Fluorescence resonance energy transfer at the single-molecule level

Fluorescence resonance energy transfer (FRET) is a powerful spectroscopic method for measuring distances in the 2–8 nm range. Often, conformational changes and molecular interactions are difficult or impossible to synchronize, or too rare or transient to detect using ensemble FRET. Single-molecule FRET (smFRET) opens new opportunities to probe biomolecular conformational changes or interactions that are missing in static snapshots provided by traditional structural biology tools, as well as to measure the kinetics of these dynamics on various timescales and under physiological conditions, including inside cells. Advances in labelling technologies, combining smFRET with optical and magnetic tweezers and Bayesian inference-based and information theory-based analysis tools are revealing rich biomolecular dynamics. We also discuss the challenges and opportunities in integrating dynamics into traditionally static structural biology approaches, extending smFRET into cells and tissues, advancing technical innovations and democratizing the practice of smFRET.