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

Abstract

Direct RNA sequencing with a commercial nanopore platform was used to sequence RNA containing uridine (U), pseudouridine (ψ) or N1-methylpseudouridine (m1ψ) in >100 different 5-nucleotide contexts. The base calling data for ψ or m1ψ were similar but different from U allowing their detection. Understanding the nanopore signatures for ψ and m1ψ enabled a running start T7 RNA polymerase assay to study the selection of UTP versus ψTP or m1ψTP 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 m1ψTP 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 m1ψTP 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 m1ψ.