Conformational Stability & Dynamics of G-Quadruplexed DNA & Ligand Interactions

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Guanine-rich DNA tandem repeat telomeric sequences located at the end of the chromosomes can assume highly stable G-quadruplex structures through Hoogsteen base pairing of the guanine residues. These secondary DNA structures can inhibit the activity of telomerase, an enzyme that is important for tumorigenesis. There is intense interest in developing so-called G-quadruplex interactive agents (QIAs), which are able to stabilize the G-quadruplex structure as potential chemotherapeutics. However, advancement of this promising area of research has been hindered by a general lack of knowledge of the fundamental rules that govern G-quadruplex formation, conformation and dynamics, properties which can influence productive and selective QIA ligand interactions. Hence there a critical need for understanding the basic chemical and physical characteristics of the G-quadruplex. The aims of this research application are: (1) to examine the conformational stability at specific sites within the G-quadruplex and to assess the effects of QIA binding on these parameters; and 2) to examine the underlying dynamics of intramolecular G-quadruplex folding and unfolding. To achieve these aims we have prepared a series of fluorescently labeled human telomeric sequences, with replacement of a single guanine residue by a fluorescent nucleoside analog at varying postions within the G-quadruplex. The conformation and dynamics of the G-quadruplex will be studied using state-of-the-art fluorescence methodologies. Isothermal titration calorimetry (ITC) will be used to examine thermodynamic quantities involved in QIA ligand binding; and electrophoresis and UV analyses to confirm quadruplex formation. The information provided by the combination of these characterization methods will allow us to map the dynamic structure of the quadruplex and determine the impact of QIA binding. The broad, long-term objective of the proposed research is to understand the fundamental "rules" that govern the conformation and dynamics of G-quadruplexed DNA, and to begin to understand the effects of specific ligand binding on them for ultimate applications in the rational design of QIAs.