Engineering of a DNA Polymerase for Direct m 6 A Sequencing - Université de Lorraine Accéder directement au contenu
Article Dans Une Revue Angewandte Chemie International Edition Année : 2017

Engineering of a DNA Polymerase for Direct m 6 A Sequencing

Résumé

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.

Dates et versions

hal-01801461 , version 1 (28-05-2018)

Identifiants

Citer

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⟩
398 Consultations
0 Téléchargements

Altmetric

Partager

Gmail Facebook X LinkedIn More