Tetherless, precise and extended observation of single-molecule FRET in an Anti-Brownian trap

Hugh Wilson; Robert S. Windeler; Quan Wang

doi:10.1117/12.2508631

4 March 2019 Tetherless, precise and extended observation of single-molecule FRET in an Anti-Brownian trap

Hugh Wilson, Robert S. Windeler, Quan Wang

Proceedings Volume 10884, Single Molecule Spectroscopy and Superresolution Imaging XII; 108840A (2019) https://doi.org/10.1117/12.2508631
Event: SPIE BiOS, 2019, San Francisco, California, United States

Abstract

A comprehensive understanding of biomolecules calls for the ability to observe single-molecule dynamics at the nanometer scale without constraints. Single-molecule Förster resonance energy transfer (smFRET) is a powerful tool for probing nanoscale dynamics, but existing modalities have limitations. Solution based confocal measurements are restricted by the short (~1ms) diffusion limited observation time. Surface immobilized measurements can extend the observation window, but at the expense of the molecule’s translational and rotational degrees of freedom. Moreover, there is always a concern that immobilization may perturb the biomolecule’s function. We overcome these limitations by combining smFRET optics with the capability to isolate individual molecules in solution using an Anti-Brownian ELectrokinetic (ABEL) trap. Our new platform, ABEL-FRET, enables photon-by-photon recording of smFRET trajectories over tens of seconds in solution, without tethering the molecule to a surface. We first demonstrate ABELFRET using short (~10bp) DNA rulers and achieve near shot-noise limited precision of ΔE~0.01 for 5,000 photons, which enables resolution of single base pair differences in a mixture of FRET-labeled dsDNA molecules. We also demonstrate the capability to make simultaneous measurements of donor fluorescence lifetime and smFRET.

Conference Presentation

Citation Download Citation

Hugh Wilson, Robert S. Windeler, and Quan Wang "Tetherless, precise and extended observation of single-molecule FRET in an Anti-Brownian trap", Proc. SPIE 10884, Single Molecule Spectroscopy and Superresolution Imaging XII, 108840A (4 March 2019); https://doi.org/10.1117/12.2508631

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