Engineering of a DNA Polymerase for Direct m6A Sequencing

Publication type:
Journal article
Metadata:
Autoren
  • Joos Aschenbrenner
  • Stephan Werner
  • Virginie Marchand
  • Martina Adam
  • Yuri Motorin
  • Mark Helm
  • Andreas Marx
Autoren-URL
https://www.webofscience.com/api/gateway?GWVersion=2&SrcApp=fis-test-1&SrcAuth=WosAPI&KeyUT=WOS:000419110500004&DestLinkType=FullRecord&DestApp=WOS_CPL
DOI
10.1002/anie.201710209
eISSN
1521-3773
Externe Identifier
  • Clarivate Analytics Document Solution ID: FR5ML
  • PubMed Identifier: 29115744
ISSN
1433-7851
Ausgabe der Veröffentlichung
2
Zeitschrift
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION
Schlüsselwörter
  • DNA polymerases
  • enzyme engineering
  • epitranscriptomics
  • N-6-methyladenosine
  • RNA modification
Paginierung
417 - 421
Datum der Veröffentlichung
2018
Status
Published
Titel
Engineering of a DNA Polymerase for Direct m<SUP>6</SUP>A Sequencing
Sub types
  • Article
Ausgabe der Zeitschrift
57

Data source: Web of Science (Lite)

Other metadata sources:
Abstract
<jats:title>Abstract</jats:title><jats:p>Methods for the detection of RNA modifications are of fundamental importance for advancing epitranscriptomics. <jats:italic>N</jats:italic><jats:sup>6</jats:sup>‐methyladenosine (m<jats:sup>6</jats: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<jats:sup>6</jats:sup>A‐containing RNA prior to sequencing, since m<jats:sup>6</jats: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<jats:sup>6</jats:sup>A sequencing. Therefore, we developed a screen to evolve an RT‐active KlenTaq DNA polymerase variant that sets a mark for <jats:italic>N</jats:italic><jats:sup>6</jats:sup>‐methylation. We identified a mutant that exhibits increased misincorporation opposite m<jats:sup>6</jats:sup>A compared to unmodified A. Application of the generated DNA polymerase in next‐generation sequencing allowed the identification of m<jats:sup>6</jats:sup>A sites directly from the sequencing data of untreated RNA samples.</jats:p>
Autoren
  • Joos Aschenbrenner
  • Stephan Werner
  • Virginie Marchand
  • Martina Adam
  • Yuri Motorin
  • Mark Helm
  • Andreas Marx
DOI
10.1002/anie.201710209
eISSN
1521-3773
ISSN
1433-7851
Ausgabe der Veröffentlichung
2
Zeitschrift
Angewandte Chemie International Edition
Sprache
en
Online publication date
2017
Paginierung
417 - 421
Datum der Veröffentlichung
2018
Status
Published
Herausgeber
Wiley
Herausgeber URL
http://dx.doi.org/10.1002/anie.201710209
Datum der Datenerfassung
2023
Titel
Engineering of a DNA Polymerase for Direct m<sup>6</sup>A Sequencing
Ausgabe der Zeitschrift
57

Data source: Crossref

Abstract
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.
Addresses
  • Department of Chemistry, Konstanz Research School Chemical Biology, University of Konstanz, Universitätsstraße 10, 78457, Konstanz, Germany.
Autoren
  • Joos Aschenbrenner
  • Stephan Werner
  • Virginie Marchand
  • Martina Adam
  • Yuri Motorin
  • Mark Helm
  • Andreas Marx
DOI
10.1002/anie.201710209
eISSN
1521-3773
Externe Identifier
  • PubMed Identifier: 29115744
  • PubMed Central ID: PMC5768020
Funding acknowledgements
  • European Research Council: ERC Advanced Grant 339834
  • European Research Council: 339834
  • Deutsche Forschungsgemeinschaft: SPP 1784
  • Carl-Zeiss-Stiftung:
Open access
true
ISSN
1433-7851
Ausgabe der Veröffentlichung
2
Zeitschrift
Angewandte Chemie (International ed. in English)
Schlüsselwörter
  • DNA-Directed DNA Polymerase
  • RNA, Messenger
  • Adenosine
  • Protein Engineering
  • Reverse Transcriptase Polymerase Chain Reaction
  • DNA Methylation
  • High-Throughput Nucleotide Sequencing
Sprache
eng
Medium
Print-Electronic
Online publication date
2017
Open access status
Open Access
Paginierung
417 - 421
Datum der Veröffentlichung
2018
Status
Published
Publisher licence
CC BY-NC
Datum der Datenerfassung
2017
Titel
Engineering of a DNA Polymerase for Direct m<sup>6</sup> A Sequencing.
Sub types
  • brief-report
  • Research Support, Non-U.S. Gov't
  • Journal Article
Ausgabe der Zeitschrift
57

Files

https://onlinelibrary.wiley.com/doi/pdfdirect/10.1002/anie.201710209 https://europepmc.org/articles/PMC5768020?pdf=render

Data source: Europe PubMed Central

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.
Autoren
  • Joos Aschenbrenner
  • Stephan Werner
  • Virginie Marchand
  • Martina Adam
  • Yuri Motorin
  • Mark Helm
  • Andreas Marx
Autoren-URL
https://www.ncbi.nlm.nih.gov/pubmed/29115744
DOI
10.1002/anie.201710209
eISSN
1521-3773
Externe Identifier
  • PubMed Central ID: PMC5768020
Funding acknowledgements
  • European Research Council: 339834
Ausgabe der Veröffentlichung
2
Zeitschrift
Angew Chem Int Ed Engl
Schlüsselwörter
  • DNA polymerases
  • N6-methyladenosine
  • RNA modification
  • enzyme engineering
  • epitranscriptomics
  • Adenosine
  • DNA Methylation
  • DNA-Directed DNA Polymerase
  • High-Throughput Nucleotide Sequencing
  • Protein Engineering
  • RNA, Messenger
  • Reverse Transcriptase Polymerase Chain Reaction
Sprache
eng
Country
Germany
Paginierung
417 - 421
Datum der Veröffentlichung
2018
Status
Published
Datum, an dem der Datensatz öffentlich gemacht wurde
2019
Titel
Engineering of a DNA Polymerase for Direct m6 A Sequencing.
Sub types
  • Journal Article
  • Research Support, Non-U.S. Gov't
Ausgabe der Zeitschrift
57

Data source: PubMed

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