Abstract
The optimisation and further expansion of methods for the synthesis of oligonucleotide conjugates is receiving increased attention due to their importance for further advancement of therapeutic and diagnostic nucleic acid-based applications. Current methodologies, particularly those relying on maleimide-type linkers, are often hampered by linker instability. Herein, we present a versatile method for the direct functionalisation of readily available amino-modified oligonucleotides (AONs), where a 5-hydroxy-1,5-dihydro-2H-pyrrol-2-ones (5HP2O) Michael acceptor is directly formed in a rapid and efficient manner on a free primary amine. The methodology demonstrates broad applicability, tolerating various amino-modifiers and their positions within different oligonucleotide types, including DNA, LNA, PNA, and phosphorothioate-modified oligonucleotide strands. Most importantly, the possibility to introduce an additional second orthogonal reactive handle uniquely enables a direct single-site dual-functionalisation (Michael acceptor and click handle) of AONs for the assembly of complex constructs, as exemplified by the synthesis of a fluorescent peptide-oligonucleotide construct.
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The authors declare that the main data supporting the findings of this study are available within the article and its Supplementary Information files. Extra data are available from the corresponding author upon request.
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Acknowledgements
This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie Grant Agreement No. 956070 (OLIGOMED) (J.H.M., M.L., A.M., M.B.). E.C. acknowledges a FWO fellowship (12B1923N). We thank Dr. Chloe Howells and Prof. Eugen Stulz for LCMS measurement of crude functionalisation reactions. We thank Pieter Surmont and Prof. Frederic Lynen for HRMS Orbitrap measurement of purified oligonucleotide constructs. We thank Jan Goeman for LCMS measurements and Stijn Tanghe for general lab support. We thank the NMR expertise centre (Ghent University) for providing support and access to its NMR infrastructure. The 400 MHz NMR used in this work has been funded by a grant/project of the Research Foundation Flanders (FWO I006920N) and the ‘Bijzonder Onderzoeksfonds’ (BOF.BAS.2022.0023.01).
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Jan H. Meffert was responsible for the overall conceptualisation of the study, designed and conducted all experiments including condition screening, oligonucleotide mono- and dual-functionalisation, product analysis and small molecule synthesis, and wrote the manuscript, with support from the other authors. Enrico Cadoni provided PNA constructs (P1 to P4). Mónica Lopes, Martin Bollmark and UIf Tedebark provided phosphorothioated amine oligonucleotides (D2-4). Annemieke Madder was responsible for the funding and overall guidance, contributed to the conceptualisation and assisted in manuscript writing, proofreading and correction.
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Annemieke Madder has a pending patent application relating to 5HP2Os application for bioconjugation (Bioconjugation reagent and methods, WO2020174086A2, Annemieke MADDER, Ewout DE GEYTER, Eirini ANTONATOU, Sabina SMOLEN, Dimitris Kalaitzakis, Georgios VASSILIKOGIANNAKIS, Europe&US). All other authors declare no competing interest.
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Meffert, J.H., Lopes, M., Cadoni, E. et al. Versatile introduction of multifunctional Michael-acceptor moieties on amino-oligonucleotides for bioconjugation purposes. Commun Chem (2026). https://doi.org/10.1038/s42004-025-01882-8
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DOI: https://doi.org/10.1038/s42004-025-01882-8
