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Engineering of a DNA Polymerase for Direct m 6 A Sequencing
Author(s) -
Aschenbrenner Joos,
Werner Stephan,
Marchand Virginie,
Adam Martina,
Motorin Yuri,
Helm Mark,
Marx Andreas
Publication year - 2018
Publication title -
angewandte chemie international edition
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.831
H-Index - 550
eISSN - 1521-3773
pISSN - 1433-7851
DOI - 10.1002/anie.201710209
Subject(s) - polymerase , microbiology and biotechnology , dna sequencing , biology , dna , rna , dna nanoball sequencing , rna polymerase ii , multiple displacement amplification , chemistry , gene , genetics , computational biology , polymerase chain reaction , gene expression , promoter , genomic library , base sequence , dna extraction
Methods for the detection of RNA modifications are of fundamental importance for advancing epitranscriptomics. N 6 ‐methyladenosine (m 6 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 6 A‐containing RNA prior to sequencing, since m 6 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 6 A sequencing. Therefore, we developed a screen to evolve an RT‐active KlenTaq DNA polymerase variant that sets a mark for N 6 ‐methylation. We identified a mutant that exhibits increased misincorporation opposite m 6 A compared to unmodified A. Application of the generated DNA polymerase in next‐generation sequencing allowed the identification of m 6 A sites directly from the sequencing data of untreated RNA samples.