STRUCTURAL INVESTIGATION OF DNA QUADRUPLEXES BY NMR

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The goal of my PhD thesis project was to obtain information about the biophysical characteristics of a potentially biologically significant higher-order DNA structural motif. We have been interested in structures of DNA quadruplexes whose core architecture is based on guanine quartets (G-quartets). It has been suggested that G-quartet structures may form at the natural eukaryotic chromosome ends (telomeres) and contribute to protein binding, regulation, or resistance to degradation. Telomeres are usually composed of simple tandem repeats of guanine-rich sequences associated with telomeric binding proteins. Telomeric sequences are typically found to be highly conserved across phylogenetically diverse organisms. More recently, however, many budding yeast telomeres have been shown to have more divergent sequences both in length and in base composition. Oligonucleotides having one to two repeats of the yeast telomeric sequences are usually less stable thermodynamically compared to those with more conserved sequences, such as ones from ciliate protozoa. My thesis project involved the characterization of several quadruplex-forming guanine-rich oligonucleotides from budding yeast telomeric and related sequences. Solution nuclear magnetic resonance (NMR) and other spectroscopic methods were used to understand their physical chemical features, the factors contributing to the stability of the complexes, and to determine high resolution solution structures of those oligonucleotides. The recent improvement of our NMR spectrum analysis tool, Sparky, has been very useful for our structural determination process. The capability of the program to visualize structural models and inter-proton distances using MidasPlus within Sparky accelerated tis process enormously. The incorporation of graphical tools to the structural determination process, spectrum analysis (Sparky), distance calculation (MARDIGRAS), structure elucidation (Amber, X-PLOR) was exciting and it has already helped the iterative nature of this process.