Base Pairing and Functional Insights into -Methylcytidine (mC) in RNA.

-methylcytidine (mC) is present in both eukaryotic tRNA and mRNA and plays critical roles in many biological processes. We report the synthesis of the mC phosphoramidite building block and its containing RNA oligonucleotides. The base-pairing stability and specificity studies show that the mC modification significantly disrupts the stability of the Watson-Crick C:G pair. Further mC 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 mC:G base pairing pattern in an RNA duplex. More importantly, the biochemical investigation of mC using reverse transcription shows that -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 mC in RNA and a foundation for developing new molecular tools for mapping mC in different RNA contexts and exploring the biochemical and biomedical potentials of mC in the design and development of RNA based therapeutics.