Single-Molecule FRET at 10 MHz Count Rates

A bottleneck in many studies utilizing single-molecule Forster Resonance Energy Transfer (smFRET) is the attainable photon count rate as it determines the temporal resolution of the experiment. As many biologically relevant processes occur on timescales that are hardly accessible with currently achievable photon count rates, there has been considerable effort to find strategies to increase the stability and brightness of fluorescent dyes. Here, we use DNA nanoantennas to drastically increase the achievable photon count rates and to observe fast biomolecular dynamics in the small volume between two plasmonic nanoparticles. As a proof of concept, we observe the coupled folding and binding of two intrinsically disordered proteins which form transient encounter complexes with lifetimes on the order of 100 s. To test the limits of our approach, we also investigated the hybridization of a short single-stranded DNA to its complementary counterpart, revealing a transition path time of 17 s at photon count rates of around 10 MHz, which is an order-of-magnitude improvement when compared to the state of the art. Concomitantly, the photostability was increased, enabling many seconds long megahertz fluorescence time traces. Due to the modular nature of the DNA origami method, this platform can be adapted to a broad range of biomolecules, providing a promising approach to study previously unobservable ultrafast biophysical processes.