An Optimized Chemical-Genetic Method for Cell-Specific Metabolic Labeling of RNA

Tissues and organs are composed of diverse cell types, which poses a major challenge for cell-specific gene expression profiling. Current metabolic labeling methods rely on the inability of mammalian cells to incorporate exogenous pyrimidine analogs, which are then co-opted by ectopically-expressed enzymes. We demonstrate that mammalian cells can incorporate uracil analogs and characterize the enzymatic pathways responsible for high background incorporation. To overcome these limitations, we developed a novel small-molecule/enzyme pair consisting of uridine-cytidine kinase 2 (UCK2) and 2’-azidouridine (2’AzUd). We demonstrate that 2’AzUd is only incorporated in UCK2-expressing cells and characterize selectivity mechanisms using molecular dynamics and X-ray crystallography. Furthermore, this pair can be used to purify and track RNA from specific cellular populations, making it ideal for high-resolution cell-specific RNA labeling. Overall, these results reveal novel aspects of mammalian salvage pathways and serve as a new benchmark for designing, characterizing and evaluating cell-specific biomolecule labeling methodologies.