Synthesis of N⁴ -Methylcytidine (m⁴ C) and N⁴ ,N⁴ -Dimethylcytidine (m⁴ ₂ C) 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 (m⁴ ₂ C) residues for base-pairing, structural, and enzymatic activity studies. The two key m⁴ C and m⁴ ₂ 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 (T_m ) 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 m⁴ ₂ C phosphoramidite Basic Protocol 3: Synthesis and purification of m⁴ C and m⁴ ₂ C containing RNA oligonucleotides.