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Engineering of a DNA Polymerase for Direct m 6 A Sequencing

Abstract : Methods for the detection of RNA modifications are of fundamental importance for advancing epitranscriptomics. N6 -methyladenosine (m6 A) is the most abundant RNA modification in mammalian mRNA and is involved in the regulation of gene expression. Current detection techniques are laborious and rely on antibody-based enrichment of m6 A-containing RNA prior to sequencing, since m6 A modifications are generally "erased" during reverse transcription (RT). To overcome the drawbacks associated with indirect detection, we aimed to generate novel DNA polymerase variants for direct m6 A sequencing. Therefore, we developed a screen to evolve an RT-active KlenTaq DNA polymerase variant that sets a mark for N6 -methylation. We identified a mutant that exhibits increased misincorporation opposite m6 A compared to unmodified A. Application of the generated DNA polymerase in next-generation sequencing allowed the identification of m6 A sites directly from the sequencing data of untreated RNA samples.
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Contributor : JULIE GONNET Connect in order to contact the contributor
Submitted on : Monday, May 28, 2018 - 2:30:04 PM
Last modification on : Thursday, May 19, 2022 - 1:52:03 PM

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Joos Aschenbrenner, Stephan Werner, Virginie Marchand, Martina Adam, Yuri Motorin, et al.. Engineering of a DNA Polymerase for Direct m 6 A Sequencing. Angewandte Chemie International Edition, 2017, 57 (2), pp.417 - 421. ⟨10.1002/anie.201710209⟩. ⟨hal-01801461⟩



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