Monitoring Small Molecule and G-Quadruplex Interactions and Kinetics Using Single Molecule FRET

Telomestatin and oxazole telomestatin derivatives (OTD) are small molecules stabilizing G-quadruplex (GQ) structures and are prominent due to their anti-cancer drug potential. Despite the observation of enhanced thermodynamic stability imparted by such small molecules on GQ, the underlying dynamics of small molecule-GQ interactions are not known. To have a better understanding of these interactions we employed single molecule Förster resonance energy transfer (smFRET) to study the system, where we utilized a Cy5-labeled OTD (L1Cy5-7OTD). These studies demonstrate that interactions of this small molecule with GQ are dynamic in terms of binding kinetics and possibly in terms of rotational freedom. The Cy5 fluorophore has enabled monitoring and quantifying binding, dwell, and dissociation of a single L1Cy5-7OTD molecule as it interacted with GQ, which to our knowledge has not been demonstrated for any GQ stabilizing small molecule before. We show that L1Cy5-7OTD remains bound to GQ for tens of seconds, with significantly longer dwell times and higher binding frequencies for more stable GQ. In addition, we propose L1Cy5-7OTD to have at least two preferred primary binding orientations and is able to transition between these orientations while it remains stacked on a G-tetrad.