Farfret: Extending the FRET Range in Single-Molecule Measurements with Multiple Acceptors

Abstract

Measurements of Forster resonance energy transfer (FRET) efficiencies at the single-molecule level have become a powerful and invaluable tool as a spectroscopic nanoruler in quantitative studies of biomolecular structures and dynamics for distances between 2 to 10 nm. However, many biological macromolecular complexes and interactions exceed this relatively short range accessible to FRET, thus limiting the great potential of single-molecule FRET methodologies in biological applications. Although recent advances in super-resolution microscopy have paved the way for measuring distances down to 20 nm lateral resolution, there still remains a gap for measuring distances between 10-20 nm.Here, we present a method termed farFRET that bridges these two scales and extends the conventional range of FRET beyond 10 nm by using the enhanced energy transfer probability of multiple acceptors, the so-called ‘antenna effect’ (Bojarski et al. J. Phys. Chem. B, 2011, 115, 10120). We combine single-molecule FRET confocal microscopy with pulsed interleaved excitation (PIE) measurements and time-correlated single-photon counting (TCSPC). This allows us to simultaneously measure FRET, stoichiometry and fluorescence lifetime to be able to distinguish farFRET populations with different number of active acceptors.We show the possibilities of this method on a model system of dsDNA. Our experimental findings are supported by extensive Monte-Carlo simulations. Therefore, a precise distance measurement that surpasses the 10 nm limit is readily available for various future applications.