Engineering of a DNA Polymerase for Direct m A Sequencing.
Fiche publication
Date publication
janvier 2018
Journal
Angewandte Chemie (International ed. in English)
Auteurs
Membres identifiés du Cancéropôle Est :
Dr MARCHAND Virginie
Tous les auteurs :
Aschenbrenner J, Werner S, Marchand V, Adam M, Motorin Y, Helm M, Marx A
Lien Pubmed
Résumé
Methods for the detection of RNA modifications are of fundamental importance for advancing epitranscriptomics. N -methyladenosine (m 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 A-containing RNA prior to sequencing, since m 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 A sequencing. Therefore, we developed a screen to evolve an RT-active KlenTaq DNA polymerase variant that sets a mark for N -methylation. We identified a mutant that exhibits increased misincorporation opposite m A compared to unmodified A. Application of the generated DNA polymerase in next-generation sequencing allowed the identification of m A sites directly from the sequencing data of untreated RNA samples.
Mots clés
DNA polymerases, N6-methyladenosine, RNA modification, enzyme engineering, epitranscriptomics
Référence
Angew. Chem. Int. Ed. Engl.. 2018 Jan 8;57(2):417-421