DSE-FRET: A new anticancer drug screening assay for DNA binding proteins.
Bottom Line: Evaluation of two NF-κB inhibitors, Evans Blue and dehydroxymethylepoxyquinomicin ([-]-DHMEQ), was carried out using p50 and p52 (another form of NF-κB), and IC50 values were obtained.The DSE-FRET technique also detected the differential effect of (-)-DHMEQ on p50 and p52 inhibition.These data indicate that DSE-FRET can be used for high throughput screening of anticancer drugs targeted to DNA-binding proteins.
Affiliation: Department of Cellular and Molecular Biology, Graduate School of Biomedical & Health Science, Hiroshima University, Hiroshima, Japan; Japanese Red Cross Kanto-koshinetsu Block Blood Center, Tokyo, Japan.Show MeSH
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Mentions: The principle of DSE-FRET is illustrated in Figure 1. Two partially double-stranded DNA probes, named D1 and D2, are used. Each probe has a double-stranded region containing a protein binding site and two single-stranded tails. One strand of D1 is labeled with a fluorophore at the 5′-end and the other is labeled with a quencher at the 3′-end. The fluorophore and quencher are placed at the same end of the double-stranded region; therefore, the fluorescence of the fluorophore is quenched. The tails of D1 are complementary to those of D2, so that D1 hybridizes with D2 to form a four-way structure. As the double-stranded regions of the two probes have identical sequences, the junction of the structure migrates spontaneously, followed by irreversible dissociation to give two fully double-stranded duplexes. In other words, strand exchange occurs between D1 and D2. As a result, the quencher-labeled strand of D1 is exchanged for its non-labeled counterpart in D2; therefore, fluorescence is restored. DNA-binding proteins bind to the duplex and block strand exchange, thereby suppressing the fluorescence elevation.
Affiliation: Department of Cellular and Molecular Biology, Graduate School of Biomedical & Health Science, Hiroshima University, Hiroshima, Japan; Japanese Red Cross Kanto-koshinetsu Block Blood Center, Tokyo, Japan.