Fluorescence Resonance Energy Transfer in Calf Thymus DNA from a Long-Lifetime Metal-Ligand Complex to Nile Blue

We extended the measurable time scale of DNA dynamics to submicrosecond using a long-lifetime metal-ligand complex, $[Ru(phen)_2(dppz)]^{2+}$ (phen=1,10-phenanthroline, dppz=dipyrido[3,2-a:2',3'-c]phenazine) (RuPD), which displays a mean lifetime near 350 ns. We partially characterized the fluorescence resonance energy transfer (FRET) in calf thymus DNA from RuPD to nile blue (NB) using frequency-domain fluorometry with a high-intensity, blue light-emitting diode (LED) as the modulated light source. There was a significant overlap of the emission spectrum of the donor RuPD with the absorption spectrum of the acceptor NB. The F$\ddot{o}$rster distance ($R_0$) that was calculated from the spectral overlap was $33.4\;{\AA}$. We observed dramatic decreases in the steady-state fluorescence intensities of RuPD when the NB concentration was increased. The intensity decays of RuPD were matched the closest by a triple exponential decay. The mean decay time of RuPD in the absence of the acceptor NB was 350.7 ns. In a concentration-dependent manner, RuPD showed rapid intensity decay times upon adding NB. The mean decay time decreased to 184.6 ns at $100\;{\mu}M$ NB. The FRET efficiency values that are calculated from the mean decay times increased from 0.107 at $20\;{\mu}M$ NB to 0.474 at $100\;{\mu}M$ NB concentration. The use of FRET with a long-lifetime metal-ligand complex donor is expected to offer the opportunity to increase the information about the structure and dynamics of nucleic acids.