Splint-free circular RNA synthesis via RNA secondary structure-guided ligation.

Area of Science:

• Molecular Biology
• RNA Therapeutics
• Biotechnology

Background:

• Circular RNA (circRNA) is a unique RNA structure with potential therapeutic applications.
• Current in vitro circRNA synthesis methods, like the PIE system, can introduce immunogenic sequences.
• A need exists for efficient and clean in vitro circRNA production methods.

Purpose of the Study:

• To develop an alternative, efficient in vitro method for synthesizing circular RNA (circRNA).
• To enable high-yield circRNA production for targeted gene expression.
• To overcome immunogenicity issues associated with existing methods.

Main Methods:

• Designed linear RNA molecules with predicted secondary structures to form a terminal nick.
• Utilized T4 RNA ligase 2 (T4 Rnl2) for enzymatic ligation to seal the nick and create circRNA.
• Validated functional gene expression from the synthesized circRNA in mammalian cells.

Main Results:

• Achieved high efficiency in ligase-mediated in vitro RNA circularization.
• Demonstrated successful functional protein expression from the synthesized circRNA constructs.
• Established a splint-free, secondary-structure-guided strategy for circRNA synthesis.

Conclusions:

• The developed ligase-mediated, secondary-structure-guided, splint-free strategy is an efficient alternative for circRNA production.
• This method enables both high-yield synthesis and validated gene expression in mammalian cells.
• This approach offers a cleaner alternative to existing methods, potentially reducing immunogenicity.