechanical, kinetic, and thermodynamic properties of the interaction between ligands and macromolecules of biological relevance.
Hanbin Mao, Ph.D. (Advisor)
Biochemistry; Biophysics; Chemistry; Molecular Biology; Physical Chemistry
Human Telomeric G-quadruplex, Single Molecule Studies, Optical Tweezers, G-quadruplex and Ligand Interactions, Mechanochemistry of G-quadruplex DNA, G-triplex DNA, Folding Dynamics of G-quadruplex DNA, Misfolded G-quadruplex

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Citations

  • Koirala, D. P. (2014). Mechanochemistry, Transition Dynamics and Ligand-Induced Stabilization of Human Telomeric G-Quadruplexes at Single-Molecule Level [Doctoral dissertation, Kent State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=kent1397919270

    APA Style (7th edition)

  • Koirala, Deepak. Mechanochemistry, Transition Dynamics and Ligand-Induced Stabilization of Human Telomeric G-Quadruplexes at Single-Molecule Level . 2014. Kent State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=kent1397919270.

    MLA Style (8th edition)

  • Koirala, Deepak. "Mechanochemistry, Transition Dynamics and Ligand-Induced Stabilization of Human Telomeric G-Quadruplexes at Single-Molecule Level ." Doctoral dissertation, Kent State University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=kent1397919270

    Chicago Manual of Style (17th edition)

kent1397919270
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