The assay is based on fluorescence resonance energy transfer (FRET), which is a powerful technique for detecting the molecular proximity of fluorescently labelled molecules. The process involves the transfer of energy from a donor fluorophore in an excited state that is attached to one type of molecule, to a nearby acceptor fluorophore that is attached to another. The fluorophores are chosen so that the emission wavelength of the donor is the same as the excitation wavelength of the acceptor. Detection of fluorescent light at the emission wavelength of the acceptor molecule indicates that the donor has excited the acceptor, and that the labelled molecules are therefore less than 10-nm apart.
Although FRET is already used in protein co-localization studies, Heyduk and Heyduk have adapted this technique to detect DNA–protein binding. They designed two DNA fragments, the molecular beacons, each of which contained half of a full DNA-binding sequence labelled with either a fluorescent donor or acceptor. The efficiency of spontaneous combination of the two DNA fragments is low. However, in the presence of a suitable DNA-binding protein, the high affinity of the protein for the binding-site sequence drives the DNA fragments to combine, bringing the donor and acceptor fluorophores into close proximity, and thereby resulting in a strong FRET signal. Molecular beacons were designed and shown to work successfully for several different proteins.
This is a preview of subscription content, access via your institution
