Single-molecule FRET for probing nanoscale biomolecular dynamics

Single-molecule spectroscopy is a powerful method for studying the physics of molecular systems, particularly biomolecules, such as proteins and nucleic acids. By avoiding ensemble averaging, single-molecule techniques can resolve structural distributions and fluctuations even for complex and conformationally heterogeneous systems; they also reveal the close link between biological function and the statistical mechanics of the underlying processes. The combination of single-molecule fluorescence detection with F & ouml;rster resonance energy transfer has become an essential tool for probing biomolecular dynamics on timescales ranging from nanoseconds to days. This Review briefly outlines the state of the art of single-molecule F & ouml;rster resonance energy transfer spectroscopy and then highlights some of the most important physics-based developments that are expected to further expand the scope of the technique. Key areas of progress include improved time resolution, access to nonequilibrium dynamics and synergies with advances in data analysis and simulations. These developments create new opportunities for attaining a comprehensive understanding of the dynamics and functional mechanisms of biological processes at the nanoscale. The combination of single-molecule fluorescence detection with F & ouml;rster resonance energy transfer provides a powerful probe of biomolecular dynamics on timescales ranging from nanoseconds to days. This Review outlines single-molecule F & ouml;rster resonance energy transfer spectroscopy with a focus on dynamics and highlights future developments and enhanced capabilities. The functions of biological macromolecules depend on changes in their conformations across 24 orders of magnitude in time.Single-molecule F & ouml;rster resonance energy transfer can be used to probe biomolecular dynamics on nanometre-length scales across timescales from nanoseconds to days.An important challenge is to increase the time resolution for measurements of rapid dynamics and nonequilibrium processes.Nanophotonics, microfluidic mixing and advances in data analysis and molecular simulations are particularly promising strategies for extending the scope of single-molecule F & ouml;rster resonance energy transfer.