Fluorescence anisotropy microplate assay for analysis of steroid receptor-DNA interactions.
To analyze the interactions of steroid/nuclear hormone receptors with their DNA response elements, we used ultra low-volume microplates to develop a simple and rapid fluorescence anisotropy assay. The novel fluorescence anisotropy microplate assay (FAMA) was applied to the binding of estrogen and progesterone receptors (ER and PR, respectively) to their respective DNA response elements. The FAMA offers exceptional flexibility in its ability to test a variety of binding conditions and DNA response elements in real time. This assay can differentiate between, and quantitate, sequence-specific and nonspecific binding of receptors to DNA and offers the possibility of true solution analysis of the interaction of coregulators with the estrogen response element (ERE)-ER complex. To test suitability for screening large compound libraries, we demonstrated that the FAMA generates stable signals for more than 4 hours, is insensitive to inhibition by dimethyl sulfoxide (DMSO), and works well in 384-well plates. We analyzed inhibition of receptor-DNA interaction by several zinc chelators and demonstrated zinc dependence and a generally higher sensitivity to inhibition for PR-progesterone response element (PRE) interactions than for ER-ERE interactions. The FAMA is the first system suitable for screening large compound libraries to identify novel compounds that antagonize (or stimulate) binding of steroid receptors to their DNA response elements.
Wang
Stanley Y
SY
University of Illinois, Urbana, IL, USA.
Ahn
Bonnie S
BS
Harris
Rebecca
R
Nordeen
Steven K
SK
Shapiro
David J
DJ
D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D005454:Fluorescence Polarization / Q000295:instrumentation* / Q000379:methods; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation* / Q000379:methods; D011987:Receptors, Steroid / Q000378:metabolism*; D020218:Response Elements
D011987:Receptors, Steroid; D004247:DNA