Expanding APEX2 Substrates for Proximity-Dependent Labeling of Nucleic Acids and Proteins in Living Cells

摘要

The subcellular organization of biomolecules such as proteins and nucleic acids is intimately linked to their biological functions. APEX2, an engineered ascorbate peroxidase that enables proximity-dependent labeling of proteins in living cells, has emerged as a powerful tool for deciphering the molecular architecture of various subcellular structures. However, only phenolic compounds have thus far been employed as APEX2 substrates, and the resulting phenoxyl radicals preferentially react with electron-rich amino acid residues. This narrow scope of substrates could potentially limit the application of APEX2. In this study, we screened a panel of aromatic compounds and identified biotin-conjugated arylamines as novel probes with significantly higher reactivity towards nucleic acids. As a demonstration of the spatial specificity and depth of coverage in mammalian cells, we applied APEX2 labeling with biotin-aniline (Btn-An) in the mitochondrial matrix, capturing all 13 mitochondrial messenger RNAs and none of the cytoplasmic RNAs. APEX2-mediated Btn-An labeling of RNA is thus a promising method for mapping the subcellular transcriptome, which could shed light on its functions in cell physiology.