High-resolution and tether-free single-molecule FRET with hydrodynamic profiling

Hugh Wilson; Quan Wang

doi:10.1117/12.2567676

20 August 2020 High-resolution and tether-free single-molecule FRET with hydrodynamic profiling

Hugh Wilson, Quan Wang

Proceedings Volume 11463, Optical Trapping and Optical Micromanipulation XVII; 1146307 (2020) https://doi.org/10.1117/12.2567676
Event: SPIE Nanoscience + Engineering, 2020, Online Only

Abstract

Single-molecule Förster resonance energy transfer (smFRET) is a powerful tool for probing nanoscale conformation and dynamics, but existing modalities have limitations. Solution-based confocal measurements have a short (~1ms), diffusion-limited observation time and extending the observation window by immobilization restricts the molecule’s translational and rotational degrees of freedom. We overcome these limitations by combining smFRET with the capability to isolate individual molecules in solution using an Anti-Brownian ELectrokinetic (ABEL) trap. Our platform, ABEL-FRET, enables photon-by-photon recording of smFRET over tens of seconds in solution and achieves near shot-noise limited resolution in FRET efficiency for short (10-30bp) DNA rulers. We further demonstrate that combining high-resolution smFRET spectroscopy with simultaneous inference of single-molecule diffusivity offers an expanded view of biomolecules and their complexes, filling a gap in the single-molecule toolkit.

Conference Presentation

Citation Download Citation

Hugh Wilson and Quan Wang "High-resolution and tether-free single-molecule FRET with hydrodynamic profiling", Proc. SPIE 11463, Optical Trapping and Optical Micromanipulation XVII, 1146307 (20 August 2020); https://doi.org/10.1117/12.2567676

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