Ashley F Tsue, Evan E Kania, Diana Q Lei, Rose Fields, Christopher D McGann, Daphnée M Marciniak, Elliot A Hershberg, Xinxian Deng, Maryanne Kihiu, Shao-En Ong, Christine M Disteche, Sita Kugel, Brian J Beliveau, Devin K Schweppe, David M Shechner
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引用次数: 0
Abstract
RNA molecules form complex networks of molecular interactions that are central to their function and to cellular architecture. But these interaction networks are difficult to probe in situ. Here, we introduce Oligonucleotide-mediated proximity-interactome MAPping (O-MAP), a method for elucidating the biomolecules near an RNA of interest, within its native context. O-MAP uses RNA-fluorescence in situ hybridization-like oligonucleotide probes to deliver proximity-biotinylating enzymes to a target RNA in situ, enabling nearby molecules to be enriched by streptavidin pulldown. This induces exceptionally precise biotinylation that can be easily optimized and ported to new targets or sample types. Using the noncoding RNAs 47S, 7SK and Xist as models, we develop O-MAP workflows for discovering RNA-proximal proteins, transcripts and genomic loci, yielding a multiomic characterization of these RNAs' subcellular compartments and new regulatory interactions. O-MAP requires no genetic manipulation, uses exclusively off-the-shelf parts and requires orders of magnitude fewer cells than established methods, making it accessible to most laboratories.
期刊介绍:
Nature Methods is a monthly journal that focuses on publishing innovative methods and substantial enhancements to fundamental life sciences research techniques. Geared towards a diverse, interdisciplinary readership of researchers in academia and industry engaged in laboratory work, the journal offers new tools for research and emphasizes the immediate practical significance of the featured work. It publishes primary research papers and reviews recent technical and methodological advancements, with a particular interest in primary methods papers relevant to the biological and biomedical sciences. This includes methods rooted in chemistry with practical applications for studying biological problems.