Photo-Facilitated Detection and Sequencing of 5-Formylcytidine RNA

5-Formylcytidine (f⁵ C) is one of the epigenetic nucleotides in tRNA. Despite the evident importance of f⁵ 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 f⁵ 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 f⁵ C with a limit of detection at 0.58 nM. This method altered the hydrogen bonding activities of f⁵ C and modulated its reverse transcription signature in its sequencing profile. We showed that f⁵ 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 f⁵ C-containing RNA.