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Nanopore sequencing for N1-methylpseudouridine in RNA reveals sequence-dependent discrimination of the modified nucleotide triphosphate during transcription.
- Source :
-
Nucleic acids research [Nucleic Acids Res] 2023 Feb 28; Vol. 51 (4), pp. 1914-1926. - Publication Year :
- 2023
-
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.<br /> (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)
Details
- Language :
- English
- ISSN :
- 1362-4962
- Volume :
- 51
- Issue :
- 4
- Database :
- MEDLINE
- Journal :
- Nucleic acids research
- Publication Type :
- Academic Journal
- Accession number :
- 36727474
- Full Text :
- https://doi.org/10.1093/nar/gkad044