Synthesis of point-modified mRNA
- Publication type:
- Journal article
- Metadata:
-
- 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
Data source: Web of Science (Lite)
- Other metadata sources:
-
- 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
Data source: 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
- 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
Data source: 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
Data source: PubMed
- Beziehungen:
- Property of