Date of Award

Spring 2013

Document Type

Thesis

Terms of Use

© 2013 Steven P. Barrett. All rights reserved. This work is freely available courtesy of the author. It may only be used for non-commercial, educational, and research purposes. For all other uses, including reproduction and distribution, please contact the copyright holder.

Degree Name

Bachelor of Arts

Department

Chemistry & Biochemistry Department

First Advisor

Liliya A. Yatsunyk

Abstract

G-quadruplexes (GQs) are noncauonical secondary structures of DNA that can adopt a multitude of different topologies. Here, we explore the factors that drive the formation of these structures and favor the specific topologies they adopt. Our discussion of this topic begins with an investigation of the interactions of N-methyl mesoporphyrin IX (NMM), a small aromatic molecule, with Tel22, a 22-nucleotide DNA sequence that serves as a model for the putative quadruplex formed by the human telomeric repeat sequence. NMM is exceptionally selective for GQ DNA over all other types of nucleic acid secondary structure and specifically prefers the parallel GQ topology, inducing an isomerization in Tel22 from the mixed hybrid structure to the all-parallel form. Thus, we were interested in understanding the molecular basis of this specificity. Our recently obtained crystal structure of the NMM-Tel22 complex indicates that the N-methyl group plays a large role in this selectivity by distorting the porphyrin core of NMM, thereby optimizing its interactions with GQ DNA and precluding its association with double stranded B-form sequences. NMM's peripheral side chains are largely unresolved in the crystal structure while the N-methyl group has well-defined electron density, suggesting either a large degree of thermal motion or the binding of 4 distinct NMM regioisomers. Here, we detail the separation and spectroscopic characterization of these isomers. Our preliminary data suggest that all four isomers are able to interact with Tel22, although there appears to be significant contamination of these samples. In the next chapter of this work, we explore the bimolecular quadruplex formation between a strand of DNA and its complement. Using a variety of different tactics, we attempted to induce GQ formation in engineered sequences endowed with what is known as duplex derived interstrand quadruplex forming potential (ddiQFP). Our efforts were mostly unsuccessful likely due to the extremely high thermal stability of the competing duplex structure and the difficulty of interpreting our spectroscopic data. In the final chapter of this work, we investigate quadruplex formation at mitochondrial DNA (mtDNA) sites associated with DNA deletions. Three of these sequences showed stable quadruplex formation under physiological conditions, indicating that GQs may be responsible for these breakpoints in vivo. In low salt conditions, the addition of lead (Pb²⁺) to two of these sequences appears to select for a small population of stable GQs and even induce GQ formation in one sequence. This suggests an additional mechanism for the toxicological effects of lead on mitochondrial structure and function. Because of the disparate nature of these topics, the conclusions and future directions will be discussed at the end of each chapter rather than together.

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