Non-canonical nucleosides: Biomimetic triphosphorylation, incorporation into mRNA and effects on translation and structure.

Autor: Benčić P; Institute of Pharmaceutical Sciences, University of Freiburg, Germany., Keppler M; Institute of Pharmaceutical Sciences, University of Freiburg, Germany., Kuge M; Institute of Pharmaceutical Sciences, University of Freiburg, Germany., Qiu D; Institute of Organic Chemistry, University of Freiburg, Germany., Schütte LM; Institute of Pharmaceutical Sciences, University of Freiburg, Germany., Häner M; Institute of Organic Chemistry, University of Freiburg, Germany., Strack K; Institute of Pharmaceutical Sciences, University of Freiburg, Germany., Jessen HJ; Institute of Organic Chemistry, University of Freiburg, Germany., Andexer JN; Institute of Pharmaceutical Sciences, University of Freiburg, Germany., Loenarz C; Institute of Pharmaceutical Sciences, University of Freiburg, Germany.
Jazyk: angličtina
Zdroj: The FEBS journal [FEBS J] 2023 Oct; Vol. 290 (20), pp. 4899-4920. Date of Electronic Publication: 2023 Jun 26.
DOI: 10.1111/febs.16889
Abstrakt: Recent advances in mRNA therapeutics demand efficient toolkits for the incorporation of nucleoside analogues into mRNA suitable for downstream applications. Herein, we report the application of a versatile enzyme cascade for the triphosphorylation of a broad range of nucleoside analogues, including unprotected nucleobases containing chemically labile moieties. Our biomimetic system was suitable for the preparation of nucleoside triphosphates containing adenosine, cytidine, guanosine, uridine and non-canonical core structures, as determined by capillary electrophoresis coupled to mass spectrometry. This enabled us to establish an efficient workflow for transcribing and purifying functional mRNA containing these nucleoside analogues, combined with mass spectrometric verification of analogue incorporation. Our combined methodology allows for analyses of how incorporation of nucleoside analogues that are commercially unavailable as triphosphates affect mRNA properties: The translational fidelity of the produced mRNA was demonstrated in analyses of how incorporated adenosine analogues impact translational recoding. For the SARS-CoV-2 frameshifting site, analyses of the mRNA pseudoknot structure using circular dichroism spectroscopy allowed insight into how the pharmacologically active 7-deazaadenosine destabilises RNA secondary structure, consistent with observed changes in recoding efficiency.
(© 2023 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)
Databáze: MEDLINE
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