Structure, dynamics and kinetics of folding of G-quadruplex nucleic acids

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In this application, we intend to elucidate the energy folding landscape of G-quadruplex structures using a combination of biophysical methods, including in particular real time NMR spectroscopy. Experimental findings will guide molecular dynamics simulations by J. Sponer (Brno University). Sophisticated chemical and biochemical approaches will be developed and applied for the preparation of isotope labeled DNA and caged DNAs.The structures of G-quadruplexes are very polymorphic and their overall folding can vary in terms of strands orientation, geometry of the loops and of the glycosidic torsion angles. There is increasing evidence for a regulatory role of G-quadruplex structures in many biological processes. In fact, G-quadruplex are found at the 3'-overhang of telomeres and in the promoter regions of many oncogenes and are nowadays considered a novel target in anticancer therapy. However, more information is needed to fully understand the structure and the folding dynamics of G-quadruplexes and to unravel the details of its interaction with ligands and protein binding partners. In fact, the G-quadruplex energy landscape is far from being completely understood. The data reported until now suggest a very rugged energy landscape with multiple energy minima and an overall slow folding kinetics that follows a kinetic partition mechanism.Real-time NMR is optimally suited to obtain kinetic information at atomic resolution.We will use real-time NMR to investigate the folding kinetics of DNA and RNA G-quadruplexes, employing two different approaches. The first approach allows us to trigger the G-quadruplex refolding by injection of KCl directly in the NMR tube using a rapid mixing device. The second approach relies on the photocaging of selected guanine residues (work performed in the Heckel group) to block the oligonucleotide in a specific conformation or to maintain it unfolded. The refolding is triggered by illumination of the photocaged DNA/RNA directly in the NMR tube with a quartz fiber connected to a laser. In particular, the photocaging approach will be used to investigate the process of G-register exchange in G-quadruplex forming sequences containing more than 3 guanine residues per G-tract. These data will be complemented with activation energies derived by UV-vis hysteresis experiments (Mittermaier group). The information will be then the base for future MD simulations (Sponer group) that will provide details on the fast folding intermediates that cannot be detected and characterized by NMR. Furthermore, we aim at optimizing an enzymatic method based on the rolling circle amplification (RCA) for the production of 13C,15N labeled single stranded DNA sequences in mg amount. The availability of 13C,15N labeled DNA will allow us to monitor the folding kinetics with 2D NMR experiments as well as to characterize at high resolution the structure of selected G-quadruplexes.