Single-molecule acceptor rise time (smART) FRET for nanoscale distance sensitivity

The structure, dynamics, and binding of individual biomolecules have been extensively investigated using single-molecule Förster resonance energy transfer (smFRET) as a ‘spectroscopic ruler.’ The FRET efficiency between a fluorophore pair is used to measure distances in the several nanometer range. Existing approaches to detect closer distances come at the expense of sensitivity to longer distances. Here, we introduce single-molecule acceptor rise-time (smART) FRET that spans closer and longer distances. The acceptor rise time encodes the FRET rate, which scales polynomially with distance and thus has a steep dependence that expands the working range by 50%. High precision and accuracy is achieved through the spectroscopic separation between the rise time and the photophysical fluctuations that obfuscate other FRET readouts. Using the nanoscale sensitivity, we resolved the architectures of DNA bound to the single-stranded binding protein from E. coli , demonstrating the ability of smART FRET to elucidate the complex behaviors of biomolecules. ### Competing Interest Statement The authors have declared no competing interest.