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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\u2010canonical 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\u2010CoV\u20102 frameshifting site, analyses of the mRNA pseudoknot structure using circular dichroism spectroscopy allowed insight into how the pharmacologically active 7\u2010deazaadenosine destabilises RNA secondary structure, consistent with observed changes in recoding efficiency.<\/jats:p>","DOI":"10.1111\/febs.16889","type":"journal-article","created":{"date-parts":[[2023,6,17]],"date-time":"2023-06-17T09:34:42Z","timestamp":1686994482000},"page":"4899-4920","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":16,"title":["Non\u2010canonical nucleosides: Biomimetic triphosphorylation, incorporation into mRNA and effects on translation and structure"],"prefix":"10.1111","volume":"290","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-3363-431X","authenticated-orcid":false,"given":"Patricia","family":"Ben\u010di\u0107","sequence":"first","affiliation":[{"name":"Institute of Pharmaceutical Sciences University of 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