Distance determination in protein-DNA complexes using fluorescence resonance energy transfer

Fluorescence resonance energy transfer (FRET) provides distance information between a donor and an acceptor dye in the range of 10-100 A. Knowledge of the exact positions of some dyes (e.g., fluorescein, rhodamine, or Cy3) with respect to nucleic acids and DNA design enables us to translate these data into precise structural information using molecular modeling. Here we describe this in vitro approach from the design and synthesis of the DNA FRET samples to the fluorescence spectroscopy methods and analysis. Advances in the preparation of dye-labeled nucleic acid molecules and modern techniques like the measurement of FRET in vivo lead to an increased importance of FRET studies in structural and molecular biology.