Location of cyanine-3 on double-stranded DNA: Importance for fluorescence resonance energy transfer studies

Fluorescence resonance energy transfer provides valuable long-range distanc e information about macromolecules in solution. Fluorescein and Cy3 are an important donor-acceptor pair of fluorophores; the characteristic Forster l ength for this pair on DNA is 56 Angstrom, 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-comp lementary DNA duplex 5'-labeled with Cy3 shows that the fluorophore is stac ked 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 fro m FRET measurements are measured. Using the FRET efficiencies for the serie s of DNA duplexes as restraints, we have determined an effective position f or the fluorescein, which is maximally extended laterally from the helix. T he knowledge of the fluorophore positions can now be used for more precise interpretation of FRET data from nucleic acids.