Date on Master's Thesis/Doctoral Dissertation

12-2012

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Biochemistry and Molecular Biology

Committee Chair

Chaires, Jonathan Bradford

Author's Keywords

Quadruplex; unfolding; telomere; polymorphism; spectroscopy; biophysical

Subject

Telomere; Quadruplex nucleic acids

Abstract

G-quadruplexes are thought to have biological importance, with studies based on small molecule interactions and quadruplex-interactive antibodies demonstrating their potential for formation in vivo. One potential biological function of quadruplex structures is the regulation and maintenance of telomeres. Telomeres are nucleoprotein complexes involved in chromosome stability. Human telomeres are composed of the repeated DNA sequence, 5'- d(TIAGGG), that terminates in a 3' single-stranded overhang. DNA sequences with homology to the human telomere are capable of quadruplex formation in vitro. Specifically, sequences containing four-guanine stretches (e.g. 5'-d(AGGG(TIAGGGb)) are capable of forming at least five distinct unimolecular structures. Which structure is favored is believed to be linked to solvent composition and the addition of 3'- and 5'-flanking residues. This dissertation provides an essential biophysical investigation of the polymorphic equilibrium displayed by human telomeric quadruplexes. Multiple biophysical techniques are utilized to assemble a thermodynamic description of the influences of hydration and molecular crowding on conformational selection and elucidate complex unfolding mechanisms with unique intermediate states. This dissertation provides the first application of phasor diagrams in the study of quadruplexes. Phasor diagrams are shown to be sensitive to alterations in quadruplex structure (i.e. folding and unfolding) by monitoring changes in the complex lifetime distribution of 2-aminopurine. This dissertation contains the first multi-faceted biophysical investigation of the underlying mechanism of co-solvent driven conformational changes of human telomeric quadruplexes. The thermodynamic study illustrates that quadruplexes are stabilized by dehydration, a behavior opposite that of canonical duplex structures. Additionally, the ability of PEGs to drive the conformational selection of a parallel quadruplex through differential binding is clarified, addressing unsubstantiated claims that the propeller form is the most biologically relevant conformation. Finally, an in-depth thermodynamic investigation of the thermal unfolding of human telomeric quadruplexes is conducted. Multiple spectroscopic techniques are used to evaluate the thermal unfolding process and characterize potential intermediates states. This dissertation work is the first to apply spectroscopic deconvolutions to demonstrate that human telomeric quadruplexes unfold through sequential mechanisms requiring intermediate species. These results are highlighted by the recovery of an intermediate species whose biophysical description is best characterized by an ensemble of triple-helical conformations.

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