Engineering of a DNA Polymerase for Direct m6A Sequencing

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ASCHENBRENNER, Joos, Stephan WERNER, Virginie MARCHAND, Martina ADAM, Yuri MOTORIN, Mark HELM, Andreas MARX, 2018. Engineering of a DNA Polymerase for Direct m6A Sequencing. In: Angewandte Chemie International Edition. 57(2), pp. 417-421. ISSN 0570-0833. eISSN 1521-3773. Available under: doi: 10.1002/anie.201710209

@article{Aschenbrenner2018-01-08Engin-41793, title={Engineering of a DNA Polymerase for Direct m6A Sequencing}, year={2018}, doi={10.1002/anie.201710209}, number={2}, volume={57}, issn={0570-0833}, journal={Angewandte Chemie International Edition}, pages={417--421}, author={Aschenbrenner, Joos and Werner, Stephan and Marchand, Virginie and Adam, Martina and Motorin, Yuri and Helm, Mark and Marx, Andreas} }

terms-of-use Marchand, Virginie Aschenbrenner, Joos Adam, Martina Methods for the detection of RNA modifications are of fundamental importance for advancing epitranscriptomics. N<sup>6</sup>-methyladenosine (m<sup>6</sup>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 m<sup>6</sup>A-containing RNA prior to sequencing, since m<sup>6</sup>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 m<sup>6</sup>A sequencing. Therefore, we developed a screen to evolve an RT-active KlenTaq DNA polymerase variant that sets a mark for N<sup>6</sup>-methylation. We identified a mutant that exhibits increased misincorporation opposite m<sup>6</sup>A compared to unmodified A. Application of the generated DNA polymerase in next-generation sequencing allowed the identification of m<sup>6</sup>A sites directly from the sequencing data of untreated RNA samples. Werner, Stephan Engineering of a DNA Polymerase for Direct m<sup>6</sup>A Sequencing Adam, Martina Werner, Stephan Motorin, Yuri Aschenbrenner, Joos eng 2018-03-15T08:34:42Z Marx, Andreas Motorin, Yuri 2018-01-08 Helm, Mark Marchand, Virginie Helm, Mark 2018-03-15T08:34:42Z Marx, Andreas

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