The basic theory of FRET has been discussed above. FRET is arguably one of the most powerful of all fluorescent technologies. It can be used in almost all assay formats and allow the user to monitor interactions occurring at macromolecular distances, 1 –10 nm. Upon energy transfer that depends on R^-6, where R is the distance between the donor and the acceptor, the donor’s lifetime and quantum yield is reduced and the acceptor fluorescence is increased, or sensitized. When using FRET in most applications, the donor and acceptor dyes are different, in these cases FRET can be detected by the fluorescence emission of the acceptor or by quenching of donor fluorescence. The dyes used in FRET must have spectral overlap so pairs of dyes typically used are Cy3/Cy5 and Cy5/Cy5.5 (Ref. 20). Using these types of dyes it has been possible to examine a wide range of molecular interactions such as enzyme assays, immunoassays and other binding events.

The enzyme, metallo-endoproteinase, (Asp-N) is used to cleave an 8 amino acid peptide on the N-terminal side of aspartic acid (D). The assay has been configured in a quench format (figure 23). The N-terminus of the peptide has been labelled with either Cy3 or Cy3B and the C-terminus with the quencher Cy5Q. This means that following illumination at Cy3 excitation wavelengths the intact peptide cannot generate a fluorescent signal at Cy3 emission wavelengths due to quenching by Cy5Q. However following proteolytic digestion of the peptide the quench effect is removed. This means that excitation at Cy3 wavelengths now results in emission at Cy3 wavelengths. The assay format could, in principle, be adapted for use with other more therapeutically relevant proteases such as the matrix metalloproteinases (MMP), which are known to be involved in diseases such as arthritis, cancer and multiple sclerosis.

The second application involves the interaction between the p65 subunit of the transcription factor, NF-kB and its DNA binding site (figure 24). An anti-GST antibody labelled with Cy3 (approx. 7-12 dyes per molecule) is allowed to interact with an affinity purified GST fusion protein of p65 and GST (p65GST). A double-stranded DNA (dsDNA) sequence of 19 base pairs which contains the NF-kB binding site is singly labelled with Cy5 at the 5’ end of the coding sequence. This is then incubated with the Cy3 labelled antibody and p65GST. This reaction is done either in the presence or absence of unlabelled non-specific or specific competitor dsDNA. In the absence of either competitor, binding by p65-GST results in FRET between the Cy3 donor molecules (when excited at Cy3 ex wavelengths) located on anti-GST and Cy5 acceptor molecule on dsDNA (measurements taken at Cy5 em wavelength). The inclusion of a large excess of unlabelled dsDNA sequences as competitors have been investigated. The specificity of the FRET reaction has been examined using either the specific NF-kB sequence (same as substrate) or unrelated sequences containing no consensus binding sequence. NF-kB is of great relevance to the pharmaceutical sector due to its ability to regulate a number of genes involved in various immune and inflammatory responses. As such NF-kB has been implicated in several disease states including various viral infections (HIV), arthritis and cancer.

One of the original and most important applications of FRET is the field of molecular beacons. Molecular beacons are oligonucleotide probes that can detect the presence of specific nucleic acids in solution. (Tyagi and Kramer Ref. 26). Molecular beacons are hairpin-shaped molecules with an internally quenched fluorophore whose fluorescence is restored when they bind to a target nucleic acid . The loop section of the molecule is a probe sequence that is complementary to a target nucleic acid molecule. The stem is formed by the annealing of complementary arm sequences on the ends of the probe sequence. A fluorescent dye is covalently linked to the end of one arm and a quencher is attached to the end of the other arm. The close proximity of the dye and quencher causes the fluorescence of the dye to be quenched by energy transfer. When the probe hybridises to a target molecule, it forms a hybrid that is longer and more stable than the stem. This, means that the molecular beacon undergoes a conformational change that forces the stem apart, and so the fluorescent dye and quencher are forced apart, this in turns restores the ability of the dye to fluoresce.

• Time-resolved fluorescence energy transfer (TRFRET)
  
  Fluorescence resonance energy transfer (FRET) in which a fluorescent donor molecule transfers energy via a non-radiative dipole-dipole interaction to an acceptor molecule (which is usually a fluorescent molecule), is a standard spectroscopic technique. Homogeneous time resolved fluorescence energy transfer can be used to overcome many of the problems encountered with conventional chelates. The technique of TRFRET is based on fluorescence energy transfer and the use of long-lived fluorescent molecules. This involves using a lanthanide as a donor and a conventional fluor as an acceptor molecule. Typically this system would be excited in the UV and the lanthanide, which would emit at ~600 nm for europium or 450 nm for terbium, would transfer the energy directly to the acceptor molecule if it was in close proximity, following a binding event. This uses the strength of the lanthanide to overcome background fluorescence as well as energy transfer to produce a homogeneous format. The use of chelates of the rare earth elements as labels offers advantages particularly the signal to noise ratio, over conventional fluorophores
  
  For an example of this technique please click here