[18] Fluorescence resonance energy transfer and nucleic acids

Fluorescence resonance energy transfer (FRET) is a spectroscopic process by which energy is passed nonradiatively among molecules over long distances. The “donor” molecule, which must be a fluorophore, absorbs a photon and transfer this energy nonradiatively to the “acceptor” molecule. Energy can be transferred over distances on the order of common macromolecular dimensions. This chapter discusses the applications of FRET to nucleic acid molecules conjugated to dye molecules; but in general, the donor-acceptor pairs can be free in solution, one or both bound to a macromolecule, or be an inherent part of the structure. The chapter discusses FRET specifying the physical parameters that are available from a variety of fluorescence measurements. In the chapter, some basic considerations and experimental procedures are described that are necessary to make reliable FRET measurements. It also discusses some difficulties that can arise in the experimental proceedings and data analysis and the sources of experimental error are pointed out.