Tetherless, Precise and Extended Observation of Single-Molecule FRET in an Anti-Brownian Trap

Abstract

A comprehensive understanding of biomolecules calls for the ability to observe single-molecule dynamics at the nanometer scale without constraints. Single-molecule Forster 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.