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Philip Bolton






Guanine rich DNA and RNA can form higher order structures known as G-quadruplex, which consist of stacked units of Hoogsteen paired four guanines and are further stabilized by K+ and Na+ ions. G-quadruplexes are present in telomeres and promoter region of oncogenes and play significant regulatory role in cancer signaling pathways. Perturbation of the cancer signaling pathway by quadruplex specific ligands can be a promising anticancer drug design strategy. Structural investigation of the properties of ligand-quadruplex interactions can provide important insights for structure based rational drug design and development.

We developed an enhanced hydroxyl radical footprinting protocol that allows determination of structures of ligand-quadrplex complexes at nucleotide resolution. The protocol revealed that NSC-176319, a quinolinium derivative, binds specifically to the two TT loops of the thrombin binding aptamer, TBA. NMR was used to demonstrate that NSC-176319 disrupts the base pairing of T4 and T13, validating the footprinting results. We applied this protocol further to other biologically relevant quadruplexes and found ten drug like molecules that exhibit binding to the loop and tetrad regions of these quadruplexes. CD spectroscopy was used to study the effects of these ligands on structure and thermal stability quadruplexes.

While investigating the interactions of ligands with quadruplex at various temperatures, we discovered the presence of two slow conformational transitions in TBA. One of these transitions consists of the TT loop residues T4 and T13. The enthalpic contribution to the free energy barrier for this transition is twice as high as the entropic contribution. The presence of NSC-176319 and higher potassium decreases the rate of this transition. The other transition consists at least the G1 and G14 residues that are in the quartets. The enthalpic contribution to the free energy barrier for this transition is similar to the entropic contribution. The rate for this transition is not affected by the concentration of potassium in the range of 10 to 40 mM.



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