ABEL-FRET: tether-free single-molecule FRET with hydrodynamic profiling

Single-molecule Forster resonance energy transfer (smFRET) has become a versatile and widespread method to probe nanoscale conformation and dynamics. However, current experimental modalities often resort to molecule immobilization for long observation times and do not always approach the resolution limit of FRET-based nanoscale metrology. Here we present ABEL-FRET, an immobilization-free platform for smFRET measurements with ultrahigh resolving power in FRET efficiency. Importantly, single-molecule diffusivity is used to provide additional size and shape information for hydrodynamic profiling of individual molecules, which, together with the concurrently measured intramolecular conformation through FRET, enables a holistic and dynamic view of biomolecules and their complexes. ABEL-FRET combines anti-Brownian trapping for tether-free observation of biomolecules in solution with smFRET. ABEL-FRET offers ultrahigh resolution of FRET efficiency and enables simultaneous hydrodynamic profiling of molecular composition.

Automated summary

ABEL-FRET is an immobilization-free platform with ultrahigh resolution in FRET efficiency, providing additional size and shape information through single-molecule diffusivity for a holistic and dynamic view of biomolecules.