RNA modifications play important roles in RNA structures and regulation of gene expression and translation. We report the first RNA modification on the phosphate, the RNA phosphorothioate (PS) modification, discovered in both prokaryotes and eukaryotes. The PS modification is also first reported on nucleic acids of eukaryotes. The GpsG modification exists in the Rp configuration and was quantified with liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). By knocking out the DndA gene in E. coli, we show the Dnd clusters that regulate DNA PS modification may also play roles in RNA PS modification. We also show that the GpsG modification locates on rRNA in E. coli, L. lactis, and HeLa cells, and it is not detected in rRNA-depleted total RNAs from these cells.

N 3-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 N 3-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.

The N4-methylation of cytidine (m4C and m42C) in RNA plays important roles in both bacterial and eukaryotic cells. In this work, we synthesized a series of m4C and m42C modified RNA oligonucleotides, conducted their base pairing and bioactivity studies, and solved three new crystal structures of the RNA duplexes containing these two modifications. Our thermostability and X-ray crystallography studies, together with the molecular dynamic simulation studies, demonstrated that m4C retains a regular C:G base pairing pattern in RNA duplex and has a relatively small effect on its base pairing stability and specificity. By contrast, the m42C modification disrupts the C:G pair and significantly decreases the duplex stability through a conformational shift of native Watson-Crick pair to a wobble-like pattern with the formation of two hydrogen bonds. This double-methylated m42C also results in the loss of base pairing discrimination between C:G and other mismatched pairs like C:A, C:T and C:C. The biochemical investigation of these two modified residues in the reverse transcription model shows that both mono- or di-methylated cytosine bases could specify the C:T pair and induce the G to T mutation using HIV-1 RT. In the presence of other reverse transcriptases with higher fidelity like AMV-RT, the methylation could either retain the normal nucleotide incorporation or completely inhibit the DNA synthesis. These results indicate the methylation at N4-position of cytidine is a molecular mechanism to fine tune base pairing specificity and affect the coding efficiency and fidelity during gene replication.