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RNA. 2016 Nov;22(11):1785-1792. Epub 2016 Sep 22.

A proximity-dependent assay for specific RNA-protein interactions in intact cells.

Zhang W1, Xie M2,3, Shu MD2,3, Steitz JA2,3,4, DiMaio D1,3,4,5.

Author information

1
Department of Genetics, Yale School of Medicine, New Haven, Connecticut 06520-8005, USA.
2
Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06520-208024, USA.
3
Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06520-208024, USA.
4
Yale Cancer Center, New Haven, Connecticut 06520-8028, USA.
5
Department of Therapeutic Radiology, Yale School of Medicine, New Haven, Connecticut 06520-208040, USA.

Abstract

The proximity ligation assay (PLA) is an immune staining method that detects protein-protein interactions in fixed cells. We describe here RNA-PLA, a simple adaptation of this technology that allows the detection of specific RNA-protein interactions in fixed cells by using a DNA oligonucleotide that hybridizes to a target RNA in combination with an antibody that recognizes the protein bound to the target RNA. Stable and transient RNA-protein interactions can be readily detected by generation of a fluorescent signal in discrete compartments in intact fixed cells with high specificity. We demonstrate that this approach requires the colocalization of the binding protein and its RNA target in the same cellular compartment, use of an oligonucleotide complementary to the target RNA, and the presence of a binding site for the protein in the target RNA.

KEYWORDS:

PLA; RNA–protein complex; RNP; gene expression; small nuclear RNA

PMID:
27659050
PMCID:
PMC5066630
DOI:
10.1261/rna.058248.116
[Indexed for MEDLINE]
Free PMC Article

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