Mol. Cell. 59, 858–866 (2015)

Bioorthogonal chemistry approaches, which often include metabolic tagging with a chemical handle and its subsequent modification using a chemoselective reaction, have made it possible to specifically label biomolecules in cells. Many of these tools focus on protein labeling, but methods for tagging other biomolecules have enabled technologies including 'omic-'scale analysis of cellular nucleic acids. One approach for the reversible isolation of cellular RNAs involves metabolic labeling of transcripts with 4-thiouridine (s4U) nucleotides and selective disulfide formation between thiolated bases and a biotin affinity tag. Duffy et al. now report an enhancement of this method that allows them to robustly track microRNA (miRNA) turnover within cells. To optimize the disulfide exchange reaction between the s4U-modified RNA and the capture reagent, the authors used a methylthiosulfonate-activated form of biotin (MTS-biotin) and demonstrated that it outperformed the original reagent, efficiently modifying s4U nucleotides with high yields and in short reaction times. Profiling metabolically labeled RNAs from HEK293T cells with MTS-biotin revealed that the reagent provided broader coverage of the transcriptome and an enhanced representation of short RNA sequences than the earlier protocol. The greater efficiency of MTS-biotin enabled the authors to measure the levels of miRNAs in HEK293T cells without perturbing transcription or miRNA biogenesis pathways. Time-dependent labeling and deep sequencing revealed changes in cellular miRNA abundance, confirmed the existence of a subset of known miRNAs with fast-turnover kinetics and further identified two miRNAs whose turnover rates could not be characterized by other methods. The chemical insights of the current study have substantially improved this s4U-based RNA profiling method, enhancing its utility for future explorations of RNA dynamics in cells.