Site-Specifically Modified Circular Ribonucleic Acid Serves as Multitarget miRNA Sponge with Low Immunogenicity

Area of Science:

• Biotechnology
• RNA Therapeutics
• Molecular Biology

Background:

• MicroRNAs (miRNAs) regulate gene expression and are targets for therapy.
• Linear miRNA sponges have limitations like low stability and high immunogenicity.
• Artificial circular RNAs (circRNAs) offer better stability but still face immunogenicity challenges.

Purpose of the Study:

• To develop a method for synthesizing site-specifically modified circRNAs with reduced immunogenicity.
• To investigate the impact of pseudouridine modification on circRNA immunogenicity and function.
• To enable long-term coregulation of multiple miRNAs using engineered circRNAs.

Main Methods:

• Developed a DNA-templated RNA ligation method for synthesizing modified circRNAs.
• Incorporated multiple miRNA binding sites into the circRNAs.
• Introduced pseudouridine modifications (30%) into the synthetic circRNAs.

Main Results:

• The developed method allows for high-yield, site-specific synthesis of modified circRNAs.
• Pseudouridine modification (30%) effectively reduced circRNA immunogenicity, irrespective of modification site or pattern.
• Engineered circRNAs demonstrated long-term coregulation of multiple miRNAs.

Conclusions:

• Site-specifically modified circRNAs can be precisely synthesized using the developed DNA-templated ligation method.
• Pseudouridine modification is a viable strategy to mitigate circRNA immunogenicity for therapeutic applications.
• These advancements pave the way for developing next-generation circRNA-based therapeutics with balanced properties.