Dabcyl quencher

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],... 

Fig. 23 (a) DNAzyme-based sensor design with two dabcyl quenchers and a FAM fluorophore (top) and mechanism of operation (bottom), (b) Fluorescence response before and after complete cleavage through Pb2+ inset contains the corresponding image of the DNAzyme probe in the absence (left) and presence of Pb2+ (after 2 min of reaction time, right). (Reprinted with permission from [150]. Copyright 2003 American Chemical Society)... 

Figure 24 The pH-sensitive nature of the C-quadruplex in the presence of the complanentary 17-base sequence yields a YES gate that is easily reset. F = rhodamine green fluorophore (Xex = 504 nm A,em = 534 nm) and Q = dabcyl quencher.

Tsourkas et al. (2003) reported dual FRET molecular beacon assays, where the donor probe was labeled with either Eu3+ or Tb3+ complex of DTPA-csl24-ethylenediamine (and no quenchers attached). For the Eu3+ complex, the acceptor probe was Cy5-labeled (and no quenchers attached) and for the Tb complex, the acceptor probe was labeled with Cy3 or ROX as a fluorophore and with dabcyl as a quencher. They demonstrated that these pairs of probes detected DNA targets ( 50-mer) with high S/N. 

All data obtained with Tecan Ultra Evolution MTP reader. The following excitation and emission wavelengths were used EDANS and AMC 350 and 500 nm RhllO 485 and 535 nm TAMRA 535 and 595 nm PT14 405 and 450 nm. 4 = primary cleavage site confirmed by MS. AMC = aminomethylcoumarin. RhllO = rhodamine 110. yE = glutamic acid attached to RhllO via its carbonic acid in side chain. EDANS = fluorophore 5-[(2-aminoethyl)amino]naphthalene-l-sulphonic acid. DABCYL = 4-(4-dimethylaminophenylazo)benzoic acid quencher. BTN = biotin. PT14 = acridone-based fluorescence lifetime label. 

Abz was combined with a broad variety of non-fluorescent acceptors such as p-nitrobenzyl for leucine aminopeptidase (Carmel et al., 1977), pNA for trypsin (Bratanova and Petkov, 1987), 4-ni-trophenylalanine [Phe(NC>2)] for HIV protease (Toth and Marshall, 1990), and V-(2,4-di n itrophenyl) ethylenediamine (EDDnp) for thermolysin and trypsin (Nishino et al., 1992). Lecaille et al. (2003) described a FRET quench assay based on a specific substrate for cathepsin K labeled with Abz and EDDnp. This substrate is not cleaved by the other Cl cysteine cathepsins and serine proteases in contrast to methoxycoumarin (Mca)-based substrates described earlier (Aibe et al., 1996 Xia et al., 1999) and merely covered the non-primed site of the scissile bond. The 5-[(2-aminoethyl)amino] naphthalene-l-sulfonic acid (EDANS) compound is a second example of a fluorescence donor historically used for many FRET quench-based protease assays, e.g., in combination with tryptophan as a quencher in an ECE activity assay (Von Geldren et al., 1991). The FRET-1 example in Table 2.2 shows the typical dynamic range that can be achieved with an EDANS/DABCYL-based assay. 

Custom modifications have previously been developed whereby a non-fluorescent chromophore can be attached to the DNA sequence to provide a strong SE(R)RS signature which is indicative of the DNA sequence present. This has been done previously using DABCYL, phthalocyanines and black hole quenchers (BHQs) as well as specifically designed simple azo dyes which contain moieties to aid in their binding ability to metal surfaces such as the benzotriazole motif which has been shown to be very effective at complexing onto silver nanoparticles [12, 13, 40, 41]. 

Fig. 9 Implementation of a protease assay with fluorescence quenching as readout. The quenching is based on energy transfer between the fluorophore EDANS (E) and the quencher DABCYL (D).

The fluorescence properties of Peptide 1 and Ip was investigated. Energy transfer from EDANS to dabcyl, quenching by dabcyl, was reduced by phosphorylation of Ser (Fig. 2). Peptide main chain was stretched by increase in hydrophilicity, and the distance between the fluorescence dye and quencher increased. 

Since some DNAzymes depend on specific metal ions for activity, they can be employed for the detection of those ions, hi the first example, a previously reported [59] DNAzyme was labeled with a Dabcyl fluorescence quencher at the 3 -end and the corresponding RNA substrate with a TAMRA fluorophore at the 5 -end [60]. Upon addition of Pb " ions, the substrate was cleaved, resulting in dissociation from the DNA enzyme strand. This led to spatial separation of the fluorophore-quencher pair, resulting in fluorescence (Fig. 5). The sensor system was over 80 times more responsive to Pb than to other metal ions, and had a quantifiable detection range of 10 nM to 4 pM. A similar strategy was developed for the detection of Ctf by a DNAzyme that oxidatively cleaves DNA [61]. The system showed a dynamic range of 35 nM to 20 pM and had a metal ion selectivity of a factor of 2000 for Cu over other metal ions. A comparable system was reported for the detection of the uranyl cation (UO/ ), with millionfold selectivity over other metal ions and parts-per-trillion sensitivity. 

Internally quenched fluorogenic substrate probes have been shown to be useful in a variety of contexts, from conventional enzymology and inhibitor evaluation, to searches for important new therapeutic protease targets. The development of automated methods for synthesis of these substrate probes has enabled access to a wide range of substrate sequences, and facilitated detailed studies of enzyme-substrate sub-site interactions. The selection of the Edans-Dabcyl fLuorophore-quencher pair has led to the synthesis of efficiently quenched peptide substrates containing more than 30 amino add residues. The overall versat bty of these substrate probes ensures widespread future application to studies of many new and existing proteases of interest. 

A non-fluorescent tripartite ensemble consisting of three DNA aptamer units (A, B, and C) was created (Figure 16.6). The 5-fluorescein (F) and quencher (Q, dabcyl), upon recognition of ATP by one of the aptamer units, enhanced fluorescence due to release of a dabcyl-appended aptamer. This structure-switching ATP reporter... 

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