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DSE-FRET: A new anticancer drug screening assay for DNA binding proteins.

Miyagi T, Shiotani B, Miyoshi R, Yamamoto T, Oka T, Umezawa K, Ochiya T, Takano M, Tahara H - Cancer Sci. (2014)

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.

View Article: PubMed Central - PubMed

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.

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Specificity of DSE-FRET. (a) 2.5 μL of 2000 nM competitor was mixed with 2.5 μL of 800 nM p50 and incubated for 30 min. Then, 5 μL of 20 nM NF-D1 was added and incubated for a further 30 min. Finally, 40 μL of 10 nM NF-D2 was added and fluorescence was measured: NF-D1 and NF-D2 without p50 (white bar), without competitor (black bar), and with competitors (gray bars). Horizontally and diagonally striped bars indicate non-specific probes (NFs-D1 + NFs-D2) without and with p50, respectively. (b) Sequences of the competitor upper strand are shown: underline, nuclear factor-κB (NF-κB) binding site; bold type, substituted nucleotide in binding site. Error bars represent ±3 SD (n = 4).
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fig03: Specificity of DSE-FRET. (a) 2.5 μL of 2000 nM competitor was mixed with 2.5 μL of 800 nM p50 and incubated for 30 min. Then, 5 μL of 20 nM NF-D1 was added and incubated for a further 30 min. Finally, 40 μL of 10 nM NF-D2 was added and fluorescence was measured: NF-D1 and NF-D2 without p50 (white bar), without competitor (black bar), and with competitors (gray bars). Horizontally and diagonally striped bars indicate non-specific probes (NFs-D1 + NFs-D2) without and with p50, respectively. (b) Sequences of the competitor upper strand are shown: underline, nuclear factor-κB (NF-κB) binding site; bold type, substituted nucleotide in binding site. Error bars represent ±3 SD (n = 4).

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.


DSE-FRET: A new anticancer drug screening assay for DNA binding proteins.

Miyagi T, Shiotani B, Miyoshi R, Yamamoto T, Oka T, Umezawa K, Ochiya T, Takano M, Tahara H - Cancer Sci. (2014)

Specificity of DSE-FRET. (a) 2.5 μL of 2000 nM competitor was mixed with 2.5 μL of 800 nM p50 and incubated for 30 min. Then, 5 μL of 20 nM NF-D1 was added and incubated for a further 30 min. Finally, 40 μL of 10 nM NF-D2 was added and fluorescence was measured: NF-D1 and NF-D2 without p50 (white bar), without competitor (black bar), and with competitors (gray bars). Horizontally and diagonally striped bars indicate non-specific probes (NFs-D1 + NFs-D2) without and with p50, respectively. (b) Sequences of the competitor upper strand are shown: underline, nuclear factor-κB (NF-κB) binding site; bold type, substituted nucleotide in binding site. Error bars represent ±3 SD (n = 4).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4317927&req=5

fig03: Specificity of DSE-FRET. (a) 2.5 μL of 2000 nM competitor was mixed with 2.5 μL of 800 nM p50 and incubated for 30 min. Then, 5 μL of 20 nM NF-D1 was added and incubated for a further 30 min. Finally, 40 μL of 10 nM NF-D2 was added and fluorescence was measured: NF-D1 and NF-D2 without p50 (white bar), without competitor (black bar), and with competitors (gray bars). Horizontally and diagonally striped bars indicate non-specific probes (NFs-D1 + NFs-D2) without and with p50, respectively. (b) Sequences of the competitor upper strand are shown: underline, nuclear factor-κB (NF-κB) binding site; bold type, substituted nucleotide in binding site. Error bars represent ±3 SD (n = 4).
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.

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.

View Article: PubMed Central - PubMed

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
Related in: MedlinePlus