Affiliation:
1. Dept. of Chemistry, University of Utah , Salt Lake City , UT 84112-0850, USA
Abstract
AbstractDirect 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.
Funder
National Institutes of Health
Publisher
Oxford University Press (OUP)
Cited by
6 articles.
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