Chemical modification of cytidine in noncoding RNAs plays a key role in regulating translation and disease. However, the distribution and dynamics of many of these modifications remain unknown due to a lack of sensitive site-specific sequencing technologies. Researchers at the National Cancer Institute, Frederick have developed a protonation-dependent sequencing reaction for the detection of 5-formylcytidine (5fC) and 5-carboxycytidine (5caC) in RNA. First, the researchers evaluate how protonation combined with electron-withdrawing substituents alters the molecular orbital energies and reduction of modified cytidine nucleosides, highlighting 5fC and 5caC as reactive species. Next, they apply this reaction to detect these modifications in synthetic oligonucleotides as well as endogenous human transfer RNA (tRNA). Finally, they demonstrate the utility of our method to characterize a patient-derived model of 5fC deficiency, where it enables facile monitoring of both pathogenic loss and exogenous rescue of NSUN3-dependent 5fC within the wobble base of human mitochondrial tRNAMet. These studies showcase the ability of protonation to enhance the reactivity and sensitive detection of 5fC in RNA and more broadly provide a molecular foundation for using optimized sequencing reactions to better understand the role of oxidized RNA cytidine residues in diseases.
(a) Schematic for assessing reaction-based detection of 5caC and 5fC in RNA using protonationassisted cyanoborohydride sequencing. (b) pH-dependent reaction of 5fC in RNA with NaCNBH3 as assessed by dot blot. (c) pH-dependent reaction of 5caC in RNA NaCNBH3 as assessed by dot blot. (d) Misincorporation signals in RNAs containing C (top), 5fC (middle), and 5caC (bottom) following treatment with NaCNBH3 (pH 1), reverse transcription, PCR, and Sanger sequencing.