Base Pairing and Functional Insights into N³-Methylcytidine (m³C) in RNA

N³-methylcytidine (m³C) is present in both eukaryotic tRNA and mRNA and plays critical roles in many biological processes. We report the synthesis of the m³C phosphoramidite building block and its containing RNA oligonucleotides. The base-pairing stability and specificity studies show that the m³C modification significantly disrupts the stability of the Watson-Crick C:G pair. Further m³C decreases the base pairing discrimination between C:G and the other mismatched C:A, C:U, and C:C pairs. Our molecular dynamic simulation study further reveals the detailed structural insights into the m³C:G base pairing pattern in an RNA duplex. More importantly, the biochemical investigation of m³C using reverse transcription in vitro shows that N³-methylation specifies the C:A pair and induces a G to A change using HIV-1-RT, MMLV-RT, and MutiScribe-RT enzymes, all with relatively low replication fidelity. For other reverse transcriptases with higher fidelity like AMV-RT, the methylation could completely shut down DNA synthesis. Our work provides detailed insights into the thermostability of m³C in RNA and a foundation for developing new molecular tools for mapping m³C in different RNA contexts and exploring the biochemical and biomedical potentials of m³C in the design and development of RNA based therapeutics.