Synthesis of point-modified mRNA

Publikationstyp:
Zeitschriftenaufsatz
Metadaten:
Autoren
  • Jasmin Hertler
  • Kaouthar Slama
  • Benedikt Schober
  • Zeynep Ozrendeci
  • Virginie Marchand
  • Yuri Motorin
  • Mark Helm
Autoren-URL
https://www.webofscience.com/api/gateway?GWVersion=2&SrcApp=fis-test-1&SrcAuth=WosAPI&KeyUT=WOS:000849783300001&DestLinkType=FullRecord&DestApp=WOS_CPL
DOI
10.1093/nar/gkac719
eISSN
1362-4962
Externe Identifier
  • Clarivate Analytics Document Solution ID: 7U1BJ
  • PubMed Identifier: 36062567
ISSN
0305-1048
Ausgabe der Veröffentlichung
20
Zeitschrift
NUCLEIC ACIDS RESEARCH
Datum der Veröffentlichung
2022
Status
Published
Titel
Synthesis of point-modified mRNA
Sub types
  • Article
Ausgabe der Zeitschrift
50

Datenquelle: Web of Science (Lite)

Andere Metadatenquellen:
Abstract
<jats:title>Abstract</jats:title> <jats:p>Synthetic mRNA has recently moved into the focus of therapeutic and vaccination efforts. Incorporation of modified nucleotides during in vitro transcription can improve translation and attenuate immunogenicity, but is limited to triphosphate nucleotides which are accepted by RNA polymerases, and their incorporation is either random or complete. In contrast, site-specific modification, herein termed ‘point modification’ in analogy to point mutations, holds significant technical challenge. We developed fundamental techniques for isolation of long, translatable and internally point-modified mRNAs. Enabling concepts include three-way-one-pot splint ligations, and isolation of mRNA by real-time elution from agarose gels. The use of blue light permitted visualization of mRNA in pre-stained gels without the photochemical damage associated with the use of hard UV-radiation. This allowed visualization of the mRNA through its migration in the agarose gel, which in turn, was a prerequisite for its recovery by electroelution into precast troughs. Co-eluting agarose particles were quantified and found to not be detrimental to mRNA translation in vitro. Translation of EGFP-coding mRNA into functional protein was quantified by incorporation of 35S-labelled methionine and by in-gel EGFP fluorescence. This enabled the functional analysis of point modifications, specifically of ribose methylations in the middle of a 1371 nt long mRNA.</jats:p>
Autoren
  • Jasmin Hertler
  • Kaouthar Slama
  • Benedikt Schober
  • Zeynep Özrendeci
  • Virginie Marchand
  • Yuri Motorin
  • Mark Helm
DOI
10.1093/nar/gkac719
eISSN
1362-4962
ISSN
0305-1048
Ausgabe der Veröffentlichung
20
Zeitschrift
Nucleic Acids Research
Sprache
en
Online publication date
2022
Paginierung
e115 - e115
Datum der Veröffentlichung
2022
Status
Published
Herausgeber
Oxford University Press (OUP)
Herausgeber URL
http://dx.doi.org/10.1093/nar/gkac719
Datum der Datenerfassung
2022
Titel
Synthesis of point-modified mRNA
Ausgabe der Zeitschrift
50

