Chemical circularization of in vitro transcribed RNA for exploring circular mRNA design
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 p...
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Nature Portfolio
2025-07-01
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| Series: | Nature Communications |
| Online Access: | https://doi.org/10.1038/s41467-025-61775-1 |
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| author | 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 Jacek Jemielity |
| author_facet | 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 Jacek Jemielity |
| author_sort | Malgorzata Wasinska-Kalwa |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-51a68436b7d44e3e93dfcf9ee6acfcc8 |
| institution | Kabale University |
| issn | 2041-1723 |
| language | English |
| publishDate | 2025-07-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Nature Communications |
| spelling | doaj-art-51a68436b7d44e3e93dfcf9ee6acfcc82025-08-20T03:46:20ZengNature PortfolioNature Communications2041-17232025-07-0116111510.1038/s41467-025-61775-1Chemical circularization of in vitro transcribed RNA for exploring circular mRNA designMalgorzata Wasinska-Kalwa0Adam Mamot1Karol Czubak2Katarzyna Frankowska3Adam Ado Rajkiewicz4Tomasz Spiewla5Marcin Warminski6Zofia Pilch7Marta Szulc-Gasiorowska8Kacper Siekan9Andrzej Dziembowski10Dominika Nowis11Jakub Golab12Joanna Kowalska13Jacek Jemielity14Centre of New Technologies, University of WarsawCentre of New Technologies, University of WarsawLaboratory of Experimental Medicine, Medical University of WarsawCentre of New Technologies, University of WarsawCentre of New Technologies, University of WarsawCentre of New Technologies, University of WarsawDivision of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of WarsawDepartment of Immunology, Medical University of WarsawCentre of New Technologies, University of WarsawGenome Engineering Facility, International Institute of Molecular and Cell BiologyLaboratory of RNA Biology, International Institute of Molecular and Cell BiologyLaboratory of Experimental Medicine, Medical University of WarsawDepartment of Immunology, Medical University of WarsawDivision of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of WarsawCentre of New Technologies, University of WarsawAbstract 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.https://doi.org/10.1038/s41467-025-61775-1 |
| spellingShingle | 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 Jacek Jemielity Chemical circularization of in vitro transcribed RNA for exploring circular mRNA design Nature Communications |
| title | Chemical circularization of in vitro transcribed RNA for exploring circular mRNA design |
| title_full | Chemical circularization of in vitro transcribed RNA for exploring circular mRNA design |
| title_fullStr | Chemical circularization of in vitro transcribed RNA for exploring circular mRNA design |
| title_full_unstemmed | Chemical circularization of in vitro transcribed RNA for exploring circular mRNA design |
| title_short | Chemical circularization of in vitro transcribed RNA for exploring circular mRNA design |
| title_sort | chemical circularization of in vitro transcribed rna for exploring circular mrna design |
| url | https://doi.org/10.1038/s41467-025-61775-1 |
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