Multiomic proximity labeling in vivo by D-amino-acid-activated peroxidase reaction

Understanding the spatial organization of biomolecules in living organisms is a central but challenging goal in biology. Here, we introduce peroxidase reactions activated by D-amino acids (PRADA), which uses an engineered oxidase to convert nonproteinogenic D-amino acids into H2O2 for in situ activation of a genetically fused peroxidase. PRADA enables versatile proximity-based chemistries, including protein labeling, RNA labeling and functional polymer assembly, with minimal toxicity and background. We demonstrate that PRADA is broadly applicable inside living organisms, including flies, worms, fish and mice. By further leveraging the unique RNA reactivity of PRADA, we developed a mutational profiling sequencing strategy for spatiotemporally resolved mapping of RNA secondary structure. Application of this multifaceted PRADA platform in a mouse xenograft model offered insights into its mitochondrial proteome, transcriptome and RNA structurome, revealing RNA folding as a regulatory mechanism in mitochondrial gene expression. Collectively, PRADA holds promise to uncover the intricate organization of biomolecules in vivo.