Probing the interaction between two single molecules: fluorescence resonance energy transfer between a single donor and a single acceptor.

TLDR

Classical ensemble FRET equations are adapted for single-molecule measurements. The study extends FRET sensitivity to single molecules by measuring energy transfer between a single donor and a single acceptor fluorophore. Near-field scanning optical microscopy captures dual-color images and spectra of donor–acceptor pairs linked by short DNA, while photodestruction dynamics assess transfer presence and efficiency, with ensemble equations adapted for single-molecule analysis. Single-pair FRET reveals dynamic molecular events and enables monitoring of nanometer-scale conformational changes that are obscured in ensemble measurements.

Abstract

We extend the sensitivity of fluorescence resonance energy transfer (FRET) to the single molecule level by measuring energy transfer between a single donor fluorophore and a single acceptor fluorophore. Near-field scanning optical microscopy (NSOM) is used to obtain simultaneous dual color images and emission spectra from donor and acceptor fluorophores linked by a short DNA molecule. Photodestruction dynamics of the donor or acceptor are used to determine the presence and efficiency of energy transfer. The classical equations used to measure energy transfer on ensembles of fluorophores are modified for single-molecule measurements. In contrast to ensemble measurements, dynamic events on a molecular scale are observable in single pair FRET measurements because they are not canceled out by random averaging. Monitoring conformational changes, such as rotations and distance changes on a nanometer scale, within single biological macromolecules, may be possible with single pair FRET.