Validation of an isomorphic fluorescent analogue of guanosine as a unique tool to site specifically and dynamically characterize the conformations and molecular interactions of RNA G quadruplexes. – GQfluodynint

G quadruplexes (GQs) are non-canonical nucleic acid structures that consist of at least two stacks of G quartets held together by stacking interactions, Hoogsteen base pairs and cation binding. GQ topologies can be categorized based on the relative direction of their four strands. RNA GQs mainly adopt the parallel topology, but non-parallel topologies have also been described. GQ forming sequences are widely distributed in mRNAs and non-coding RNAs, where they regulate gene expression. In vivo, RNA GQs are thought to be largely unfolded, due to the activity of RNA binding proteins (RBP), such as the human helicase DHX36 and the CCHC-type zinc finger nucleic acid binding protein (CNBP). Therefore, RNA GQs are probably transient structures converted by RBPs between their folded and unfolded states. To explore their structure and dynamics as well as their interaction with proteins, a limited set of techniques has been used. While X-ray crystallography and NMR spectroscopy can provide atomically resolved structures, they need high concentrations and give only limited dynamic information (X-ray) or are limited by the molecule size (NMR). Fluorescence techniques are well complementary, being able to monitor molecular interactions and dynamics on a wide time range and at low concentration, but suffering from the need of introducing external labels. Numerous fluorescent purine surrogates have been developed, but they generally destabilize the GQ structure or are highly quenched. In this context, our objective is to validate and apply tzG, an isomorphic fluorescent analogue of G from the isothiazolo[4,3-d]-pyrimidine family, developed by one partner of the project, as a unique tool to site-specifically characterize the conformations, dynamics and molecular interactions of RNA GQs. To reach this aim, we assembled an international multidisciplinary consortium of experts, all highly recognized in their respective fields. The project will be divided in four work packages (WP) and a fifth organizational one (WP0). The aim of WP1 is to synthesize the tzG-labelled GQ forming sequences needed in this project and characterize their structure(s). The effect of tzG on GQ topology and stability will be determined using CD, thermal denaturation experiments and NMR spectroscopy in both K+ and Na+ salt conditions. For sequences with well-resolved NMR spectra, the 3D structure will be then determined, to obtain fine structural details of the impact of tzG insertion in GQ structures. In WP2, we will perform a thorough steady-state and time-resolved fluorescence characterization of the tzG-labelled RNA GQs in both Na+ and K+ salt conditions. Interpretation of the obtained data using a combined MD/QM approach is expected to provide a full picture of the underlying photophysics of tzG in GQs that will be critical to rationalize its spectroscopic properties in GQs and interpret their changes on interaction with RBPs. In WP3, we will validate tzG as a key tool for studying RNA GQs by applying it to decipher the GQ unfolding/refolding mechanism of DHX36 and CNBP proteins, through a combination of NMR, fluorescence spectroscopy and stopped-flow techniques. Finally, using the comprehensive data set of WP1–3, the aim of WP4 will be to refine the methods and interpretative models adopted in the project and, in particular to develop a excitonic Hamiltonian for the study of photoactivated dynamics in multichromophoric systems, including charge transfer processes. Through this study, we anticipate tzG to become the first validated tool to faithfully monitor any substituted G in RNA GQs, and thus lead to breakthroughs in the understanding of GQ properties and RBP mechanisms of action.