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Dual FRET assay for detecting receptor protein interaction with DNA.

Krusiński T, Ozyhar A, Dobryszycki P - Nucleic Acids Res. (2010)

Bottom Line: The effectiveness of the test depends on the exclusive binding of three DNA fragments in the presence of two proteins, and the monitoring of the process depends upon observing the quenching of two independent fluorescence donors.The response element consists of two binding sites (half-sites) for the DNA binding domains (DBDs).We have shown that protein-protein interactions mediate cooperative binding of the ecdysteroid receptor DBDs to a hsp27(pal) response element.

View Article: PubMed Central - PubMed

Affiliation: Wroclaw University of Technology, Faculty of Chemistry, Division of Biochemistry, Wybrzeze Wyspiańskiego 27, 50-370 Wrocław, Poland.

ABSTRACT
We present here a new assay that is based on the idea of the molecular beacon. This assay makes it possible to investigate two proteins interacting with DNA at two binding sites that are close to each other. The effectiveness of the test depends on the exclusive binding of three DNA fragments in the presence of two proteins, and the monitoring of the process depends upon observing the quenching of two independent fluorescence donors. As a model we used the components of the heterodimeric ecdysteroid receptor proteins ultraspiracle (Usp) and ecdysone receptor (EcR) from Drosophila melanogaster and a response element from the promoter of the hsp27 gene. The response element consists of two binding sites (half-sites) for the DNA binding domains (DBDs). We have shown that protein-protein interactions mediate cooperative binding of the ecdysteroid receptor DBDs to a hsp27(pal) response element. The analysis of the microscopic dissociation constants obtained with the DMB led to the conclusion that there was increased affinity of UspDBD to the 5' half-site in the presence of EcRDBD when the 3' half-site was occupied, and increased affinity of EcRDBD to the 3' half-site when the 5' half-site was occupied.

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The noneffective placement of the donor–quencher pair, FL at the position 24 of the sequence of the regulatory element (numbering according to Figure 1A); DAB at the position 23, respectively, (filled circles). Second fluorophore Cy5.5 at the position 41 is quenched by BHQ-3 -42 (open squares). Titration of the M fragment (10 nM) labeled with fluorescein (λEX = 497 nm, λEM = 518 nm) and Cy5.5 (λEX = 675 nm, λEM = 691 nm) with equimolar amounts of the L and R duplex (L/R) in the absence of protein monitored at 518 nm (filled circles) and 691 nm (open squares).
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Figure 2: The noneffective placement of the donor–quencher pair, FL at the position 24 of the sequence of the regulatory element (numbering according to Figure 1A); DAB at the position 23, respectively, (filled circles). Second fluorophore Cy5.5 at the position 41 is quenched by BHQ-3 -42 (open squares). Titration of the M fragment (10 nM) labeled with fluorescein (λEX = 497 nm, λEM = 518 nm) and Cy5.5 (λEX = 675 nm, λEM = 691 nm) with equimolar amounts of the L and R duplex (L/R) in the absence of protein monitored at 518 nm (filled circles) and 691 nm (open squares).

Mentions: The key for successful dual FRET assay application is the proper choice of fluorophore labels and their position in the DNA sequence. If FL and Cy5.5 are chosen as a pair, there is sufficient separation of the emission and absorption spectra (Supplementary Figure 1S). The donor/quencher pairs FL/ DAB and Cy5.5/BHQ3 also fulfilled the conditions needed for distinct fluorescence quenching. In control experiments, we used oligonucleotides with different positions of fluorescence dyes. The DAB was placed at the 22 position of the full-length 64-bp sequence, FL at the 24 position, Cy5.5 at the 41 position and BHQ-3 at the 45 position (numbering according to Figure 1A). This was the most effective placement and we continued to use these positions in further experiments. When the donor (FL-24) and quencher (DAB-23) probes were too close, quenching of the donor dye was blocked (Figure 2, full circles) probably due to sterically blocked DNA association, while the second donor (Cy5.5-41) was effectively quenched by (BHQ-3-42) (Figure 2, open squares). When there was too long of a distance between the labels (FL-24, DAB-20), the fluorescence quenching considerably decreased (not shown).Figure 2.


