Phosphate backbone epitranscriptomics: discovery of natural RNA phosphorothioates and their writer machinery

In contrast, phosphate backbone modifications, and particularly phosphorothioates (PS), a replacement of a non-bridging oxygen with a sulfur group, are central to RNA therapeutics, enhancing stability and bioavailability. While PS modifications are known on DNA, they were never reliably detected on natural RNA.

Here, we establish sequencing- and mass spectrometry–based workflows that quantitatively map RNA PS at single-nucleotide resolution. Across diverse archaeal species, we identify highly specific PS at conserved positions in ribosomal RNAs (rRNAs) and transfer RNA (tRNA) hotspots, including in proximity to the peptidyltransferase center. Across most targets PS deposition was highly dynamic with sulfur availability, and in a subset of targets PS deposition was temperature-dependent.