Date on Master's Thesis/Doctoral Dissertation

8-2014

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Biochemistry and Molecular Biology

Committee Chair

Trent, John O.

Committee Co-Chair (if applicable)

Chaires, Jonathan B.

Committee Member

Chaires, Jonathan B.

Committee Member

Bates, Paula J.

Committee Member

Darling, Douglas S.

Committee Member

Dean, William L.

Subject

Quadruplex nucleic acids; DNA

Abstract

In the cell, guanine-rich nucleic acids can self-assemble into unique four stranded tertiary structures known as G-quadruplexes. G-quadruplex formation in the telomere leads inhibits telomerase, an enzyme activated in cancer cells to maintain the telomere and allowing for cancer cells to achieve immortality. G-quadruplex formation in the promoters and 5’-untranslated regions regulates the expression of many oncogenes. Furthermore, G-quadruplex formation during cellular replication promotes genomic instability, a characteristic which enables tumor development. Because of their implication in cancer, G-quadruplex structures have emerged as attractive drug targets for anti-tumor therapeutics. In the current dissertation work, we present three experimental approaches to investigate G-quadruplex structures, biophysical properties, small molecule interaction, and the thermodynamics of G-quadruplex formation. Current approaches to study G-quadruplex structures often employ sequence modifications or changes to the experimental condition, as a way of resolving the structural polymorphism associated with many G-quadruplex-forming sequences, to select for a single conformation for high-resolution structural studies. Our strategy for resolving G-quadruplex structural polymorphism is superior in that the experimental approaches do not result in drastic perturbation of the system. In the first approach, we employed size exclusion chromatography to separate a mixture of G-quadruplex structures formed from a G-quadruplex-forming sequence. We demonstrated that it is possible to isolate distinct species of G-quadruplex structures for further biophysical studies. In the second approach, we employed hydrodynamic bead modeling to study the structural polymorphism of a G-quadruplex-forming sequence. We showed that properties calculated from models agreed with experimentally determined values and could be used to predict the folding of G-quadruplex-forming oligonucleotides whose high-resolution structures are ambiguous or not available. In our third approach, we presented a virtual screening platform that was successful in identifying a new Gquadruplex-interacting small molecule. The results of the virtual screen were validated with extensive biophysical testing. Our target for the virtual screen was a G-quadruplex structure generated in silico, which represents one approach to receptor-based drug discovery when high-resolution structures of the binding site are not available. Taken together, our three approaches represent a new paradigm for drug discovery from guaninerich sequence to anti-cancer drugs.

Download

Share

COinS