ABSTRACT Ribozymes for site‐specific RNA modification provide an elegant approach for the installation of diverse functional groups, fluorophores, affinity tags, or crosslinkers at defined positions within an RNA of interest. There is increasing interest in expanding the ribozyme toolbox, since recently reported in vitro selected ribozymes have been mostly limited to labeling at adenosine sites, either by alkylation of the nucleobase or phosphodiester formation at the 2’‐OH group. Here we report a cytidine‐specific alkyltransferase ribozyme (CSAR) that uses O 6 ‐benzylguanines as alkyl group donors. CSAR is the first ribozyme that catalyzes direct alkylation of the exocyclic amino group of a nucleobase and generates N 4 ‐alkylated cytidine in a defined sequence context of a short RNA hairpin loop. In combination with tuning the electronic parameters of the transferred benzyl group, CSAR enables highly efficient cytidine alkylation for the installation of bioorthogonal functional groups.
Dorinova et al. (2026) studied this question.