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The cellular RNA pool in animals arises from two separate genomes stored in the nucleus and multiple mitochondria. Chemical methods to track nascent RNA synthesis are unable to distinguish between these two with stringency. Herein, we report that spatially restricting bioorthogonal nucleoside biosynthesis enables, for the first time, selective metabolic labeling of the RNA transcribed in the mitochondria. We envision that this approach could open the door for heretofore-impossible analyses of mitochondrial RNA. Beyond our results revealed herein, our approach provides a roadmap for researchers to begin to design strategies to examine biomolecules within subcellular compartments.

Spatially Restricting Bioorthogonal Nucleoside Biosynthesis Enables Selective Metabolic Labeling of the Mitochondrial Transcriptome