Monitoring conformational dynamics of a single molecule by selective fluorescence spectroscopy

A recently developed, real-time spectroscopic technique, burst-integrated fluorescence lifetime (BIFL), is shown to be well suited for monitoring the individual molecular conformational dynamics of a single molecule diffusing through the microscopic, open measurement volume (approximate to 10 fl) of a confocal epi-illuminated set-up, In a highly diluted aqueous solution of 20-mer oligonucleotide strand of DNA duplex labeled with the environment-sensitive fluorescent dye tetramethylrhodamine (TMR), fluorescence bursts indicating traces of individual molecules 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 complementary autocorrelation analysis was performed on the time-dependent fluctuations in fluorescence lifetime and intensity, The consistent results strongly support the hypothesized three-state model of the conformational dynamics of the TMR-DNA duplex with a polar, a nonpolar, and a quenching environment of TMR.