FRET (Fluorescence Resonance Energy Transfer) is based on the transfer of energy between two fluorophores, a donor and an acceptor, when in close proximity. Molecular interactions between biomolecules can be assessed by coupling each partner with a fluorescent label and by detecting the level of energy transfer.
When two entities come close enough to each other, excitation of the donor by an energy source (e.g. a flash lamp or a laser) triggers an energy transfer towards the acceptor, which in turn emits specific fluorescence at a given wavelength (Figure 1). The donor and acceptor can be grafted covalently onto multiple partners that can associate, among others, two dimerizing proteins, two DNA strands, an antigen and an antibody, or a ligand and its receptor.
Because of these spectral properties, FRET, a donor-acceptor complex, can be detected without the need for physical separation from the unbound partners. Fully homogeneous assays do not require separation steps such as centrifuging, washing, filtration, or magnetic partitioning.
Traditional FRET chemistries are hampered by background fluorescence from sample components such as buffers, proteins, chemical compounds and cell lysate. This type of background fluorescence is extremely transient (with a lifetime in the nanosecond range) and can therefore be eliminated using time-resolved methodologies.
Figure 1: The detection of specific fluorescence is a sign that a FRET process has taken place, caused by the proximity of the two interacting partners.
HTRF combines standard FRET technology with time-resolved measurement of fluorescence, eliminating short-lived background fluorescence. As shown in Figure 2, introducing a time delay of approximately 50 to 150 µ secondsbetween the system excitation and fluorescence measurement allows the signal to be cleared of all non-specific short-lived emissions. In contrast, HTRF acceptors emit long-lived fluorescence when engaged in a FRET process. Therefore, long-lived emissions signify energy transfer due to the proximity of the labeled biomolecules.
Of course, FRET is governed by the physics of molecular proximity, which only allows this phenomenon to occur when the distance between the donor and the acceptor is short enough. In practice, FRET systems are characterized by the Förster's radius (R0) distance at which FRET efficiency is 50%. For HTRF R0 lies between 50 and 90 Å, depending on the acceptor used. Nevertheless, this distance is theoretical, as it does not take into account spatial molecular rearrangements. Therefore, a variety of HTRF assays involving molecular complexes of different sizes have been implemented.
They include assessment of small phosphorylated peptides, immunoassays for quantifying large glycoproteins such as thyroglobulin, and indirect detection (via secondary antibodies) of tagged complexes such as CD28/CD86 (Figure 3), and therefore a broad range of possible molecular distances.
Figure 2: The energy pulse from the excitation source (flash lamp, laser) is immediately followed by a time delay, allowing interfering short-lived fluorescence (compounds, proteins, medium…) to decay.
Figure 3: Two HTRF assays theoretically involving very different donor-acceptor distances. Detecting a phosphorylated biotinylated-peptide (short distance), and CD28/CD86 association quantified by anti-tag conjugates (long distance).