Chemical circularization of in vitro transcribed RNA for exploring circular mRNA design

Malgorzata Wasinska-Kalwa, Adam Mamot, Karol Czubak, Katarzyna Frankowska, Adam Ado Rajkiewicz, Tomasz Spiewla, Marcin Warminski, Zofia Pilch, Marta Szulc-Gasiorowska, Kacper Siekan, Andrzej Dziembowski, Dominika Nowis, Jakub Golab, Joanna Kowalska () and Jacek Jemielity ()
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Malgorzata Wasinska-Kalwa: University of Warsaw
Adam Mamot: University of Warsaw
Karol Czubak: Medical University of Warsaw
Katarzyna Frankowska: University of Warsaw
Adam Ado Rajkiewicz: University of Warsaw
Tomasz Spiewla: University of Warsaw
Marcin Warminski: University of Warsaw
Zofia Pilch: Medical University of Warsaw
Marta Szulc-Gasiorowska: University of Warsaw
Kacper Siekan: International Institute of Molecular and Cell Biology
Andrzej Dziembowski: International Institute of Molecular and Cell Biology
Dominika Nowis: Medical University of Warsaw
Jakub Golab: Medical University of Warsaw
Joanna Kowalska: University of Warsaw
Jacek Jemielity: University of Warsaw

Nature Communications, 2025, vol. 16, issue 1, 1-15

Abstract: Abstract Circularization is an important step for therapeutic messenger RNA (mRNA) enhancements. Current enzymatic and ribozymatic-based circularization methods face limitations including sequence constraints, purification challenges, and sub-optimal biological activity. Chemical strategies, while promising, have been restricted to short RNA sequences. Here, we report a method for chemically circularized in vitro transcribed RNAs of various lengths (chem-circRNAs; 35–4000 nt) with circularization efficiencies reaching up to 60%. This approach leverages a 5′ ethylenediamine modification and a periodate-oxidized 3′ end to drive intramolecular reductive amination. We demonstrate that this method is applicable to various sequences and modification compatible. We report the effective separation methods of chem-circRNAs from their linear precursors. We show that protein-coding chem-circRNAs are translationally active in cells and exhibit increased durability, like enzymatically circularized mRNAs. Furthermore, our method allows incorporation of functional modifications, including endocyclic N7-methylguanosine cap and N1-methylpseudouridine, enabling access to chemically defined translationally active circRNAs for therapeutic applications.

Date: 2025
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DOI: 10.1038/s41467-025-61775-1

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