4-Thiouridine-Enhanced Peroxidase-Generated Biotinylation of RNA

Peroxidase-generated proximity labeling is in widespread use to study subcellular proteomes and the protein interaction networks in living cells, but the development of subcellular RNA labeling is limited. APEX-seq has emerged as a new method to study subcellular RNA in living cells, but the labeling of RNA still has room to improve. In this work, we describe 4-thiouridine (s⁴ U)-enhanced peroxidase-generated biotinylation of RNA with high efficiency. The incorporation of s⁴ U could introduce additional sites for RNA labeling, enhanced biotinylation was observed on monomer, model oligo RNA and total RNA. Through the s⁴ U metabolic approach, the in vivo RNA biotinylation efficiency by peroxidase catalysis was also dramatically increased, which will benefit RNA isolation and study for the spatial transcriptome.