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.
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.
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