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Monitoring conformational dynamics of a single molecule by selective fluorescence spectroscopy

Monitoring conformational dynamics of a single molecule by selective fluorescence spectroscopy

Proceedings Of The National Academy Of Sciences Of The United States Of America. 95(4): 1556-1561,. 17

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 (apprxeq10 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 BIFL 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.

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Accession: 009048300

Download citation: RISBibTeXText

PMID: 9465054

DOI: 10.2307/44322

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