Single-pair fluorescence resonance energy transfer on freely diffusing molecules: Observation of Forster distance dependence and subpopulations

Photon bursts from single diffusing donor-acceptor labeled macromolecules w ere used to measure intramolecular distances and identify subpopulations of freely diffusing macromolecules in a heterogeneous ensemble. By using DNA as a rigid spacer, a series of constructs with varying intramolecular donor -acceptor spacings were used to measure the mean and distribution width of fluorescence resonance energy transfer (FRET) efficiencies as a function of distance. The mean single-pair FRET efficiencies qualitatively follow the distance dependence predicted by Forster theory. Possible contributions to the widths of the FRET efficiency distributions are discussed, and potentia l applications in the study of biopolymer conformational dynamics are sugge sted. The ability to measure intramolecular (and intermolecular) distances for single molecules implies the ability to distinguish and monitor subpopu lations of molecules in a mixture with different distances or conformationa l states. This is demonstrated by monitoring substrate and product subpopul ations before and after a restriction endonuclease cleavage reaction. Dista nce measurements at single-molecule resolution also should facilitate the s tudy of complex reactions such as biopolymer folding. To this end, the dena turation of a DNA hairpin was examined by using single pair FRET.