Characterizing The Equilibrium Blinking Behavior Of Fluorogens With Fluorogen Activating Proteins (FAPs)

Abstract

We have recently developed FAPs (fluorogen activating proteins) that can specifically activate the fluorogenic dyes thiazole orange (TO) and malachite green (MG) with nanomolar affinities. Upon binding to FAPs, the otherwise dark fluorogens display thousands of fold increase in fluorescence intensity. The reversible interaction between the fluorogen and the FAP allows the same FAP to bind and activate fresh fluorogens in solution after one fluorogen dissociates from the FAP. Therefore, the binding and unbinding reactions lead to on-and-off (blinking) fluorescent signals at the single molecule level. As a result, this FAP-fluorogen system is ideal for generating a renewable, photostable signal and providing high photon flux. Since the localization accuracy is in principle limited by the number of photons collected from fluorescent probes, our module holds great promise for achieving super-resolution imaging (See abstract by Lidke, K.A. et al.). In this study, we characterize the blinking behavior of FAP-fluorogen pairs at equilibrium. We show that the on-time is highly dependent on the excitation power density and the off-time is controlled by the free dye concentration and therefore, is limited by diffusion. By adjusting the dye concentration in solution and the intensity of incident power, the on and off rates of the binding reaction are controllable and could be optimized to resolve the objects in time and space. Clones of the FAP with point mutations show different blinking behaviors, which suggests that these amino-acid residues could play an important role in FAP-fluorogen binding and indicates that structural differences of the FAP at the binding interface can change the photochemical and photophysical properties of fluorogen.