PNA-DNA Heteroquadruplex Formation: Optimization of PNA Structure, Recognition by Cyanine Dyes and Evaluation of Molecular Crowding Effects

Guanine (G)-quadruplex as a DNA secondary structure has been widely studied for over five decades. Today, the growing recognition of the vital roles of G-quadruplex structures in regulating gene expression and telomere biology have spurred considerable efforts to design or discover synthetic molecules that can bind to quadruplex with high affinity and selectivity. Quadruplex-binding ligands might find applications in chemical biology, where they could report the presence or alter the biological function of the quadruplex structure. This thesis provides detailed discussion on the biophysical characterization of binding of G-rich peptide nucleic acid (PNA) in targeting DNA G-quadruplexes. It also represents our continuous effort in the development of sequence-selective PNA probes as a viable G-quadruplex binder. The thesis is made up of four chapters which include one introductory chapter and three chapters that report experimental results along with the future directions for each chapter.;In chapter 1, we provide relevant introduction of DNA chemistry and conformation as it relates to DNA/RNA G-quadruplexes. We provide a detailed discussion on the discovery of the DNA double helix as well as G-quadruplex. These discussions include the biophysical chemistry of G-quadruplex, biology of G-quadruplex, G-quadruplex interaction with small molecules as well as discussion on PNA as a quadruplex binder.;The Armitage lab has pioneered the development of fluorescent probes for cell imaging and other biological applications. One of such dyes is cyanine dyes. Current contribution involved the use of fluorescence, CD and UV-vis techniques in the study of the interaction of carbocyanine dyes with DNA targets. In Chapter 2, we describe interaction of three carbocyanine dyes that bind noncovalently to PNA-DNA heteroquadruplexes. These dyes can exhibit variable fluorescence enhancement upon binding, depending on loop composition of the PNA strand. Circular dichroism (CD) was used to study the structures of the formed quadruplex upon binding to the dyes. Part of these results were published as part of: Mohammed, H.S., Delos Santos, J.O. and Armitage, B.A. Artificial DNA:PNA & XNA, 2011, 2:2, 1-7.;Chapter 3 reports our results on the optimization of PNA structure to enhance affinity and selectivity for two DNA targets. This includes the synthesis and characterization of nine G-rich PNA probes with varying loop region including basic, abasic and amino acid linkers. The optical spectroscopic techniques utilized in this chapter are UV melting temperature studies to characterize the stability of the PNA-DNA heteroquadruplexes and circular dichroism (CD) to study the structure of the formed complex. A manuscript summarizing results of this work will be prepared soon.;Most experimental analysis of the binding interactions between quadruplex-binding ligands and G-quadruplexes are done in dilute solution. However, the cellular environment where these molecules are expected to function is highly crowded with biomolecules (e.g. nucleic acids, proteins). It is therefore vital to evaluate the effectiveness of these ligands under more physiologically relevant conditions to improve our understanding of the biological effects of quadruplex-ligand interactions and in the design/development of quadruplex-based drugs. Chapter 4 of this dissertation provides discussion on the results of our work on the ability of PNA, to bind to DNA quadruplex target under both dilute and simulated molecular crowding conditions created by the water-soluble polymer, polyethylene glycol with average molecular weight of 200 (PEG200). A manuscript of these results is currently in preparation.;Supplemental CD, UV-vis, and fluorescence spectra are provided in the Appendix section of chapters II through IV of this thesis.