Fluorophores and quenchers

When compared to fluorescent proteins, fluorophores and quenchers of fluorescence (short quenchers) are small molecules with sizes varying from 1 to 10 A. They are the main building blocks for constructing small molecule FRET probes. As molecular entities, they might influence the performance of the probe to a great extent. Their fluorescent properties will determine the sensitivity and dynamic range of the sensor. The success of the probe for a specific application will depend on the selection of the right fluorophores... 

Baird and Escott [49] suggested that the departures from the Stern— Volmer law could be explained if there was competition between the excited fluorophors and quenchers. However, because the concentration of excited fluorophors is 103—106 times less than the quencher concentration, to an excellent approximation the excited fluorophor molecules are statistically independent. Baird et al. [50] have also developed a more detailed analysis of fluorescence quenching based upon the Wilemski and Fixman [51] approach (see Chap. 9). They wrote many-body equations... 

The duplex probe configuration provides for a close proximity of the donor/acceptor pair and may lead to two mechanisms of quenching FRET and direct transfer (contact-mediated) quenching [78]. Placement of the fluorophore and quencher toward the centre of the duplex probe sequence may further add to the quenching efficiency as the ends of duplexes are known to breathe and are not as tightly bound as internal base pairs. Duplex probes are relatively easy to synthesize as the fluorophore and quencher moieties do not have to be incorporated into the same strand. 

The most common quenchers are oxygen, acrylamide, iodide, and cesium ions. The kq value increases with probability of collisions between the fluorophore and quencher. Oxygen is a small and uncharged molecule, so it can diffuse easily. Therefore, the bimolecular diffusion constant kq observed for oxygen in solution is the most important between all cited quenchers. 

As with any assay, conditions for a QES assay must be optimized in terms of buffer composition, particularly pH and potential salt and metal ion effects. Eurther-more, the specificity of cleavage should be verified by an independent method, such as reverse-phase HPLC. Given the aromatic nature of both fluorophore and quencher, even low levels of QFS and cleavage products can be easily detected by UV absorbance. This method will also determine whether any absence of fluorescence is due to lack of cleavage or to a low fluorescent yield, as occurred with some of the ECE substrates discussed in Note 1. 

Molecular beacons (MB) are stem-loop hairpin oligonucleotide structures that have a fluorescent dye at one end and a fluorescence quencher at the other. In the hairpin state, the quencher and fluorophore are in close proximity and therefore there is no fluorescence from the probe. However, when the MB binds to a complementary oligonucleotide as a duplex then the fluorophore and quencher are separated and the fluorophore can emit fluorescence. They are particularly useful in monitoring reactions with time, e.g., in PCR, " rolling circle amplification, hybridisation, telomerase activity, ligation... 

Huorescence quenching requires a close approach of the quencher to the fluorophore and hence it can be used for smdying various structural problems and dynamie processes. When both the fluorophore and quencher are dissolved in a solution, the time-resolved data report on the rate of diffusion. When the... 

In the previous section we desciibed quenching that resulted from diffusive encounters between the fluorophore and quencher during the lifetime of the excited state. This is a time-dependent process. Quenching can also occur as a result of the formation of a nonfluorescent complex between the fluorophore and quencher. When this complex absorbs light, it immediately returns to the ground state without emission of a photon (Figure 8.1). 

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