Year: 2025
Type: article
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. Circular RNAs offer advantages for mRNA therapies but are challenging to produce. Here, the authors develop a chemical method to generate circular RNAs with internal cap structures, enabling cap-dependent translation, chemical modification, and enhanced stability.
Source: Nature Communications
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $6,990
OpenAlex ID: W4412365300