Protonation-Dependent Sequencing of 5-Formylcytidine in RNA.
Biochemistry
; 61(7): 535-544, 2022 04 05.
Article
em En
| MEDLINE
| ID: mdl-35285626
ABSTRACT
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. Here, we report a protonation-dependent sequencing reaction for the 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 transfer RNA (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 more broadly provide a molecular foundation for using optimized sequencing reactions to better understand the role of oxidized RNA cytidine residues in diseases.
Texto completo:
1
Bases de dados:
MEDLINE
Assunto principal:
RNA
/
Citidina
Limite:
Humans
Idioma:
En
Revista:
Biochemistry
Ano de publicação:
2022
Tipo de documento:
Article
País de afiliação:
Estados Unidos