A Photo-triggered Wittig-Cyclization for Detecting and Sequencing 5-Formylcytidine in RNA

The simultaneous detection of a specific post-transcriptional modification that decorates the natural RNA can provide comprehensive information to better understand cellular and molecular functions of this variation. 5-Formylcytidine f5C is one of the natural nucleotides that was discovered in the tRNA of many species during the oxidative demethylation of 5-methylcytidine m5C. Despite the evident importance of f5C in playing an important role in gene expression regulation, little is known about the exact amount and positions of this modified cytidine in RNAs, as well as its exact functions. To capture this type of information, we performed a site-specific dual functionalization of f5C with semi-stabilized ylide cyanomethylene triphenylphosphorane that can simultaneously seizure the C5-formyl and N4-amino groups in the cytidine residue under light irradiation. The photochemical labelling with f5C-containing oligonucleotides imparts a high selectivity towards f5C and allows distinction from structurally similar 5-formyluridine f5U. We implemented a detection strategy of f5C modification in RNA based on the fluorescence signal of the cyclization product 4,5-pyridin-2-amine-cytidine paC, which exhibited a relatively high fluorescence quantum yield. The results could clearly identify f5C with a limit of detection LOD at 0.58 nM. The chemical labelling was capable of altering the hydrogen bonding activity of the parent cytidine and enabled the modulation of the reverse transcription signature of f5C in sequencing profile. We showed that using this Pan-Seq strategy, f5C can be detected from tRNA segments with a single-base resolution. Taken together, this approach represents a sensitive, robust, antibody-free and applicable detection ad sequencing to f5C RNA.