Chemistry of installing epitranscriptomic 5-modified cytidines in RNA oligomers

Studies of 5-hydroxymethylcytidine (hm⁵C), 5-formylcytidine (f⁵C) and 5-carboxycytidine (ca⁵C) modifications as products of the 5-methylcytidine (m⁵C) oxidative demethylation pathway in cellular mRNAs constitute an important element of the new epitranscriptomic field of research. The dynamic process of m⁵C conversion and final turnover to the parent cytidine is considered a post-transcriptional layer of gene-expression regulation. However, the regulatory mechanism associated with epitranscriptomic cytidine modifications remains largely unknown. Therefore, oligonucleotides containing m⁵C oxidation products are of great value for the next generation of biochemical, biophysical, and structural studies on their function, metabolism, and contribution to human diseases. Herein, we summarize the synthetic strategies developed for the incorporation of hm⁵C, f⁵C and ca⁵C into RNA oligomers by phosphoramidite chemistry, including post-synthetic C5-cytidine functionalization and enzymatic methods.