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 Förster distance (R ) that was calculated from the spectral overlap was 33.4 Å. 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 μM NB. The FRET efficiency values that are calculated from the mean decay times increased from 0.107 at 20 μM NB to 0.474 at 100 μM 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.