Fluorescence-based tools to probe G-quadruplexes in cell-free and cellular environments

Biophysical and biochemical investigations provide compelling evidence connecting the four-stranded G-quadruplex (GQ) structure with its role in regulating multiple cellular processes. Hence, modulating the function of GQs by using small molecule binders is being actively pursued as a strategy to develop new chemotherapeutic agents. However, sequence diversity and structural polymorphism of GQs have posed immense challenges in terms of understanding what conformation a G-rich sequence adopts inside the cell and how to specifically target a GQ motif amidst several other GQ-forming sequences. In this context, here we review recent developments in the applications of biophysical tools that use fluorescence readout to probe the GQ structure and recognition in cell-free and cellular environments. First, we provide a detailed discussion on the utility of covalently labeled environment-sensitive fluorescent nucleoside analogs in assessing the subtle difference in GQ structures and their ligand binding abilities. Furthermore, a detailed discussion on structure-specific antibodies and small molecule probes used to visualize and confirm the existence of DNA and RNA GQs in cells is provided. We also highlight the open challenges in the study of tetraplexes (GQ and i-motif structures) and how addressing these challenges by developing new tools and techniques will have a profound impact on tetraplex-directed therapeutic strategies.