Synthesis and Purification of N3 -Methylcytidine (m3 C) Modified RNA Oligonucleotides.

This protocol describes a step-by-step chemical synthesis approach to prepare N3 -methylcytidine (m3 C) and its phosphoramidite. The method for synthesizing m3 C starts from commercially available cytidine, and proceeds via N3 -methylation in the presence of MeI, which generates the N3 -methylcytidine (m3 C) nucleoside, followed by the installation of several protecting groups at sites that include the 5'-hydroxyl group (4,4'-dimethoxytrityl protection), the 4-amino group (benzoyl protection), and the 2'-hydroxyl group (tert-butyldimethylsilyl, TBDMS, protection). Standard phosphoramidite chemistry is applied to prepare the final m3 C phosphoramidite for solid-phase synthesis of a series of RNA oligonucleotides. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of N3 -methylcytidine (m3 C) and its phosphoramidite Basic Protocol 2: Automated synthesis of m3 C modified RNA oligonucleotides.