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Protonation-dependent sequencing of 5-formylcytidine in RNA.
Biochemistry 61, 535–544 (2022)
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.
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Publication type
Article: Journal article
Document type
Scientific Article
ISSN (print) / ISBN
0006-2960
e-ISSN
1520-4995
Journal
Biochemistry
Quellenangaben
Volume: 61,
Issue: 7,
Pages: 535–544
Publisher
American Chemical Society (ACS)
Non-patent literature
Publications
Reviewing status
Peer reviewed
Institute(s)
Institute of Neurogenomics (ING)
Grants
CCR NIH HHS
NIH HHS
NCI NIH HHS
NIH HHS
NCI NIH HHS