Fluorescence energy transfer in one dimension: frequency-domain fluorescence study of DNA-fluorophore complexes

Fluorescence resonance energy transfer among linear DNA bound fluorophores was carried out to study the process in one dimension. The donor fluorescence intensity decays in the case of energy transfer in one dimension are stretched exponential and show exp[-(t/tau)1/6] time dependence, which results in an initial more rapid decay and subsequent slower decay at long times when compared to those in higher dimensions. DNA-bound 4',6'-diamidino-2-phenyl indole (DAPI), acridine orange (AO), and ethidium bromide (EB) were used as donors. The acceptors were in the case of DAPI AO and EB; in the case of AO nile blue (NB), methylene blue (MB), and crystal violet (CV); and NB, MB, and oxazine 750 in the case of EB. As expected, the donor intensity decays became highly heterogeneous upon energy transfer and were characterized by the simultaneous presence of both highly and marginally quenched donors. The intensity decays for all three donors in the presence of various acceptors are satisfactorily described by the Förster model of energy transfer in one dimension. The intensity decays also allow for clear rejection of a two- or three-dimensional model. The experimentally recovered critical Förster distances (R0) ranged between 37 A in the case of DAPI and EB to 70 A in the case of AO and CV donor-acceptor pairs. These recovered R0 values compare reasonably with those calculated from spectral properties if we use values of 1.25 for k2, and 1.5 for the refractive index of DNA. The k2 value will be even higher, between 1.5 and 2.0, if the consensus DNA refractive index of 1.75 is used. These k2 values strongly suggest that the dipoles of the acceptor chromophores when bound to DNA are not randomly oriented but are aligned preferentially in plane.