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 (m(1)psi) in >100 different 5-nucleotide contexts. The base calling data for psi or m(1)psi were similar but different from U allowing their detection. Understanding the nanopore signatures for psi and m(1)psi enabled a running start T7 RNA polymerase assay to study the selection of UTP versus psi TP or m(1)psi TP competing mixtures in all possible adjacent sequence contexts. A significant sequence context dependency was observed for T7 RNA polymerase with insertion yields for psi TP versus UTP spanning a range of 20-65%, and m(1)psi TP 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)psi TP 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)psi.

Automated summary

Direct RNA sequencing using a commercial nanopore platform was utilized to sequence RNA molecules with uridine (U), pseudouridine (psi) or N1-methylpseudouridine (m(1)psi) in various contexts. The base calling data for psi and m(1)psi differed from U, allowing their detection. The study examined the selection of UTP, psi TP, or m(1)psi TP by T7 RNA polymerase in different adjacent sequence contexts, finding a significant dependency on sequence context.