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) generated by in vitro transcription (IVT). The base calling data as well as the ionic currents and dwell times for U, Ψ, or m 1 Ψ as they translocated through the helicase and nanopore proteins identified diagnostic signatures for Ψ and m 1 Ψ; however, the two modifications yielded similar patterns although both were different from U. Understanding the nanopore signatures for Ψ and m 1 Ψ enabled a running start T7 RNA polymerase assay to study how competing mixtures of UTP with ΨTP or m 1 ΨTP lead to nucleotide selection in all possible adjacent sequence contexts. For UTP vs. ΨTP, ΨTP was favorably incorporated in singly-modified contexts, while doubly-modified contexts found high yields of ΨTP insertion on the 5′ side and lower yields on the 3′ side. For UTP vs. m 1 ΨTP, UTP was favorably selected except in 5′-XA (X = U or m 1 Ψ) where the ratio was determined by their relative NTP concentrations. Experiments with chemically-modified triphosphates and DNA templates designed based on the structure of T7 RNA polymerase provide a model to explain the observations. These results may aid in future efforts that employ IVT to make therapeutic mRNAs with sub-stochiometric amounts of m 1 Ψ.