MONITORING CONFORMATIONAL DYNAMICS OF A SINGLE-MOLECULE BY SELECTIVE FLUORESCENCE SPECTROSCOPY

A recently developed, real-time spectroscopic technique, burst-integra ted fluorescence lifetime (BIFL), is shown to be well suited for monit oring the individual molecular conformational dynamics of a single mol ecule diffusing through the microscopic, open measurement volume (appr oximate to 10 fl) of a confocal epi-illuminated set-up, In a highly di luted aqueous solution of 20-mer oligonucleotide strand of DNA duplex labeled with the environment-sensitive fluorescent dye tetramethylrhod amine (TMR), fluorescence bursts indicating traces of individual molec ules are registered and further subjected to selective burst analysis, The two-dimensional BIEL data allow the identification and detection of different temporally resolved conformational states, A complementar y autocorrelation analysis was performed on the time-dependent fluctua tions in fluorescence lifetime and intensity, The consistent results s trongly support the hypothesized three-state model of the conformation al dynamics of the TMR-DNA duplex with a polar, a nonpolar, and a quen ching environment of TMR.