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: We examined the specificity of the assay by using double-stranded competitor DNA. NF-cpt1 is a specific competitor DNA bearing a NF-κB binding site. NF-cpt2, NF-cpt3, NF-cpt4, and NF-cpt5 are non-specific competitors with one to four nucleotides substituted in the NF-κB binding site. NF-cpt1 completely restored the fluorescence suppressed by p50 (Fig. 3). The restoration level decreased as the number of substituted nucleotides in non-specific competitors increased and NF-cpt5, with four substituted nucleotides, had little effect on p50 binding. For further evaluation of specificity, we prepared non-specific probes, NFs-D1 and NFs-D2, with nucleotide substitutions as in NF-cpt5. The fluorescence signal of the nonspecific probes was not affected by p50 (Fig. 3). Hence, we concluded that p50 suppressed strand exchange in a sequence-specific manner.
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.