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 ({Psi}), or N1-methylpseudouridine (m1{Psi}) generated by in vitro transcription (IVT). The base calling data as well as the ionic currents and dwell times for U, {Psi}, or m1{Psi} as they translocated through the helicase and nanopore proteins identified diagnostic signatures for {Psi} and m1{Psi}; however, the two modifications yielded similar patterns although both were different from U. Understanding the nanopore signatures for {Psi} and m1{Psi} enabled a running start T7 RNA polymerase assay to study how competing mixtures of UTP with {Psi}TP or m1{Psi}TP lead to nucleotide selection in all possible adjacent sequence contexts. For UTP vs. {Psi}TP, {Psi}TP was favorably incorporated in singly-modified contexts, while doubly-modified contexts found high yields of {Psi}TP insertion on the 5' side and lower yields on the 3' side. For UTP vs. m1{Psi}TP, UTP was favorably selected except in 5'-XA (X = U or m1{Psi}) 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 m1{Psi}.