First Realization of Single-Molecule Four-Color FRET

Abstract

Single-molecule FRET (Fluorescence Resonance Energy Transfer) has provided unprecedented details on fundamental processes in biology. However, information of single inter-fluorophore distances in conventional two-color FRET is insufficient to completely capture the intrinsic complexity of many biological systems. Despite recent developments of single-molecule three-color FRET techniques, there is an ever increasing demand for more advanced FRET techniques, four-color FRET, as single-molecule approaches are being expanded to include biological systems with multiple components.Here, we report single-molecule four-color FRET technique both in confocal and in total-internal-reflection fluorescence microscopies. Real-time determination of six inter-fluorophore FRET efficiencies allowed us to probe the correlated motion of four arms of the Holliday junction. The technique was also applied to assess the correlation of RecA-mediated strand exchange events at both ends of a synaptic complex. We expect that the technique will have broad applications in measuring the correlated dynamics of complex biological systems.