Synthesis of N4 -Methylcytidine (m4 C) and N4 ,N4 -Dimethylcytidine (m4 2 C) Modified RNA.

ABSTRACT

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 (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 m4 2 C phosphoramidite Basic Protocol 3 Synthesis and purification of m4 C and m4 2 C containing RNA oligonucleotides.