CONFORMATIONAL TRANSITIONS MONITORED FOR SINGLE MOLECULES IN SOLUTION

Phenomena that fan he observed for a large number of molecules may not be understood if it is not possible to observe the events On the sing le-molecule level, We measured the fluorescence lifetimes of individua l tetramethylrhodamine molecules, linked to an 18-mer deoxyribonucleot ide sequence specific for M13 DNA , by time-resolved, single-photon co unting in a confocal fluorescence microscope during Brownian motion in solution. When many molecules were observed, a biexponential fluoresc ence decay was observed with equal amplitudes. However, on tile single -molecule level, the fraction of one of tile amplitudes spanned from 0 to unity for a collection of single molecule detections. Further anal ysis by fluorescence correlation spectroscopy made on many molecules r evealed a process that obeys a stretched exponential relaxation law. T hese facts, combined with previous evidence of the quenching effect of guanosine on rhodamines, indicate that the tetramethylrhodamine molec ule senses conformational transitions as It associates and dissociates to a guanosine-rich area. Thus, our results reveal conformational tra nsitions in a single molecule in solution under conditions that are re levant for biological processes.