Fluorescence resonance energy transfer provides valuable long-range distance information about macromolecules in solution. Fluorescein and Cy3 are an important donor-acceptor pair of fluorophores; the characteristic Forster length for this pair on DNA is 56 Å, so the pair can be used to study relatively long distances. Measurement of FRET efficiency for a series of DNA duplexes terminally labeled with fluorescein and Cy3 suggests that the Cy3 is close to the helical axis of the DNA. An NMR analysis of a self- complementary DNA duplex 5'-labeled with Cy3 shows that the fluorophore is stacked onto the end of the helix, in a manner similar to that of an additional base pair. This provides a known point from which distances calculated from FRET...