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Mamot A, Sikorski PJ, Siekierska A, et al. Ethylenediamine derivatives efficiently react with oxidized RNA 3' ends providing access to mono and dually labelled RNA probes for enzymatic assays and in vivo translation. Nucleic Acids Res. 2022;50(1):e3doi: 10.1093/nar/gkab867.
Mamot, A., Sikorski, P. J., Siekierska, A., de Witte, P., Kowalska, J., & Jemielity, J. (2022). Ethylenediamine derivatives efficiently react with oxidized RNA 3' ends providing access to mono and dually labelled RNA probes for enzymatic assays and in vivo translation. Nucleic acids research, 50(1), e3. https://doi.org/10.1093/nar/gkab867
Mamot, Adam, et al. "Ethylenediamine derivatives efficiently react with oxidized RNA 3' ends providing access to mono and dually labelled RNA probes for enzymatic assays and in vivo translation." Nucleic acids research vol. 50,1 (2022): e3. doi: https://doi.org/10.1093/nar/gkab867
Mamot A, Sikorski PJ, Siekierska A, de Witte P, Kowalska J, Jemielity J. Ethylenediamine derivatives efficiently react with oxidized RNA 3' ends providing access to mono and dually labelled RNA probes for enzymatic assays and in vivo translation. Nucleic Acids Res. 2022 Jan 11;50(1):e3. doi: 10.1093/nar/gkab867. PMID: 34591964.
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Nucleic Acids Res. 2022 Jan 11;50(1):e3. doi: 10.1093/nar/gkab867.

Ethylenediamine derivatives efficiently react with oxidized RNA 3' ends providing access to mono and dually labelled RNA probes for enzymatic assays and in vivo translation.

Nucleic acids research

Adam Mamot, Pawel J Sikorski, Aleksandra Siekierska, Peter de Witte, Joanna Kowalska, Jacek Jemielity

Affiliations

  1. Centre of New Technologies, University of Warsaw, Banacha 2c Street, 02-097 Warsaw, Poland.
  2. Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Pasteura 5 Street, 02-093 Warsaw, Poland.
  3. Laboratory for Molecular Biodiscovery, KU Leuven, Campus Gasthuisberg, Herestraat 49, O&N II, 3000 Leuven, Belgium.

PMID: 34591964 DOI: 10.1093/nar/gkab867

Abstract

Development of RNA-based technologies relies on the ability to detect, manipulate, and modify RNA. Efficient, selective and scalable covalent modification of long RNA molecules remains a challenge. We report a chemical method for modification of RNA 3'-end based on previously unrecognized superior reactivity of N-substituted ethylenediamines in reductive amination of periodate-oxidized RNA. Using this method, we obtained fluorescently labelled or biotinylated RNAs varying in length (from 3 to 2000 nt) and carrying different 5' ends (including m7G cap) in high yields (70-100% by HPLC). The method is scalable (up to sub-milligrams of mRNA) and combined with label-facilitated HPLC purification yields highly homogeneous products. The combination of 3'-end labelling with 5'-end labelling by strain-promoted azide-alkyne cycloaddition (SPAAC) afforded a one-pot protocol for site-specific RNA bifunctionalization, providing access to two-colour fluorescent RNA probes. These probes exhibited fluorescence resonance energy transfer (FRET), which enabled real-time monitoring of several RNA hydrolase activities (RNase A, RNase T1, RNase R, Dcp1/2, and RNase H). Dually labelled mRNAs were efficiently translated in cultured cells and in zebrafish embryos, which combined with their detectability by fluorescent methods and scalability of the synthesis, opens new avenues for the investigation of mRNA metabolism and the fate of mRNA-based therapeutics.

© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.

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