Fluorescence-resonance energy transfer

Fluorescence resonance energy transfer (FRET), a phenomenon first described by Forster in 1948 [27], involves dipole-dipole energy transfer from the emitting fluorophore moiety (donor) to the absorbing moiety (acceptor). The rate of energy transfer (kx) for any specific donor (D) and acceptor (A) pair is given by 

The efficiency of energy transfer is the proportion of photons absorbed by the donor that are transferred to the acceptor and it is equal to relative fluorescence yield in the presence (Tda) and the absence (Fp) of acceptor [1, 2]  

Equations 10.4 and 10.5 are used to calculate distances between the donor and the acceptor. 

A vast literature pertains to the quantitative investigation of exchange processes (see, e.g., [4, 28-30] and references therein). 

For systems in which the centers are separated by a nonconductive medium (molecules or groups with saturated chemical bonds), Ptt is equal to 2.6 A . For systems in which the radical centers are linked by conducting conjugated bonds, PsE is 0.3 

According to the Forster theory of resonance energy transfer, which was proposed by T. Forster in 1959, energy transfer is efficient when 

the energy donor and acceptor are separated by a short distance (of the order of nanometers)  

photons emitted by the excited state of the donor can be absorbed directly by the acceptor. 

For donor-acceptor systems that are held rigidly either by covalent bonds or by a protein scaffold , tJt increases with decreasing distance, R, according to 

The emission and absorption spectra of molecules span a range of wavelengths, so the second requirement of the Forster theory is met when the emission spectrum of the donor molecule overlaps significantly with the absorption spectrum 

ERET allows the design of homogeneous assays in which different methods can be used to discriminate the specific signal from other signals emitted by the free donor and/or the free acceptor (Eigure 3). Most commonly used FRET readouts are based on detecting variations in fluorescence intensity a decrease in fluorescence emission from the donor,increase in fluorescence emission from the acceptor (if 

To screen chemical hbraries, readouts based on a ratio of fluorescence intensities as well as on donor fluorescence lifetime variations can be of a great advantage to correct the FRET signal from the optical variations induced by some of the highly coloured compounds that are often found in libraries. This correction can limit false-positive results in HTS however, in practise, these analyses are difficult to implement. 

The use of the donor fluorescence lifetime as an FRET readout is currently limited by several parameters  

Therefore, due to the practical limitations described above, the use of a non-fluo-rescent acceptor (usually called a quencher) is still the most popular way to use FRET in HTS applications. Most of the time, FRET is used to probe a protease activity using a peptide substrate labelled at each end of its sequence with the fluorescent donor and the quencher. In that case, the FRET readout is an increase in the donor fluorescence emission upon peptide cleavage by the protease. However, as discussed above, such FRET readouts can lead to a significant number of false positive results in HTS. 

If a suitable acceptor molecule is present, a long-range dipole-dipole interaction results in an additional relaxation term ( fret) is incorporated into equation 2.36, 

The magnitude of k-pusr is a function of many properties of the donor/acceptor system and the environment surrounding the two molecules. 

In this description the FRET rate is therefore a fimction of the refractive index of the medium between the two molecules n, the fluorescence lifetime Tp[s] and quantum efficiency Qd of the donor in the absence of FRET, Avogadro s number N, the separation of the two molecules R[cm], the normalized spectral overlap integral / [ cm j and the so-called orientation factor k. Note that different forms of equa- 

In many FRET experiments the FRET transfer efficiency Tfret is the parameter that is sought. This is defined as the ratio of the energy transfer rate to the sum of all the donor de-excitation rates. 

The transfer of energy from the donor to the acceptor results in the donor returning to the ground state and the acceptor entering an excited state. Relaxation from this state can then result in fluorescence from the acceptor. fret can therefore be calculated experimentally in a number of ways, using the relative quantum yields (4 ), fluorescence intensities (f) or lifetimes (t) of the donor molecule in the presence (indicatedby superscript A) and absence of the acceptor  

In Section 14.2, we fonnd in Eqnation 14.11 that the interaction matrix element between two chromophores in an excited triplet state decreases exponentially with distance, while the corresponding singlet matrix element (Equation 14.10) decreases more slowly as 1/R with distance. These interactions were derived a long time ago from the old quantnm theory by D. L. Dexter in the triplet case and Theodor Forster in the singlet case. The conclnsions are valid also for the case of unequal chromophores. 

