Abstract
The combination of enzymatic modification and bioorthogonal click chemistry provides a powerful approach for site-specific labeling of different classes of biomolecules in vitro and even in cellular environments. Herein, we describe a chemoenzymatic method to site specifically label 5′-capped model mRNAs independent of their sequence. A trimethylguanosine synthase was engineered to introduce alkyne, azido, or 4-vinylbenzyl moieties to the 5′-cap. These functional groups were then used for labeling using typical click reactions, such as the azide-alkyne cycloaddition or the tetrazine ligation.
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Acknowledgments
A. R. gratefully acknowledges financial support from the Emmy Noether-Programme of the Deutsche Forschungsgemeinschaft (RE 2796/2-1) and the Fonds der Chemischen Industrie. This work was partly supported by the Deutsche Forschungsgemeinschaft, DFG EXC 1003 Cells in Motion—Cluster of Excellence, Münster, Germany. We thank Prof. Hahn (University of Hamburg) for providing the DNA template for transcription. We would like to thank Prof. Birgit Dräger (University of Halle, Germany) for plasmids encoding LuxS and MTAN. J. M. H. thanks the Fonds der Chemischen Industrie for a doctoral fellowship.
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Holstein, J.M., Stummer, D., Rentmeister, A. (2016). Enzymatic Modification of 5′-Capped RNA and Subsequent Labeling by Click Chemistry. In: Rhoads, R. (eds) Synthetic mRNA. Methods in Molecular Biology, vol 1428. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3625-0_3
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DOI: https://doi.org/10.1007/978-1-4939-3625-0_3
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