Photophysics of backbone fluorescent DNA modifications: reducing uncertainties in FRET

We have investigated the photophysical properties of backbone fluorescent DNA modifications with the goal of reducing many of the sources of uncertainty commonly encountered in Forster resonance energy transfer (FRET) measurements. We show that backbone modifications constrain rotational motions, providing a way by which the orientation of the dye can be controlled in a predictable manner, and reduce the uncertainties in donor-acceptor distance associated with the flexible linkers commonly used in conjugate chemistry. Rotational rigidity also prevents undesirable dye-DNA interactions, which have been shown to affect the photophysical properties of the dye. Unusually large FRET efficiencies for donor-acceptor pairs separated by 102 A (three helical turns) were measured and attributed to the favorable relative orientation of the dipoles. The same FRET efficiency was measured for a sample in which the donor-acceptor separation was 12 A shorter, demonstrating the important role of relative orientation in FRET experiments.