NMR studies of nucleic acids as drug targets
Nogalamycin is a member of the anthracycline family of antitumour antibiotics. These are potent cytotoxic agents and are routinely used in cancer chemotherapy. Though nogalamycin is clinically insignificant, it does exhibit three distinct types of non-covalent binding to DNA. Since most other anthracyclines bind to DNA by only one or two of these mechanisms, nogalamycin is an excellent model with which to probe the interaction of this class of anti-tumour agents with DNA. Here, we investigate the binding orientation and stoichiometry of nogalamycin in adjacent TpG(CpA) (and CpG(CpG)) intercalation sites using a combination of NMR techniques and NOE-restrained molecular dynamics simulations. These methods are also employed to investigate the structure of GNA hairpin loops, which are considered to have important biological functions, and assess how their structure and stability are influenced by the introduction of nogalamycin at an adjacent site. The effect of nogalamycin on extrahelical thymine bases incorporated onto either face of the intercalation sites is also investigated in this context. Binding of quadruplex-specific antibodies to telomeric DNA in Stylonychia lemnae macronuclei has recently been detected using immunofluorescence, providing direct evidence for the formation of quadruplex DNA structures in vivo. Guanine-rich quadruplex structures have been extensively studied by NMR and x-ray crystallographic methods. Previous structural studies have failed to unambiguously resolve the conformation preferred by less-stable A-tetrads incorporated into DNA quadruplexes. Additionally, little effort has been made to address the exact number of ions bound to these adenine-containing structures. This forms the basis of our study into quadruplex DNA. Finally, we endeavour to investigate the extent of hydration of both duplex and quadruplex structures using rMD methods, and to comapre hydration patterns in the liquid- and solid-state.