Regensburg 2004 – wissenschaftliches Programm

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SYLS: Life Sciences on the Nanometer Scale - Physics Meets Biology

SYLS 3: Symposium "Life Sciences on the Nanometer Scale - Physics Meets Biology"

SYLS 3.11: Poster

Mittwoch, 10. März 2004, 16:00–18:30, B

Time-resolved Fluorescence Resonance Energy Transfer on DNA duplexes — •Petra Müller, Jürgen Köhler, and Dagmar Klostermeier — Experimental Physics IV, University of Bayreuth, Bayreuth

Fluorescence resonance energy transfer (FRET) is an established technique to determine intramolecular distances between 1 and 10 nm. In time-resolved FRET experiments, inter-fluorophore distance information is retrieved from the analysis of fluorescence emission decays of the donor fluorophore in the absence and presence of the acceptor fluorophore.

Here we report distance measurements using our home-built time-resolved FRET setup. Excitation is performed using the frequency-doubled output of a pulsed titanium:sapphire laser, and the nanosecond decay profile of the donor is measured via time-correlated single photon counting. Calibration with a series of donor-acceptor labelled DNA molecules yields inter-fluorophore distances in good agreement with calculated values based on standard DNA B-form geometry and the chemistry of fluorophore attachment. Furthermore, multiple distance distributions for various mixtures of DNA molecules of different lengths can be extracted correctly from the donor decays.

Having established the possibilities and limitations of our time-resolved FRET set-up we will now employ this technique to identify functional conformers of proteins that modulate nucleic acid structures, such as helicases and topoisomerases.