Technical Focus: Fluorescence Resonance Energy Transfer (FRET)—Note 1.2
Fluorescence resonance energy transfer (FRET) is a distance-dependent interaction between the electronic excited states of two dye molecules in which excitation is transferred from a donor molecule to an acceptor molecule without emission of a photon. The efficiency of FRET is dependent on the inverse sixth power of the intermolecular separation, |
Primary Conditions for FRET
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- Donor and acceptor molecules must be in close proximity (typically 10–100 Å).
- The absorption spectrum of the acceptor must overlap the fluorescence emission spectrum of the donor (see Figure).
- Donor and acceptor transition dipole orientations must be approximately parallel.
Figure. Schematic representation of the FRET spectral overlap integral.
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Förster Radius
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The distance at which energy transfer is 50% efficient (i.e., 50% of excited donors are deactivated by FRET) is defined by the Förster radius (R0). The magnitude of R0 is dependent on the spectral properties of the donor and acceptor dyes (see Table):
Table. Typical Values of R0.
| Donor | Acceptor | R0 (Å) |
|---|---|---|
| Fluorescein | Tetramethylrhodamine | 55 |
| IAEDANS | Fluorescein | 46 |
| EDANS | Dabcyl | 33 |
| Fluorescein | Fluorescein | 44 |
| BODIPY FL | BODIPY FL | 57 |
| Fluorescein | QSY 7 and QSY 9 dyes | 61 |
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Donor/Acceptor Pairs
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In most applications, the donor and acceptor dyes are different, in which case FRET can be detected by the appearance of sensitized fluorescence of the acceptor or by quenching of donor fluorescence. When the donor and acceptor are the same, FRET can be detected by the resulting fluorescence depolarization.
Typical values of R0 for some dye pairs are listed in the table above and more extensive compilations are in R<0> values for some Alexa Fluor dyes—Table 1.6 and R<0> values for QSY and dabcyl quenchers—Table 1.11. Note that because the component factors of R0 (see above) are dependent on the environment, the actual value observed in a specific experimental situation is somewhat variable. Extensive compilations of R0 values can be found in the literature. Nonfluorescent acceptors such as dabcyl and our QSY dyes (Molecular Probes' nonfluorescent quenchers and photosensitizers—Table 1.10) have the particular advantage of eliminating the potential problem of background fluorescence resulting from direct (i.e., nonsensitized) acceptor excitation. FRET efficiencies from several donor dyes to the QSY 7 quencher in molecular beacon hybridization probes have been calculated. Probes incorporating fluorescent donor–nonfluorescent acceptor combinations have been developed primarily for detecting proteolysis (Figure 10.10) and nucleic acid hybridization (Figure 8.113, Figure 8.114).
Figure 10.10 Principle of the fluorogenic response to protease cleavage exhibited by HIV protease substrate 1 (H2930). Quenching of the EDANS fluorophore (F) by distance-dependent resonance energy transfer to the dabcyl quencher (Q) is eliminated upon cleavage of the intervening peptide linker.
Figure 8.114 Schematic representation of wavelength-shifting molecular beacons. The molecular beacon has two fluorophores on one end — a "harvester" (green circle) and an "emitter" (red circle) — and a quencher on the other end (black circle). In the hairpin loop structure, the quencher forms a nonfluorescent complex with the harvester. Upon hybridization of the molecular beacon to a complementary sequence, quenching of the harvester fluorophore is relieved, and it transfers energy (via FRET) to the emitter, which emits fluorescence.
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Selected Applications of FRET
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- Structure and conformation of proteins
- Spatial distribution and assembly of protein complexes
- Receptor/ligand interactions
- Immunoassays
- Probing interactions of single molecules
- Structure and conformation of nucleic acids
- Real-time PCR assays and SNP detection (Figure 8.115, Figure 8.116, Figure 8.117)
- Detection of nucleic acid hybridization (Figure 8.113)
- Primer-extension assays for detecting mutations (Figure 8.116)
- Automated DNA sequencing
- Distribution and transport of lipids
- Membrane fusion assays (Lipid Mixing Assays of Membrane Fusion - Note 13.1)
- Membrane potential sensing
- Fluorogenic protease substrates
- Indicators for cyclic AMP and zinc
Figure 8.115 Schematic representation of real-time PCR with TaqMan primers. In the intact TaqMan probe, energy is transferred (via FRET) from the short-wavelength fluorophore on one end (green circle) to the long-wavelength fluorophore on the other end (red circle), quenching the short-wavelength fluorescence. After hybridization, the probe is susceptible to degradation by the endonuclease activity of a processing Taq polymerase. Upon degradation, FRET is interrupted, increasing the fluorescence from the short-wavelength fluorophore and decreasing the fluorescence from the long-wavelength fluorophore.
Figure 8.116 Schematic representation of real-time PCR with Scorpion primers. In the hairpin loop structure, the quencher (black circle) forms a nonfluorescent complex with the fluorophore (green circle). Upon extension of the amplicon, the Scorpion probe hybridizes to the newly formed complementary sequence, separating the fluorophore from the quencher and restoring the fluorescence.
Figure 8.117 Schematic representation of real-time PCR with UniPrimers. In the first round of amplification, the reverse primer, containing a special sequence tag, primes synthesis along the template. In the second round, the forward primer primes synthesis that extends through the special sequence tag, forming a complementary sequence to the tag. In the third round, the UniPrimer hybridizes to this complementary sequence via the special sequence tag. The hairpin structure of the UniPrimer ensures that the quencher (black circle) suppresses the fluorescence of the fluorophore (green circle). Finally, in the fourth round, synthesis extends through the hairpin loop, relieving the quenching of the fluorophore.
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