A Single Molecule Study of G-Quadruplex and Telomestatin Intractions

Abstract

Small molecules that interact with and stabilize G-quadruplex (GQ) structures have attracted attention due to their potential use as anti-cancer drugs and in biotechnological applications where GQ is used as a sensor. Some of these small molecules have been studied using bulk fluorescence microscopy methods as they demonstrate fluorescence enhancement or quenching upon binding to GQ structures. However, the fluorescence signal generated by these molecules has not been strong enough to enable their study using single molecule fluorescence methods, which are particularly sensitive to dynamics of the interactions and heterogeneities of the system. In this study, we used an acceptor (Cy5) labeled telomestatin, which is a prominent GQ stabilizing small molecule, and a donor (Cy3) labeled GQ construct to perform single molecule Forster resonance energy transfer (smFRET) measurements. By placing the Cy3 at different locations on the DNA, we determined that telomestatin stacks on top of a particular G-tetrad layer (top layer) for 2-30 seconds before dissociating, with a minor population lasting significantly longer times. Furthermore, our data implies that telomestatin can stack on the G-tetrad in at least two different orientations. We investigated the effects of this stacking orientation, GQ stability, and GQ conformation on the duration of stable stacking interactions. We also studied interactions of telomestatin with intermediate folding structures, before GQ structure is attained, and observed a telomestatin induced stability in these structures as well.