Synthesis of N4-Methylcytidine (m4C) and N4,N4-Dimethylcytidine (m42C) Modified RNA

This article summarizes the protocols for phosphoramidite chemistry and solid phase synthesis of RNA oligonucleotides containing N4 -methylcytidine (m4 C) and N4 ,N4 -dimethylcytidine (m42 C) residues for base-pairing, structural, and enzymatic activity studies. The two key m4 C and m42 C phosphoramidite building blocks can be synthesized starting from the partially protected cytidine nucleosides, followed by solid-phase synthesis and HPLC purification of the modified target RNA oligonucleotides. These modified RNA strands are then prepared for base pairing stability, specificity, and structural studies using UV-melting temperature (Tm ) measurements and X-ray crystallography. Functional studies are performed by reverse transcription assays in primer extension reactions employing different enzymes. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Chemical synthesis of m4 C phosphoramidite Basic Protocol 2: Synthesis of m42 C phosphoramidite Basic Protocol 3: Synthesis and purification of m4 C and m42 C containing RNA oligonucleotides.