in vivo proximity-based mapping of subcellular proteomes, transcriptomes, and RNA structuromes

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 non-proteinogenic D-amino acid 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 a unique RNA reactivity of PRADA, we develop a mutational profiling sequencing strategy for spatiotemporally-resolved mapping of RNA secondary structure. Application of this multifaceted PRADA platform in a mouse tumor model offered insights into its mitochondrial proteome, transcriptome, and RNA structurome, revealing distinct RNA folding pattern as a critical layer of mitochondrial gene expression regulation. Collectively, the application of PRADA in diverse systems holds promise to uncover the intricate organization of biomolecules in vivo.