Quenched activity-based probe

Fig. 6.13. Different designs of FRET sensors. (A) Substrates for hydrolytic enzymes. (B) Sensors for bond formation. (C) Sensors based on conformational or structural change. (D) Environmentally sensitive probes. (E) Quenched activity-based probe to monitor small molecule-enzyme interaction. (F) Small molecule-enzyme interaction using a labeled protein.

Fig. 6.14. Quenched activity based probe for the imaging of cathepsins. Upon covalent binding of the sensor to histidine and serine residues in the active site of the enzyme, the quencher is released and increased fluorescence indicates the now covalently labeled enzyme of interest. 

Blum, G., Mullins, S. R., Keren, K., Fonovic, M., Jedeszko, C., Rice, M. J., Sloane, B. F. and Bogyo, M. (2005). Dynamic imaging of protease activity with fluorescently quenched activity-based probes. Nat. Chem. Biol. 1, 203-209. 

Blum G, von Degenfeld G, Merchant MJ et al (2007) Noninvasive optical imaging of cysteine protease activity using fluorescently quenched activity-based probes. Nat Chem Biol 3 668-677... 

Another approach of energy transfer-based probe has been demonstrated for a-Amylase sensing/98 In this case amylose was doubly labeled, by fluorescein derivative (donor) and Procion Red MX8B (acceptor). As a-Amylase catalyzes the cleavage of the amylose into smaller units, the average distance between fluorescein and Procion Red increases, which reduces the degree of quenching. The rate of increase in fluorescence intensity is proportional to ee-amylase activity. 

Another important class of substrate-based probes for proteases uses two or more fluorophores, that are self-quenched when in close proximity. " Multiple fluorophores can be linked to graft polymers containing peptide substrate sequences. When these linkers are cleaved by the protease, free fluorescent monomer can be detected. This class of probes has been widely used to study the activity of the cysteine cathepsin family of proteases aeross many diverse disease models. 

A different strategy for measuring protease activity is based on the property of xanthene dyes to form H-type dimers (see Sect. 6.2.3) when they are in close proximity. These dimers are accompanied with a characteristic quenching of their fluorescence and, particularly for rhodamines, with a blue shift in the absorption spectrum [121, 122]. The probe D-NorFES-D designed to measure activity of elastase in HL-60 cells consists of an undecapeptide derivatized with one tetramethylrhodamine dye on each side. The sequence contains proline residues to create a bent structure and bring the two fluoro-phores in close proximity. Intact D-NorFES-D shows 90% of its fluorescence quenched plus a blue shift of the absorption spectrum. After addition of the serine protease elastase, an increase in the fluorescence and a bathochromic shift of the absorption spectrum is observed, resulting in an increase in the emission ratio [80],... 

Fig. 6.7. Probe based real-time PCR (a) During primer annealing the probe hybridises to a region within the target sequence and fluorescence is quenched, (h) and (c) As the primer extension proceeds, the exonuclease activity of the polymerase cleaves the reporter from the rest of the probe, (d) The reporter now fluoresces freely as it is in solution and no longer...

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