Photo‐Facilitated Detection and Sequencing of 5‐Formylcytidine RNA

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.