Base Pairing and Functional Insights intoN3-methylcytidine (m3C) in RNA

ABSTRACT N 3 -methylcytidine (m 3 C) is present in both eukaryotic tRNA and mRNA and plays critical roles in many biological processes. We report the synthesis of the m 3 C phosphoramidite building block and its containing RNA oligonucleotides. The base-pairing stability and specificity studies show that the m 3 C modification significantly disrupts the stability of the Watson-Crick C:G pair. Further m 3 C 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 m 3 C:G base pairing pattern in an RNA duplex. More importantly, the biochemical investigation of m 3 C using reverse transcription shows that N 3 -methylation specifies the C:A pair and induces a G to A mutation 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.