Esterification of Cyclic N 6 -Threonylcarbamoyladenosine During RNA Sample Preparation.

Autor: Bessler L; Institute of Pharmaceutical and Biomedical Sciences, Johannes Gutenberg University Mainz, Staudingerweg 5, 55128, Mainz, Germany., Sirleaf J; Department of Chemistry, Johannes Gutenberg University Mainz, Duesbergweg 10-14, 55128, Mainz, Germany., Kampf CJ; Department of Chemistry, Johannes Gutenberg University Mainz, Duesbergweg 10-14, 55128, Mainz, Germany., Frankowska K; Institute of Organic Chemistry, Faculty of Chemistry, Lodz University of Technology, Zeromskiego 116, 90-924, Łódź, Poland., Leszczyńska G; Institute of Organic Chemistry, Faculty of Chemistry, Lodz University of Technology, Zeromskiego 116, 90-924, Łódź, Poland., Opatz T; Department of Chemistry, Johannes Gutenberg University Mainz, Duesbergweg 10-14, 55128, Mainz, Germany., Helm M; Institute of Pharmaceutical and Biomedical Sciences, Johannes Gutenberg University Mainz, Staudingerweg 5, 55128, Mainz, Germany.
Jazyk: angličtina
Zdroj: ChemMedChem [ChemMedChem] 2024 Jul 02; Vol. 19 (13), pp. e202400115. Date of Electronic Publication: 2024 May 26.
DOI: 10.1002/cmdc.202400115
Abstrakt: The continuous deciphering of crucial biological roles of RNA modifications and their involvement in various pathological conditions, together with their key roles in the use of RNA-based therapeutics, has reignited interest in studying the occurrence and identity of non-canonical ribonucleoside structures during the past years. Discovery and structural elucidation of new modified structures is usually achieved by combination of liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) at the nucleoside level and stable isotope labeling experiments. This approach, however, has its pitfalls as demonstrated in the course of the present study: we structurally elucidated a new nucleoside structure that showed significant similarities to the family of (c)t 6 A modifications and was initially considered a genuine modification, but subsequently turned out to be an in vitro formed glycerol ester of t 6 A. This artifact is generated from ct 6 A during RNA hydrolysis upon addition of enzymes stored in glycerol containing buffers in a mildly alkaline milieu, and was moreover shown to undergo an intramolecular transesterification reaction. Our results demand for extra caution, not only in the discovery of new RNA modifications, but also with regard to the quantification of known modified structures, in particular chemically labile modifications, such as ct 6 A, that might suffer from exposure to putatively harmless reagents during the diverse steps of sample preparation.
(© 2024 The Authors. ChemMedChem published by Wiley-VCH GmbH.)
Databáze: MEDLINE
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