DNA dynamics: a fluorescence resonance energy transfer study using a long-lifetime metal-ligand complex
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Fluorescent probes bound to DNA typically display nanosecond decay times and reveal only nanosecond motions. We extend the time range of measurable DNA dynamics using [Ru(bpy)2(dppz)]2+ (bpy=2,2’-bipyridine, dppz=dipyrido[3,2-a:2’,3’-c]phenazine) (RuBD) which displays a mean lifetime near 90 ns. To test the usefulness of RuBD as a probe for diffusive processes in calf thymus DNA, we compared the efficiencies of fluorescence resonance energy transfer (FRET) using three donors which display lifetimes near 5 ns for acridine orange (AO), 22 ns for ethidium bromide (EB) and 92 ns for RuBD, with nile blue (NB) as the acceptor. The Förster distances for AO-NB, EB-NB and RuBD-NB donor-acceptor pairs were 42.3, 52.3, and 30.6 A, respectively. All three donors showed dramatic decreases in fluorescence intensities and more rapid intensity decays with increasing NB concentrations. The intensity decays of AO and EB in the presence of varying concentrations of NB were satisfactorily described by the one-dimensional FRET model without diffusion (Blumen and Manz, 1979). In the case of the long-lifetime donor RuBD, the experimental phase and modulation somewhat deviated from the recovered values computed from this model. The recovered NB concentrations and FRET efficiencies from the model were slightly larger than the expected values, however, the recovered and expected values did not show a significant difference. Thus, it is suggested that the lifetime of RuBD is too short to measure diffusive processes in calf thymus DNA.
Key wordsFluorescence resonance energy transfer Long-lifetime metal-ligand complex Diffusion in DNA Frequency-domain fluorometry
- Gratton, E., Lakowicz, J. R., Maliwal, B. P., Cherek, H. and Laczko, G., Resolution of mixtures of fluorophores using variable-frequency phase and modulation data.Biophys. J., 46, 478–486 (1984).Google Scholar
- Kang, J. S. and Lakowicz, J. R., Fluorescence resonance energy transfer in calf thymus DNA from a long-lifetime metal-ligand complex to nile blue.J. Biochem. Mol. Biol., 34, 551–558 (2001).Google Scholar