Comprehensive identification of RNA-protein interactions in any organism using orthogonal organic phase separation (OOPS)

Nat Biotechnol. 2019 Feb;37(2):169-178. doi: 10.1038/s41587-018-0001-2. Epub 2019 Jan 3.


Existing high-throughput methods to identify RNA-binding proteins (RBPs) are based on capture of polyadenylated RNAs and cannot recover proteins that interact with nonadenylated RNAs, including long noncoding RNA, pre-mRNAs and bacterial RNAs. We present orthogonal organic phase separation (OOPS), which does not require molecular tagging or capture of polyadenylated RNA, and apply it to recover cross-linked protein-RNA and free protein, or protein-bound RNA and free RNA, in an unbiased way. We validated OOPS in HEK293, U2OS and MCF10A human cell lines, and show that 96% of proteins recovered were bound to RNA. We show that all long RNAs can be cross-linked to proteins, and recovered 1,838 RBPs, including 926 putative novel RBPs. OOPS is approximately 100-fold more efficient than existing methods and can enable analyses of dynamic RNA-protein interactions. We also characterize dynamic changes in RNA-protein interactions in mammalian cells following nocodazole arrest, and present a bacterial RNA-interactome for Escherichia coli. OOPS is compatible with downstream proteomics and RNA sequencing, and can be applied in any organism.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line, Tumor
  • Cluster Analysis
  • Cross-Linking Reagents / chemistry
  • Escherichia coli
  • Glycoproteins / chemistry
  • HEK293 Cells
  • Humans
  • Nocodazole / chemistry
  • Protein Binding
  • Proteome
  • Proteomics
  • RNA / chemistry
  • RNA / isolation & purification*
  • RNA, Bacterial / chemistry
  • RNA, Long Noncoding / chemistry
  • RNA, Messenger / chemistry*
  • RNA-Binding Proteins / chemistry
  • RNA-Binding Proteins / isolation & purification*
  • Sequence Analysis, RNA
  • Thymidine / chemistry
  • Transcriptome


  • Cross-Linking Reagents
  • Glycoproteins
  • Proteome
  • RNA, Bacterial
  • RNA, Long Noncoding
  • RNA, Messenger
  • RNA-Binding Proteins
  • RNA
  • Nocodazole
  • Thymidine