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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 (m1Ψ) generated by in vitro transcription (IVT). The base calling data as well as the ionic currents and dwell times for U, Ψ, or m1Ψ as they translocated through the helicase and nanopore proteins identified diagnostic signatures for Ψ and m1Ψ; however, the two modifications yielded similar patterns although both were different from U. Understanding the nanopore signatures for Ψ and m1Ψ enabled a running start T7 RNA polymerase assay to study how competing mixtures of UTP with ΨTP or m1ΨTP 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. m1ΨTP, UTP was favorably selected except in 5′-XA (X = U or m1Ψ) 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 m1Ψ.