Single-molecule Förster resonance energy transfer (smFRET) has become a powerful nanoscopic tool in studies of biomolecular structures and nanoscale objects; however, conventional smFRET measurements are generally blind to distances above 10 nm thus impeding the study of long-distance phenomena. Here, we report the development of farFRET, a technique that extends the range in smFRET measurements beyond the 10 nm line by enhanced energy transfer using multiple acceptors. We demonstrate that farFRET can be readily employed to quantify FRET efficiencies and conformational dynamics using double-stranded DNA molecules, RecA-filament formation on single-stranded DNA and Holliday junction dynamics. farFRET allows quantitative measurements of large biomolecular complexes and nanostructures thus bridging the remaining gap to superresolution microscopy.
Keywords: Holliday junction; RecA; biomolecular complexes; confocal spectroscopy; large conformational changes; smFRET.