Datenquelle: Crossref

Abstract
Synthetic mRNA has recently moved into the focus of therapeutic and vaccination efforts. Incorporation of modified nucleotides during in vitro transcription can improve translation and attenuate immunogenicity, but is limited to triphosphate nucleotides which are accepted by RNA polymerases, and their incorporation is either random or complete. In contrast, site-specific modification, herein termed 'point modification' in analogy to point mutations, holds significant technical challenge. We developed fundamental techniques for isolation of long, translatable and internally point-modified mRNAs. Enabling concepts include three-way-one-pot splint ligations, and isolation of mRNA by real-time elution from agarose gels. The use of blue light permitted visualization of mRNA in pre-stained gels without the photochemical damage associated with the use of hard UV-radiation. This allowed visualization of the mRNA through its migration in the agarose gel, which in turn, was a prerequisite for its recovery by electroelution into precast troughs. Co-eluting agarose particles were quantified and found to not be detrimental to mRNA translation in vitro. Translation of EGFP-coding mRNA into functional protein was quantified by incorporation of 35S-labelled methionine and by in-gel EGFP fluorescence. This enabled the functional analysis of point modifications, specifically of ribose methylations in the middle of a 1371 nt long mRNA.
Addresses
  • Institute of Pharmaceutical and Biomedical Sciences, Johannes Gutenberg-Universität, Staudinger Weg 5, D-55128 Mainz, Germany.
Autoren
  • Jasmin Hertler
  • Kaouthar Slama
  • Benedikt Schober
  • Zeynep Özrendeci
  • Virginie Marchand
  • Yuri Motorin
  • Mark Helm
DOI
10.1093/nar/gkac719
eISSN
1362-4962
Externe Identifier
  • PubMed Identifier: 36062567
  • PubMed Central ID: PMC9723659
Funding acknowledgements
  • Deutsche Forschungsgemeinschaft: TRR319 RMaP
  • JGU:
  • Deutsche Forschungsgemeinschaft: HE3397/13-2
  • Deutsche Forschungsgemeinschaft: SFB 1066/A7
  • Deutsche Forschungsgemeinschaft: TP A05
Open access
true
ISSN
0305-1048
Ausgabe der Veröffentlichung
20
Zeitschrift
Nucleic acids research
Schlüsselwörter
  • Nucleotides
  • Sepharose
  • RNA, Messenger
  • Genetic Engineering
  • Methylation
Sprache
eng
Medium
Print
Open access status
Open Access
Paginierung
e115
Datum der Veröffentlichung
2022
Status
Published
Publisher licence
CC BY
Datum der Datenerfassung
2022
Titel
Synthesis of point-modified mRNA.
Sub types
  • Research Support, Non-U.S. Gov't
  • research-article
  • Journal Article
Ausgabe der Zeitschrift
50

Files

https://europepmc.org/articles/PMC9723659?pdf=render

Datenquelle: Europe PubMed Central

Abstract
Synthetic mRNA has recently moved into the focus of therapeutic and vaccination efforts. Incorporation of modified nucleotides during in vitro transcription can improve translation and attenuate immunogenicity, but is limited to triphosphate nucleotides which are accepted by RNA polymerases, and their incorporation is either random or complete. In contrast, site-specific modification, herein termed 'point modification' in analogy to point mutations, holds significant technical challenge. We developed fundamental techniques for isolation of long, translatable and internally point-modified mRNAs. Enabling concepts include three-way-one-pot splint ligations, and isolation of mRNA by real-time elution from agarose gels. The use of blue light permitted visualization of mRNA in pre-stained gels without the photochemical damage associated with the use of hard UV-radiation. This allowed visualization of the mRNA through its migration in the agarose gel, which in turn, was a prerequisite for its recovery by electroelution into precast troughs. Co-eluting agarose particles were quantified and found to not be detrimental to mRNA translation in vitro. Translation of EGFP-coding mRNA into functional protein was quantified by incorporation of 35S-labelled methionine and by in-gel EGFP fluorescence. This enabled the functional analysis of point modifications, specifically of ribose methylations in the middle of a 1371 nt long mRNA.
Date of acceptance
2022
Autoren
  • Jasmin Hertler
  • Kaouthar Slama
  • Benedikt Schober
  • Zeynep Özrendeci
  • Virginie Marchand
  • Yuri Motorin
  • Mark Helm
Autoren-URL
https://www.ncbi.nlm.nih.gov/pubmed/36062567
DOI
10.1093/nar/gkac719
eISSN
1362-4962
Externe Identifier
  • PubMed Central ID: PMC9723659
Ausgabe der Veröffentlichung
20
Zeitschrift
Nucleic Acids Res
Schlüsselwörter
  • Methylation
  • Nucleotides
  • RNA, Messenger
  • Sepharose
  • Genetic Engineering
Sprache
eng
Country
England
Paginierung
e115
PII
6691864
Datum der Veröffentlichung
2022
Status
Published
Datum, an dem der Datensatz öffentlich gemacht wurde
2022
Titel
Synthesis of point-modified mRNA.
Sub types
  • Journal Article
  • Research Support, Non-U.S. Gov't
Ausgabe der Zeitschrift
50

Datenquelle: PubMed

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