Forster s theory is behind a very nsefnl method for determining the absorption spectrum of a collection of identical chromophores within a separation distance of less than 100 A, placed in a protein, DNA, or some other scaffold. One example is the indole groups in a protein, where the interaction causes a broadening of the absorption spectrum. 

Forster s theory is also behind the FRET method (Forster resonance energy transfer or fluorescence resonance energy transfer). In this case, excitation takes place with a narrow laser pulse, corresponding to the lowest excited state of a chromo-phore. The rate of EET is determined by time-resolved fluorescence. 

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F statistic, 239, 241 False negatives, 152—153 False positives, 152—153 Fenoximone, 188 First-order kinetics, 167 Fluorescence resonance energy transfer, 182... 

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Isik N, Hereld D, Jin T (2008) Fluorescence resonance energy transfer imaging reveals that chemokine-binding modulates heterodimers of CXCR4 and CCR5 receptors. PLoS ONE... 

Toth PT, Ren D, Miller RJ (2004) Regulation of CXCR4 receptor dimerization by the chemokine SDF-lalpha and the HIV-1 coat protein gpl20 a fluorescence resonance energy transfer (FRET) study. J Pharmacol Exp Ther 310 8-17 Tran PB, Miller RJ (2005) HIV-1, chemokines and neurogenesis. Neurotox Res 8 149-158 Tran PB, Ren D, Veldhouse TJ, Miller RJ (2004) Chemokine receptors are expressed widely by embryonic and adult neural progenitor cells. J Neurosci Res 76 20-34... 

When the proteins are in close proximity the Europium-cryptate emission can be absorbed by the acceptor (such as allophycocyanin [APC], or XL) which emits at a higher wavelength. When the two proteins are far apart, no fluorescence resonance energy transfer (FRET) occurs. 

Toth PT, Ren D, Miller RJ. Regulation of CXCR4 receptor dimerization by the chemokine SDF-lalpha and the HIV-1 coat protein gpl20 a fluorescence resonance energy transfer (FRET) study. J Pharmacol Exp Ther 2004 310(1) 8-17. 

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Selvin PR (2000) The renaissance of fluorescence resonance energy transfer. Nat Struct Biol 7 730-734... 

Takakusa H, Kikuchi K, Urano Y, Kojima H, Nagano T (2003) A novel design method of ratiometric fluorescent probes based on fluorescence resonance energy transfer switching by spectral overlap integral. Chemistry 9 1479-1485... 

Giordano L, Jovin TM, Irie M, Jares-Erijman EA (2002) Diheteroarylethenes as thermally stable photoswitchable acceptors in photochromic fluorescence resonance energy transfer (pcFRET). J Am Chem Soc 124 7481-7489... 

Algar WR, Krull UJ (2008) Quantum dots as donors in fluorescence resonance energy transfer for the bioanalysis of nucleic acids, proteins, and other biological molecules. Anal Bioanal Chem 391 1609-1618... 

Following previous work, Hagiwara and collaborators [71] recently prepared 5 -terminal acridone-labeled DNAs, using the succinimidyl ester 24 of the acridone acetic acid 23 reported before [69], and evaluated their use as donors for a fluorescence resonance energy transfer (FRET) system in combination with 3 -dabcyl-tagged DNA... 

Hagiwara Y, Hasegawa T, Shoji A et al (2008) Acridone-tagged DNA as a new probe for DNA detection by fluorescence resonance energy transfer and for mismatch DNA recognition. Bioorg Med Chem 16 7013-7020... 

Oswald B, Gruber M, Bohmer M, Lehmann F, Probst M, Wolfbeis OS (2001) Novel diode laser-compatible fluorophores and their application to single molecule detection, protein labeling and fluorescence resonance energy transfer immunoassay. Photochem Photobiol 74 237-245... 

In an important extension of the above strategy, fluorescence resonance energy transfer (FRET) was used to screen room-temperature Heck reactions.41 FRET had previously been... 

FRET is often referred to as the acronym for fluorescence resonance energy transfer . This name is a misnomer, but this use has... 

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