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Epitranscriptomic RNA modifications can regulate fundamental biological processes, but we lack approaches to map modification sites and probe writer enzymes. Here we present a chemoproteomic strategy to characterize RNA 5-methylcytidine (m⁵C) dioxygenase enzymes in their native context based upon metabolic labeling and activity-based crosslinking with 5-ethynylcytidine (5-EC). We profile m⁵C dioxygenases in human cells including ALKBH1 and TET2 and show that ALKBH1 is the major hm⁵C- and f⁵C-forming enzyme in RNA. Further, we map ALKBH1 modification sites transcriptome-wide using 5-EC-iCLIP and ARP-based sequencing to identify ALKBH1-dependent m⁵C oxidation in a variety of tRNAs and mRNAs and analyze ALKBH1 substrate specificity in vitro. We also apply targeted pyridine borane-mediated sequencing to measure f⁵C sites on select tRNA. Finally, we show that f⁵C at the wobble position of tRNA-Leu-CAA plays a role in decoding Leu codons under stress. Our work provides powerful chemical approaches for studying RNA m⁵C dioxygenases and mapping oxidative m⁵C modifications and reveals the existence of novel epitranscriptomic pathways for regulating RNA function.

 

The challenge of site-selectivity must be overcome in many chemical research contexts, including selective functionalization in complex natural products and labeling of one biomolecule in a living system. Synthetic catalysts incorporating molecular recognition domains can mimic naturally-occurring enzymes to direct a chemical reaction to a particular instance of a functional group. We propose that DNA-conjugated small molecule catalysts (DCats), prepared by tethering a small molecule catalyst to a DNA aptamer, are a promising class of reagents for site-selective transformations. Specifically, a DNA-imidazole conjugate able to increase the rate of ester hydrolysis in a target ester by >100-fold compared with equimolar untethered imidazole was developed. Other esters are unaffected. Furthermore, DCat-catalyzed hydrolysis follows enzyme-like kinetics and a stimuli-responsive variant of the DCat enables programmable “turn on” of the desired reaction.