Below the FRET Limit: A New Quantitative Single-Molecule Tool for Measuring Short-Range (0-3 NM) Biomolecular Conformations

Single-molecule spectroscopy (SMS) provides a powerful tool box for investigation of biomolecular dynamics, and chiefly within this toolbox single-molecule FRET (smFRET) allows for monitoring of interactions on the 3-10 nm distance scale. More recently smFRET has been shown to be capable of measuring absolute distances within this range, however interaction distances around and below 3 nm have remain challenging due to reduced signal from dye-dye pairs of lower Förster radius, and photophysical phenomena caused by dye-dye contact. Here we develop an alternative spectroscopic approach exploiting dye quenching to provide a means of monitoring interactions over 0-3 nm. Dye-dye quenching was previously used to measure binary on/off states in a biomolecular system at the single-molecule level. Our new experiments using a series of doubly labelled duplex DNAs establish a quantitative relationship between accessible volume (AV) overlap and quenching efficiency, and our all-atom molecular dynamics simulations (with explicit dyes) provide additional insight into the mechanism of contact quenching. The quantitative relationship between AV overlap and quenching provides a link between our measurements and biomolecular structures, and is sufficiently sensitive to discriminate biomolecules in which the labelling positons vary by less than 4 Å, in cases where the FRET efficiencies remain indistinguishably high. Kinetic models involving the solution viscosity and AV overlap, quantitatively reproduce our experimental data, a feature we are now exploiting in developing single-molecule ratiometric viscosity sensors for applications in membrane dynamics and phase separation. Further molecular dynamics simulations and surface-immobilised SMS will further elucidate the quenching mechanism, facilitating the application of this method to investigate subtle biomolecular interactions over shorter distance scales than currently accessible to quantitative single-molecule techniques.