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Anal Biochem. 2006 Mar 15;350(2):222-32. Epub 2006 Jan 17.

Analysis of RNA-protein interactions by a microplate-based fluorescence anisotropy assay.

Author information

1
Department of Biochemistry, University of Illinois, 600 South Mathews Avenue, Urbana, IL 61801-3602, USA.

Abstract

Quantitative studies of RNA-protein interactions are quite cumbersome using traditional methods. We developed a rapid microplate-based fluorescence anisotropy (FA)/fluorescence polarization assay that works well, even with RNA probes >90 nucleotides long. We analyzed binding of RNA targets by vigilin/DDP1/SCP160p and by c-myc coding region instability determinant (CRD) binding protein, CRD-BP. Vigilin is essential for cell viability and functions in heterochromatin formation and mRNA decay. The CRD-BP stabilizes c-myc mRNA. Vigilin bound to a vitellogenin mRNA 3'-UTR probe with a two to three-fold lower affinity than to a Drosophila dodecasatellite ssDNA binding site and bound to the c-myc CRD with a two- to three-fold lower affinity than to the vitellogenin mRNA 3'-UTR. Competition between vigilin and CRD-BP for binding to the CRD may therefore play a role in regulating c-myc mRNA degradation. We analyzed suitability of the microplate-based FA assay for high-throughput screening for small-molecule regulators of RNA-protein interactions. The assay exhibits high reproducibility and precision and works well in 384-well plates and in 5 microl to 20 microl samples. To demonstrate the potential of this assay for screening libraries of small molecules to identify novel regulators of RNA-protein interactions, we identified neomycin and H33342 as inhibitors of binding of vigilin to the vitellogenin mRNA 3'-UTR.

PMID:
16448619
DOI:
10.1016/j.ab.2005.12.010
[Indexed for MEDLINE]

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