Dual FRET assay for detecting receptor protein interaction with DNA.

Krusiński T, Ozyhar A, Dobryszycki P - Nucleic Acids Res. (2010)

The noneffective placement of the donor–quencher pair, FL at the position 24 of the sequence of the regulatory element (numbering according to Figure 1A); DAB at the position 23, respectively, (filled circles). Second fluorophore Cy5.5 at the position 41 is quenched by BHQ-3 -42 (open squares). Titration of the M fragment (10 nM) labeled with fluorescein (λEX = 497 nm, λEM = 518 nm) and Cy5.5 (λEX = 675 nm, λEM = 691 nm) with equimolar amounts of the L and R duplex (L/R) in the absence of protein monitored at 518 nm (filled circles) and 691 nm (open squares).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2875001&req=5

Figure 2: The noneffective placement of the donor–quencher pair, FL at the position 24 of the sequence of the regulatory element (numbering according to Figure 1A); DAB at the position 23, respectively, (filled circles). Second fluorophore Cy5.5 at the position 41 is quenched by BHQ-3 -42 (open squares). Titration of the M fragment (10 nM) labeled with fluorescein (λEX = 497 nm, λEM = 518 nm) and Cy5.5 (λEX = 675 nm, λEM = 691 nm) with equimolar amounts of the L and R duplex (L/R) in the absence of protein monitored at 518 nm (filled circles) and 691 nm (open squares).
Mentions: The key for successful dual FRET assay application is the proper choice of fluorophore labels and their position in the DNA sequence. If FL and Cy5.5 are chosen as a pair, there is sufficient separation of the emission and absorption spectra (Supplementary Figure 1S). The donor/quencher pairs FL/ DAB and Cy5.5/BHQ3 also fulfilled the conditions needed for distinct fluorescence quenching. In control experiments, we used oligonucleotides with different positions of fluorescence dyes. The DAB was placed at the 22 position of the full-length 64-bp sequence, FL at the 24 position, Cy5.5 at the 41 position and BHQ-3 at the 45 position (numbering according to Figure 1A). This was the most effective placement and we continued to use these positions in further experiments. When the donor (FL-24) and quencher (DAB-23) probes were too close, quenching of the donor dye was blocked (Figure 2, full circles) probably due to sterically blocked DNA association, while the second donor (Cy5.5-41) was effectively quenched by (BHQ-3-42) (Figure 2, open squares). When there was too long of a distance between the labels (FL-24, DAB-20), the fluorescence quenching considerably decreased (not shown).Figure 2.

Bottom Line: The effectiveness of the test depends on the exclusive binding of three DNA fragments in the presence of two proteins, and the monitoring of the process depends upon observing the quenching of two independent fluorescence donors.The response element consists of two binding sites (half-sites) for the DNA binding domains (DBDs).We have shown that protein-protein interactions mediate cooperative binding of the ecdysteroid receptor DBDs to a hsp27(pal) response element.

View Article: PubMed Central - PubMed

Affiliation: Wroclaw University of Technology, Faculty of Chemistry, Division of Biochemistry, Wybrzeze Wyspiańskiego 27, 50-370 Wrocław, Poland.

ABSTRACT
We present here a new assay that is based on the idea of the molecular beacon. This assay makes it possible to investigate two proteins interacting with DNA at two binding sites that are close to each other. The effectiveness of the test depends on the exclusive binding of three DNA fragments in the presence of two proteins, and the monitoring of the process depends upon observing the quenching of two independent fluorescence donors. As a model we used the components of the heterodimeric ecdysteroid receptor proteins ultraspiracle (Usp) and ecdysone receptor (EcR) from Drosophila melanogaster and a response element from the promoter of the hsp27 gene. The response element consists of two binding sites (half-sites) for the DNA binding domains (DBDs). We have shown that protein-protein interactions mediate cooperative binding of the ecdysteroid receptor DBDs to a hsp27(pal) response element. The analysis of the microscopic dissociation constants obtained with the DMB led to the conclusion that there was increased affinity of UspDBD to the 5' half-site in the presence of EcRDBD when the 3' half-site was occupied, and increased affinity of EcRDBD to the 3' half-site when the 5' half-site was occupied.

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Related in: MedlinePlus