In FRET assays, interaction between biomolecules is measured indirectly by conjugating one of a pair of carefully selected fluorescent dyes to each of the molecules of interest. When these fluorescent dyes are held in close proximity due to binding of the biomolecules, a unique fluorescence signal is developed that specifically confirms the proximity and thus the binding reaction.
To achieve resonance energy transfer, the first fluorescent molecule (the "donor" fluor) must absorb light and transfer it through the resonance of excited electrons to the second fluorescent molecule (the "acceptor" fluor). For energy transfer to take place, the fluorescence emission wavelength of the donor must be lower than the excitation wavelength of the acceptor; that is, the process must be energetically "downhill".
The degree of proximity and the geometry of the molecules determine the extent to which energy transfer takes place. When energy transfer is complete, fluorescence of the donor molecule is completely extinguished, and all fluorescence appears at the emission wavelength of the acceptor. In practice, complete transfer is never achieved, although tandem conjugates of fluors (two fluorescent molecules conjugated to one another) can achieve significantly greater than 90% elimination of donor fluorescence.
In proximity assays, less complete quenching of donor fluorescence can be expected, but it is the appearance of acceptor fluorescence in response to excitation of the donor molecule that indicates that the molecular interaction of interest has taken place.
Phycobiliproteins are extremely useful in FRET assays because of their high quantum efficiency, and because each phycobiliprotein incorporates multiple fluorophores (individual fluorescent entities capable of absorbing and emitting quanta). Most synthetic fluors consist of a single fluorophore.
Figure 1 shows the fluorescence excitation and emission curves of two phycobiliproteins that can be useful in proximity assays; R-Phycoerythrin (RPE) as the donor, and Allophycocyanin (APC) as the acceptor. It shows the characteristics of donor-acceptor pairs that must be considered when selecting dyes for this use:
Emission spectrum overlap - Since complete quenching of donor fluorescence cannot be expected, the emission wavelength of the acceptor should be located at a wavelength where fluorescence of the donor is as low as possible, since direct fluorescence by the donor can create a significant background signal, the magnitude of which is sensitive to the extent of energy transfer (region A, Figure 1).
Excitation spectrum overlap - The acceptor molecule should be minimally excited by the wavelength employed to excite the donor. In the case of RPE, multiple excitation peaks provide a particularly suitable region for excitation in the proximity assay (region B, Figure 1).
Figure 2 illustrates the results of a proximity assay; RPE was conjugated to biotin and APC to streptavidin (SA) and the fluorescence effects of the binding of these molecules was examined. When APC-SA was added to a 4.5 µg/ml solution of RPE-biotin, fluorescence (495 nm excitation 660 nm emission) increased with sequential additions of 1.5 µg/mL of APC-SA until saturation of the available RPE-biotin became apparent (Treatment 1).
Controls demonstrate that the fluorescence response observed was due solely to proximity based on the streptavidin-biotin binding reaction. No significant fluorescence response was observed when unconjugated APC was added to the RPE-biotin (Treatment 2). When a large excess of unlabelled biotin (20 mM) was added to the RPE-biotin in advance of the addition of APC-SA, competitive binding of biotin prevented significant binding of the conjugated reactants and no significant energy transfer was observed (Treatment 3). RPE-biotin fluorescence at 660 nm for 495 nm excitation was subtracted as a blank from each value.
Thus, energy transfer-based fluorescence could only be observed when streptavidin-biotin binding occurred.
FRET finds utility in any application where the proximity of two conjugatable molecules is of interest. These include:
and many other specialized applications.