(644.12) Protonation-Dependent Sequencing of 5-Formylcytidine in RNA

Poster Board Number: A39

Courtney Link (National Cancer Institute), Supuni Thalalla Gamage (National Cancer Institute), Diamond Gallimore (National Cancer Institute), Robert Kopajtich (Helmholtz Zentrum München), Christine Evans (Frederick National Laboratory for Cancer Research), Samantha Nance (National Cancer Institute), Stephen Fox (Frederick National Laboratory for Cancer Research), Thorkell Andresson (Frederick National Laboratory for Cancer Research), Raj Chari (Frederick National Laboratory for Cancer Research), Joseph Ivanic (Frederick National Laboratory for Cancer Research), Holger Prokisch (Helmholtz Zentrum München), Jordan Meier (National Cancer Institute)

Chemical modification of cytidine in non-coding RNAs plays a key role in regulating translation and disease. However, the distribution and dynamics of many of these modifications remains unknown due to a lack of sensitive site-specific sequencing technologies. Here we report a protonation-dependent sequencing reaction for detection of 5-formylcytidine (5fC) and 5-carboxycytidine (5caC) in RNA. First, we 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, we apply this reaction to detect these modifications in synthetic oligonucleotides as well as endogenous human tRNA. Finally, we 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 provide a molecular foundation for applying optimized sequencing reactions to better understand the role of oxidized RNA cytidine nucleobases in disease.

This project has been funded in whole or in part with Federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261200800001E. This work was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research (ZIA-BC011488).