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

Chengjian Mao, Kathryn Goolsby Flavin, Stanley Wang, Robin Dodson, Jeffrey Ross, David J. Shapiro

Research output: Contribution to journalArticlepeer-review


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 μl to 20 μl 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.

Original languageEnglish (US)
Pages (from-to)222-232
Number of pages11
JournalAnalytical Biochemistry
Issue number2
StatePublished - Mar 15 2006


  • CRD-BP
  • Fluorescence anisotropy/fluorescence polarization
  • Microplate-based FA assay
  • RNA-protein interactions
  • Vigilin
  • Vitellogenin mRNA
  • c-myc mRNA

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology


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