Molecular Probes

Note 1.2 — Technical Focus: Fluorescence Resonance Energy Transfer (FRET)

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, making it useful over distances comparable with the dimensions of biological macromolecules. Thus, FRET is an important technique for investigating a variety of biological phenomena that produce changes in molecular proximity. When FRET is used as a contrast mechanism, colocalization of proteins and other molecules can be imaged with spatial resolution beyond the limits of conventional optical microscopy.

Primary Conditions for FRET

• 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.

Förster Radius

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 (R_o). The magnitude of R_o is dependent on the spectral properties of the donor and acceptor dyes (see Table):

Table. Typical Values of R_o.

Donor/Acceptor Pairs

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 R_o for some dye pairs are listed in the table above and more extensive compilations are in Table 1.6 and Table 1.11. Note that because the component factors of R_o (see above) are dependent on the environment, the actual value observed in a specific experimental situation is somewhat variable. Extensive compilations of R_o values can be found in the literature. Nonfluorescent acceptors such as dabcyl and our QSY dyes (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).

Selected Applications of FRET

• 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 (Technical Focus: Lipid-Mixing Assays of Membrane Fusion)
• Membrane potential sensing
• Fluorogenic protease substrates
• Indicators for cyclic AMP and zinc