Photo‐Facilitated Detection and Sequencing of 5‐Formylcytidine RNA

Abstract 5‐Formylcytidine (f 5 C) is one of the epigenetic nucleotides in tRNA. Despite the evident importance of f 5 C in gene expression regulation, little is known about its exact amount and position. To capture this information, we developed a modification‐specific functionalization with a semi‐stabilized ylide. The chemical labelling exhibited a high selectivity towards f 5 C and allowed distinction from similar 5‐formyluridine. We realized a detection strategy based on the fluorescence signal of the cyclization product 4,5‐pyridin‐2‐amine‐cytidine paC, which exhibited a high quantum yield. The results clearly identified f 5 C with a limit of detection at 0.58 nM. This method altered the hydrogen bonding activities of f 5 C and modulated its reverse transcription signature in its sequencing profile. We showed that f 5 C can be detected from tRNA segments with a single‐base resolution. Taken together, this approach is a sensitive, antibody‐free, and applicable detection and sequencing method for f 5 C‐containing RNA.