Oligonucleotide-directed proximity-interactome mapping (O-MAP): A unified method for discovering RNA-interacting proteins, transcripts and genomic lociin situ

Abstract

Throughout biology, RNA molecules form complex networks of molecular interactions that are central to their function, but remain challenging to investigate. Here, we introduce Oligonucleotide-mediated proximity-interactome MAPping (O-MAP), a straightforward method for elucidating the biomolecules near an RNA of interest, within its native cellular context. O-MAP uses programmable oligonucleotide probes to deliver proximity-biotinylating enzymes to a target RNA, enabling nearby molecules to be enriched by streptavidin pulldown. O-MAP induces exceptionally precise RNA-localizedin situbiotinylation, and unlike alternative methods it enables straightforward optimization of its targeting accuracy. Using the 47S pre-ribosomal RNA and long noncoding RNAXistas models, we develop O-MAP workflows for unbiased discovery of RNA-proximal proteins, transcripts, and genomic loci. This revealed unexpected co-compartmentalization ofXistand other chromatin-regulatory RNAs and enabled systematic characterization of nucleolar-chromatin interactions across multiple cell lines. O-MAP is portable to cultured cells, organoids, and tissues, and to RNAs of various lengths, abundances, and sequence composition. And, O-MAP requires no genetic manipulation and uses exclusively off-the-shelf parts. We therefore anticipate its application to a broad array of RNA phenomena.

TREX reveals proteins that bind to specific RNA regions in living cells

“…Low efficiency has been a major caveat of these pulldown-based methods, often requiring large numbers of cells per experiment for successful identification of interacting proteins (McHugh and Guttman, 2018). An alternative strategy involves the use of proximity-based labeling enzymes, such as Biotin Ligase BirA (BioID) or Ascorbate Peroxidase (APEX), which can be targeted to a specific RNA of interest (Han et al, 2020;Mukherjee et al, 2019;Ramanathan et al, 2018;Tsue et al, 2023;Zhang et al, 2020). Subsequently, the locally labeled proteins are captured and identified by MS analysis.…”

DNA O-MAP uncovers the molecular neighborhoods associated with specific genomic loci

“…We address these pressing technical limitations by introducing DNA O-MAP, a locus purification method that uses oligo-based ISH probes to recruit peroxidase activity to specific DNA intervals. DNA O-MAP builds on our previously introduced RNA O-MAP 25 and pSABER 26 techniques, which target peroxidase activity to specific RNAs and RNAs/DNA intervals for purification or visualization, respectively. Here, we describe a cost-effective and scalable bulk hybridization and biotinylation workflows capable of processing millions of cells in parallel in just a few days, and demonstrate the recovered material is compatible with sample multiplexed proteomics 27 .…”

TREX reveals proteins that bind to specific RNA regions in living cells

“…Low efficiency has been a main caveat of these methods, often requiring large numbers of cells for successful identification of interacting RBPs 13 . An alternative strategy involves the use of proximity-based labeling enzymes, such as biotin ligase BirA or ascorbate peroxidase, targeted to a specific RNA of interest [14][15][16][17][18] . Subsequently, the labeled proteins are captured and identified by MS. A notable limitation of this approach is the inclusion of indirectly associated proteins that are in close physical proximity to the targeted RNA.…”