Multiomic characterization of RNA microenvironments by oligonucleotide-mediated proximity-interactome mapping

Area of Science:

• Molecular Biology
• Genomics
• Biochemistry

Background:

• RNA molecules form complex interaction networks crucial for cellular function and architecture.
• Probing these RNA interaction networks in situ remains a significant technical challenge.

Purpose of the Study:

• To introduce Oligonucleotide-mediated proximity-interactome MAPping (O-MAP), a novel method for in situ RNA interactome elucidation.
• To characterize the subcellular localization and regulatory interactions of noncoding RNAs.

Main Methods:

• O-MAP utilizes oligonucleotide probes to deliver proximity-biotinylating enzymes to target RNAs within their native cellular context.
• Biotinylated molecules in proximity to the target RNA are enriched via streptavidin pulldown.
• Workflows were developed using 47S, 7SK, and Xist noncoding RNAs as models.

Main Results:

• O-MAP successfully identified RNA-proximal proteins, transcripts, and genomic loci.
• The method enabled multiomic characterization of RNA subcellular compartments and regulatory interactions.
• Demonstrated precise biotinylation, ease of optimization, and portability across targets and sample types.

Conclusions:

• O-MAP provides an accessible and efficient approach to map RNA interactomes in situ.
• The method requires no genetic manipulation, uses off-the-shelf components, and demands significantly fewer cells than existing techniques.
• O-MAP facilitates the discovery of novel RNA functions and regulatory mechanisms.