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

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.

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

The accuracy of crucial nuclear processes such as transcription, replication, and repair, depends on the local composition of chromatin and the regulatory proteins that reside there. Understanding these DNA-protein interactions at the level of specific genomic loci has remained challenging due to technical limitations. Here, we introduce a method termed "DNA O-MAP", which uses programmable peroxidase-conjugated oligonucleotide probes to biotinylate nearby proteins.

Programmable peroxidase-assisted signal amplification enables flexible detection of nucleic acid targets in cellular and histopathological specimens.

hybridization (ISH) is a powerful tool for investigating the spatial arrangement of nucleic acid targets in fixed samples. ISH is typically visualized using fluorophores to allow high sensitivity and multiplexing or with colorimetric labels to facilitate co-visualization with histopathological stains. Both approaches benefit from signal amplification, which makes target detection effective, rapid, and compatible with a broad range of optical systems.

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

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.

Recognition of the Diglycine C-End Degron by CRL2 Ubiquitin Ligase.

Aberrant proteins can be deleterious to cells and are cleared by the ubiquitin-proteasome system. A group of C-end degrons that are recognized by specific cullin-RING ubiquitin E3 ligases (CRLs) has recently been identified in some of these abnormal polypeptides. Here, we report three crystal structures of a CRL2 substrate receptor, KLHDC2, in complex with the diglycine-ending C-end degrons of two early-terminated selenoproteins and the N-terminal proteolytic fragment of USP1.

Automated quantitative image analysis for ex vivo metastasis assays reveals differing lung composition requirements for metastasis suppression by KISS1.

Imaging is broadly used in biomedical research, but signal variation complicates automated analysis. Using the Pulmonary Metastasis Assay (PuMA) to study metastatic colonization by the metastasis suppressor KISS1, we cultured GFP-expressing melanoma cells in living mouse lung ex vivo for 3 weeks. Epifluorescence images of cells were used to measure growth, creating large datasets which were time consuming and challenging to quantify manually due to scattering of light from outside the focal plane.