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Anal Bioanal Chem. 2012 Feb;402(5):1889-98. doi: 10.1007/s00216-011-5604-0. Epub 2012 Jan 5.

Two high-throughput screening assays for aberrant RNA-protein interactions in myotonic dystrophy type 1.

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1
NIH Chemical Genomics Center, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA.

Abstract

Myotonic dystrophy type 1 (DM1), the most prevalent form of adult muscular dystrophy, is caused by expansion of a CTG repeat in the 3' untranslated region of the DM protein kinase (DMPK) gene. The pathogenic effects of the CTG expansion arise from the deleterious effects of the mutant transcript. RNA with expanded CUG tracts alters the activities of several RNA binding proteins, including muscleblind-like 1 (MBNL1). MBNL1 becomes sequestered in nuclear foci in complex with the expanded CUG-repeat RNA. The resulting loss of MBNL1 activity causes misregulated alternative splicing of multiple genes, leading to symptoms of DM1. The binding interaction between MBNL1 and mutant RNA could be a key step in the pathogenesis of DM1 and serves as a potential target for therapeutic intervention. We have developed two high-throughput screens suitable assays using both homogenous time-resolved fluorescence energy transfer and AlphaScreen technologies to detect the binding of a C-terminally His-tagged MBNL1 and a biotinylated (CUG)(12) RNA. These assays are homogenous and successfully miniaturized to 1,536-well plate format. Both assays were validated and show robust signal-to-basal ratios and Z' factors.

PMID:
22218462
PMCID:
PMC3280409
DOI:
10.1007/s00216-011-5604-0
[Indexed for MEDLINE]
Free PMC Article
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