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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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Principles of the DSE-FRET assay. (a) In the absence of target protein, strand exchange between D1 and D2 will occur and fluorescence will be elevated. (b) In the presence of target protein, strand exchange will not occur and fluorescence will remain quenched.
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fig01: Principles of the DSE-FRET assay. (a) In the absence of target protein, strand exchange between D1 and D2 will occur and fluorescence will be elevated. (b) In the presence of target protein, strand exchange will not occur and fluorescence will remain quenched.

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.


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)

Principles of the DSE-FRET assay. (a) In the absence of target protein, strand exchange between D1 and D2 will occur and fluorescence will be elevated. (b) In the presence of target protein, strand exchange will not occur and fluorescence will remain quenched.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig01: Principles of the DSE-FRET assay. (a) In the absence of target protein, strand exchange between D1 and D2 will occur and fluorescence will be elevated. (b) In the presence of target protein, strand exchange will not occur and fluorescence will remain quenched.
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.

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