Synthesis of point-modified mRNA.
Fiche publication
Date publication
septembre 2022
Journal
Nucleic acids research
Auteurs
Membres identifiés du Cancéropôle Est :
Pr MOTORINE Iouri, Dr MARCHAND Virginie
Tous les auteurs :
Hertler J, Slama K, Schober B, Özrendeci Z, Marchand V, Motorin Y, Helm M
Lien Pubmed
Résumé
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.
Référence
Nucleic Acids Res. 2022 09 5;: