Synthesis and Purification of N -Methylcytidine (m C) Modified RNA Oligonucleotides.

This protocol describes a step-by-step chemical synthesis approach to prepare N -methylcytidine (m C) and its phosphoramidite. The method for synthesizing m C starts from commercially available cytidine, and proceeds via N -methylation in the presence of MeI, which generates the N -methylcytidine (m 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 m C phosphoramidite for solid-phase synthesis of a series of RNA oligonucleotides. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of N -methylcytidine (m C) and its phosphoramidite Basic Protocol 2: Automated synthesis of m C modified RNA oligonucleotides.