Metabolic labeling of RNAs with noncanonical nucleosides that are chemically active, followed by chemoselective conjugation with imaging probes or enrichment tags, has emerged as a powerful method for studying RNA transcription and degradation in eukaryotes. However, metabolic RNA labeling is not applicable for prokaryotes, in which the complexity and distinctness of gene regulation largely remain to be explored.
Peking University researchers report 2′-deoxy-2′-azidoguanosine (AzG) as a noncanonical nucleoside compatible with metabolic labeling of bacterial RNAs. With AzG, the researchers develop AIR-seq (azidonucleoside-incorporated RNA sequencing), which enables genome-wide analysis of transcription upon heat stress in Escherichia coli. Furthermore, AIR-seq coupled with pulse-chase labeling allows for global analysis of bacterial RNA degradation. Finally, the researchers demonstrate that RNAs of mouse gut microbiotas can be metabolically labeled with AzG in living animals. The AzG-enabled metabolic RNA labeling should find broad applications in studying RNA biology in various bacterial species.
AIR-seq for profiling RNA degradation
Workflow of pulse-chase labeling with AzG, followed by AIR-seq, for genome-wide measurement of RNA degradation.