Nanopore sequencing for N1-methylpseudouridine in RNA reveals sequence-dependent discrimination of the modified nucleotide triphosphate during transcription

Direct RNA sequencing with a commercial nanopore platform was used to sequence RNA containing uridine (U), pseudouridine (Ψ) or N1-methylpseudouridine (m(1)Ψ) in >100 different 5-nucleotide contexts. The base calling data for Ψ or m(1)Ψ were similar but different from U allowing their detection. Understanding the nanopore signatures for Ψ and m(1)Ψ enabled a running start T7 RNA polymerase assay to study the selection of UTP versus ΨTP or m(1)ΨTP competing mixtures in all possible adjacent sequence contexts. A significant sequence context dependency was observed for T7 RNA polymerase with insertion yields for ΨTP versus UTP spanning a range of 20–65%, and m(1)ΨTP versus UTP producing variable yields that differ by 15–70%. Experiments with SP6 RNA polymerase, as well as chemically-modified triphosphates and DNA templates provide insight to explain the observations. The SP6 polymerase introduced m(1)ΨTP when competed with UTP with a smaller window of yields (15–30%) across all sequence contexts studied. These results may aid in future efforts that employ RNA polymerases to make therapeutic mRNAs with sub-stoichiometric amounts of m(1)Ψ.