FRET probes probe

Also recently, Liao and collaborators [89] proposed a homogeneous noncompetitive assay of a protein in biological samples based on FRET by using its tryptophan residues as intrinsic donors and its specific fluorescent ligand as the FRET acceptor, which was defined as an analytical FRET probe. To evaluate this method, a naphthylamine derivative, namely /V-biotinyl-/V -(l -naphthylj-ethylene-diamine (BNEDA) 33 was used as an analytical FRET probe for the homogeneous noncompetitive assay of streptavidin. 

While VFPs have boosted the applications of FRET-FLIM, chemical FRET probes should not be dismissed. The advantage of chemical probes is that they are much smaller in size and that they often have much better spectral readout than VFP probes. In Chapter 6, Amanda Cobos Correa and Carsten Schultz highlight the various small molecule-based FRET probes and their use in bioimaging. 

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... 

Quenching is the reduction in fluorescence intensity and can be caused by various processes. It occurs either during the lifetime of the excited state or in the ground state. Quenching processes that happen in the excited state are collisional quenching, charge transfer reactions, or energy transfer. The latter is the basis for FRET probes but the other events happen as well under certain conditions and it is important to consider them. 

Fig. 6.11. Two types of FRET probes. (A) Ratiometric probes are formed by two fluorescent molecules that allow determination of emission ratio. (B) Quenched probes feature a donor fluorophore and a quencher. The emission increase of the donor after release of the acceptor is detected. Both types are frequently used to build proteinases probes.

While the use of FRET probes in vitro is inflicted with a limited number of complications, the use in biological samples or in living cells needs much more careful considerations since factors such as enzyme specificity, cell toxicity, and spatio-temporal resolution usually play an important role. 

Many fluorophores are sensitive to changes in the hydropho-bicity of the immediate environment. Therefore, bringing these fluorophores into a different environment may also produce a change in FRET, when a second fluorophore is affected by the emission change of the first. Fluorophores like Nile Red with changes of up to 100 nm when transferred from water to an aprotic organic solvent are principally suitable for such an approach [71], Molecular rotors have the characteristic of having a quantum yield that depends on the viscosity. Such dyes are formed by an electron donor unit and an electron acceptor unit that can rotate relative to each other upon photoexcitation with a behavior that depends on the viscosity of the environment. These dyes have been included in FRET probes for viscosity studies [53],... 

Proteases are one of the largest families of enzymes and are involved in a multitude of vital processes. Due to their biological relevance and diversity, multiple fluorescent reporters monitoring their activity have been designed and successfully applied in vitro and in vivo [112-114]. Standard small molecule FRET probes for proteases consist of an amino acid sequence flanked by a FRET pair, consisting of two fluorophores or one fluorophore and a quencher molecule. Upon cleavage of the peptide sequence, the emission of the donor fluorophore is dequenched and the intensity increases whereas the emission of the acceptor decreases and vanishes more or less completely in those cases where the acceptor is fluorescent (see Fig. 6.11). 

FRET probes have not only been generated to measure the phospholipase activity but to study its substrate specificity as well. Several substrates of PLA2 with a variety of head groups and labeled with a BODIPY dye and a Dabcyl quencher were created by Rose et al. and tested against different PLAs in cells to determine substrate specificity and intracellular localization [137], The specificity of PLA2 isoforms towards the number of double bonds in the sn2 position was evaluated with a small series of PENN derivatives. It was demonstrated that the cytosolic type V PLA2 preferred substrates with a single double bond [138],... 

Since kinases are not hydrolytic enzymes, a small molecule-based FRET probe does not seem to be a straight forward solution for this enzyme activity. Nevertheless, quite a number of fluorescent probes based on small substrate peptides have been prepared in... 

Fig. 6.18. Design and performance of the FRET probe CPF4 for measuring phosphodiesterase activity. Ri and R2 are phosphate and hydrogen or vice versa.

FRET probes based on DNA are strong potential tools to investigate cellular processes where nucleases and a set of enzymes acting on DNA play a role. Most probes of this class are not particularly... 

Variations in viscosity have been also followed with FRET probes. An innovative FRET pair constituted by a viscosity sensitive dye (CMAM) and a viscosity-independent coumarin, which acts as internal reference, demonstrated a high sensitivity for detecting viscosity changes in vitro [52],... 

Wichmann, O. and Schultz, C. (2001). FRET probes to monitor phospholipase A(2) activity. Chem. Commun. 2500-2501. 

Wichmann, O., Wittbrodt, J. and Schultz, C. (2006). A small-molecule FRET probe to monitor phospholipase A2 activity in cells and organisms. Angew. Chem. Int. Ed. 45, 508-512. 

Alternatively, proteins can be labeled selectively using amine-reactive dyes. Particularly, cysteine and lysines can be modified covalently with a variety of commercially available fluorophores including Texas Red, Oregon Green, and Cy3 [19] (see also Chapter 6 for small molecule FRET probes, and Chapter 12 depicting a variety... 

It is apparent from Table 12.1 that the selection of the most appropriate FRET probes for a certain application requires careful consideration of the photophysical properties of the dyes as well as the availability of methods for the specific labeling of the... 

Finally, it should be noted that homo-FRET, which is just the exchange of energies between the same dyes, is undetected by common spectroscopic or lifetime measurements and needs the hetero-FRET probing for its detection. The Red-Edge effect allows the easy distinguishing of the decrease of anisotropy due to FRET (static effect) from that occurring due to rotational freedom of fluorophores (dynamic effect), which does not depend on excitation wavelength. 

Fig. 14. Examples of homogeneous hybridization assay methods (F luminophore, Q quencher, D donor, A acceptor). Thick lines represent DNA strands. Open circles on DNA strands indicate a SNP/mutation site for Molecular Beacon and insertion/deletion sites for dual FRET probe and dual FRET Molecular Beacon when these methods are applied to SNP/mutation typing or deletion/insertion detection. The solid circle on die strand indicates the complementary site.

Sueda et al. (2000, 2002) reported a dual FRET probe assay, where two combinations, Eu-BHHCT-Cy 5 and Tb-BPTA-Cy 3, were tested for the donor-acceptor combination. They used a pair of 15-mer oligonucleotide probes to detect 31-34 synthetic oligonucleotide targets. As noted above, the emission intensities from Eu3+ and Tb3+ are negligibly weak at the Cy5 and Cy3 emission wavelengths (669 and 565 nm, respectively). Additionally, the initial emission intensities from Cy3 and Cy5 were quite weak when excited at the excitation wavelengths for the Eu3+ and Tb3+ chelates (340 and 325 nm, respectively). These properties enabled sensitive detection without quenchers. The detection limits were reported to be 200 pM and 30 pM for the Eu3+-Cy5 and Tb3+-Cy3 systems, respectively. 

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