PT  - JOURNAL ARTICLE
AU  - Song Mao
AU  - Phensinee Haruehanroengra
AU  - Srivathsan V. Ranganathan
AU  - Fusheng Shen
AU  - Thomas J. Begley
AU  - Jia Sheng
TI  - Base Pairing and Functional Insights into <em>N<sup>3</sup></em>-methylcytidine (m<sup>3</sup>C) in RNA
AID  - 10.1101/2020.08.27.270322
DP  - 2020 Jan 01
TA  - bioRxiv
PG  - 2020.08.27.270322
4099  - http://biorxiv.org/content/early/2020/08/28/2020.08.27.270322.short
4100  - http://biorxiv.org/content/early/2020/08/28/2020.08.27.270322.full
AB  - 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 shows that N3-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.Competing Interest StatementThe authors have declared no competing interest.