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<p>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.</p>

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

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2025年7月12日

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  • 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

摘要

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.