FRET assay

The LANCE cAMP assay is a competitive assay in which cAMP produced by the cells competes with fluorescent-labeled acceptor cAMP for a cryptate tagged donor antibody. The principal of the assay is shown in Fig. 6. On the left strepta-vidin conjugated Europium binds to biotinylated cAMP. An antibody labeled with the fluorescent dye Alexa binds to the cAMP, bringing the donor and acceptor into close proximity, and energy transfer occurs. When the cell releases cAMP, it competes with the biotin-labeled cAMP for the antibody, and a signal decrease is observed. In the TR-FRET assay the antibody is directly labeled with either Eu or Tb. In this format an increase in cAMP also causes a decrease in signal. 

In another 2009 patent application, a series of 2-pyrazolopyrimidinones were reported to be PHD inhibitors [37]. Although most of the compounds exemplified in this application lacked a carboxylic acid moiety (vide supra), compound 45 was reported as having an IC50 value of < 10 nM in a FRET assay, although no cell-based assay data were disclosed. 

In order to determine whether compounds identified in the primary HTS screen are specific, a counterscreen is required to identify and eliminate false positives that will arise in the primary screen. For protein—protein interaction screens, it is preferable to test an unrelated protein pair that uses the same mode of detection. For our purposes, we adapted a previously described TR-FRET assay that monitors the interaction between bacterial Staphylococcus aureus Dnal and phage protein 77ORF104 (Liu et al., 2004). 

Mere, L., Bennett, T., Coassin, P., Hamman, B., Rink, T., Zimmerman, S. and Nelulescu, P. (1999). Miniturized FRET assays and microfluidics Key components for ultra-high-throughput screening. Drug Discov. Today 4, 363-9. 

Fig. 6.20. Types of assays to visualize lipid trafficking in membranes. (A) Self-quenching method. Here the self-quenching is released upon transfer to unlabeled acceptor membranes that are usually in large excess. (B) FRET assays. Here the donor membrane contains a transferable lipid (green) that is quenched by FRET to a non transferable acceptor lipid (red). Upon transfer to an unlabeled acceptor membrane the green-labeled lipid becomes unquenched. 

Figure 9.51 Time-resolved FRET assay systems involve energy transfer between the lanthanide chelate and an organic dye that are brought together as two labeled molecules bind to an analyte. In this illustration, an antibody labeled with a lanthanide chelate is used along with a Cy5-labeled antibody to detect a protein target in solution. Excitation of the lanthanide label results in energy transfer and excitation of the cyanine dye only if they are held within close enough proximity to allow efficient FRET to occur. Under these conditions, excitation of the lanthanide chelate results in cyanine dye emission, which will not occur if the labeled antibodies have not bound to a target.

When used with europium or terbium ions, a carbostyril-based lanthanide chelate can be excited at 340 nm and provide sharp characteristic emission bands for transfer of energy to the appropriate acceptor fluor. Similar to the TMT chelator described previously, luminescence from terbium FRET signals well with Cy3 dyes and luminescence from europium can be used with APC or Cy5 dyes. Other fluorescent dyes that have similar excitation and emission ranges to these also can be used as acceptors in TR-FRET assays. For instance, terbium chelates can... 

Intensive effort has been devoted to the optimization of CCP structures for improved fluorescence output of CCP-based FRET assays. The inherent optoelectronic properties of CCPs make PET one of the most detrimental processes for FRET. Before considering the parameters in the Forster equation, it is of primary concern to reduce the probability of PET. As the competition between FRET and PET is mainly determined by the energy level alignment between donor and acceptor, it can be minimized by careful choice of CCP and C. A series of cationic poly(fluorene-co-phenylene) (PFP) derivatives (IBr, 9, 10 and 11, chemical structures in Scheme 8) was synthesized to fine-tune the donor/acceptor energy levels for improved FRET [70]. FI or Tex Red (TR) labeled ssDNAg (5 -ATC TTG ACT ATG TGG GTG CT-3 ) were chosen as the energy acceptor. The emission spectra of IBr, 9, 10 and 11 are similar in shape with emission maxima at 415, 410, 414 and 410 nm, respectively. The overlap between the emission of these polymers and the absorption of FI or TR is thus similar. Their electrochemical properties were determined by cyclic voltammetry experiments. The calculated HOMO and LUMO... 

Pollitt SK, Pallos J, Shao J, Desai UA, Ma AA, Thompson LM, Marsh JL, Diamond MI (2003) A rapid cellular FRET assay of polyglutamine aggregation identifies a novel inhibitor. Neuron 40(4) 685-694 Ranganathan S, Bowser, R (2003) Alterations in G(l) to S phase cell-cycle regulators during amyotrophic lateral sclerosis. Am J Pathol 162(3) 823—835... 

Finally FRET assays can be used to monitor subsequent steps such as binding of /1-arrestins to activated receptors (Bertrand et al, 2002 Krasel et al, 2005 Vilardaga et al, 2003 Violin et al., 2006). 

It is also of interest to compare our results with experiment. The experimental study most closely related to the model considered here is the SM-FRET assay by Hochstrasser and co-workers on the disulfide cross-linked two-stranded coiled-coil from the yeast transcription factor GCN4. [30,33] Our results appear to be consistent with many of the experimental observations reported in Refs. [30,33], For example, surface-immobilization in the folded state has a rather small effect on the R distribution, the folded and unfolded states are seen to correspond to narrow and broad R distributions, respectively, and conformational dynamics is seen to be characterized by a wide dynamical range in the midpoint and unfolded states. Our analysis can also help in the interpretation of the experimental results. For example, surface-... 

Kinase or phosphatase assays based on the AlphaScreen principle are similar to TR-FRET assays in that they usually require a biotinylated substrate peptide and an anti-phosphoserine or tyrosine antibody. These two reagents are sandwiched between biotin and protein A-functionalized acceptor and donor beads. A kinase assay would show an enzyme-dependent increase in antibody binding (and thus signal) over time and a phosphatase assay would show an enzyme-dependent decrease in antibody binding over time. In some cases, the phosphorylation of an epitope will block the antibody binding and thus a phosphatase assay in principle can be constructed as a signal increase assay (Von Leoprichting and Kumpf, 2004 Warner et al., 2004). 

Because of the very large variety of assay detection methods, it is difficult to cover all the parameters needed to optimize for each detection system however, it is important to mention that all the systems discussed in the assay readout technology section above must be optimized. For example, when using an antibody pair in a TR-FRET assay, it is important to find a concentration of antibodies that can trap the product efficiently at the desired time point in the reaction. 

Moshinsky, D.J. et al. 2003. A widely applicable, high-throughput TR-FRET assay for the measurement of kinase autophosphorylation VEGFR-2 as a prototype. J. Biomol. Screen. 8, 447-452. 

In general, all FRET assay formats are limited to substrates in which short distances between donor and acceptor dye do not disturb the interaction between protease and substrate. The flexibility of the peptide conformation makes the prediction of the effective distance between the dyes and consequently the prediction of the FRET effect difficult. The distance between donor and acceptor cannot be easily estimated by the mean hydrodynamic radii of the dyes. 

Figure 3 Design of a homogeneous FRET assay. The spatial proximity between the donor and the acceptor induced by the binding of the biomolecules allows the FRET to occur. Upon FRET, the donor emission is decreased while the sensitized acceptor can emit fluorescence (if a fluorescent probe is used as acceptor)...

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