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Chemical circularization of in vitro transcribed RNA for exploring circular mRNA design
Malgorzata Wasinska-Kalwa1, Adam Mamot1,2,3, Karol Czubak4
1Centre of New Technologies, University of Warsaw, Banacha 2c Street, 02-097, Warsaw, Poland.
Nature Communications
|July 12, 2025
Summary
We developed a chemical method to create circular messenger RNA (mRNA) molecules up to 4000 nucleotides long. This approach enhances mRNA stability and translational activity for therapeutic applications.
Area of Science:
- Biochemistry
- Molecular Biology
- RNA Therapeutics
Background:
- Circularization enhances messenger RNA (mRNA) therapeutic potential.
- Existing methods (enzymatic, ribozymatic, chemical) have limitations like sequence constraints, purification issues, and restricted length applicability.
- Chemical circularization has been limited to short RNA sequences.
Purpose of the Study:
- To develop a versatile chemical method for circularizing in vitro transcribed RNAs (chem-circRNAs) of various lengths.
- To achieve high circularization efficiencies and ensure compatibility with functional modifications.
- To demonstrate the translational activity and stability of chem-circRNAs in cellular systems.
Main Methods:
- Utilized a novel chemical strategy involving a 5' ethylenediamine modification and a periodate-oxidized 3' end for intramolecular reductive amination.
- Applied the method to in vitro transcribed RNAs ranging from 35 to 4000 nucleotides.
- Developed effective separation techniques to isolate chem-circRNAs from linear precursors.
- Incorporated functional modifications, including endocyclic N7-methylguanosine cap and N1-methylpseudouridine.
Main Results:
- Achieved circularization efficiencies up to 60% for chem-circRNAs.
- Demonstrated applicability across various RNA sequences and compatibility with diverse modifications.
- Successfully separated chem-circRNAs from linear counterparts.
- Showcased that protein-coding chem-circRNAs are translationally active in cells and exhibit enhanced durability compared to linear mRNA.
Conclusions:
- The developed chemical circularization method provides a robust and scalable approach for producing therapeutic mRNA.
- Chem-circRNAs exhibit improved stability and translational efficiency, making them promising candidates for RNA-based therapies.
- This method overcomes previous limitations, enabling the creation of chemically defined, functional circular RNAs.
