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Title: Nanopore sequencing for N1-methylpseudouridine in RNA reveals sequence-dependent discrimination of the modified nucleotide triphosphate during transcription
Authors: Fleming, Aaron M.
Keywords: Nanopore; N1-methylpseudouridine; RNA; nucleotide triphosphate; phiên mã; biến đổi
Issue Date: 2023
Publisher: bioRxiv
Abstract: Direct RNA sequencing with a commercial nanopore platform was used to sequence RNA containing uridine (U), pseudouridine (Ψ), or N1-methylpseudouridine (m1Ψ) generated by in vitro transcription (IVT). The base calling data as well as the ionic currents and dwell times for U, Ψ, or m1Ψ as they translocated through the helicase and nanopore proteins identified diagnostic signatures for Ψ and m1Ψ; however, the two modifications yielded similar patterns although both were different from U. Understanding the nanopore signatures for Ψ and m1Ψ enabled a running start T7 RNA polymerase assay to study how competing mixtures of UTP with ΨTP or m1ΨTP lead to nucleotide selection in all possible adjacent sequence contexts. For UTP vs. ΨTP, ΨTP was favorably incorporated in singly-modified contexts, while doubly-modified contexts found high yields of ΨTP insertion on the 5′ side and lower yields on the 3′ side.
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