Efficient circularization of protein-encoding RNAs via a novel cis-splicing system

Area of Science:

• Molecular Biology
• RNA Therapeutics
• Biotechnology

Background:

• Circular RNAs (circRNAs) offer unique advantages over linear mRNA for therapeutic applications.
• Efficient and controllable production of circRNAs is crucial for their development.
• Existing methods for circRNA synthesis often require complex designs or additional elements.

Purpose of the Study:

• To develop a novel and efficient cis-splicing system for circRNA production.
• To investigate the structural requirements for efficient circRNA self-splicing and circularization.
• To evaluate the performance of the generated circRNAs in terms of protein expression and immunogenicity.

Main Methods:

• Introduction of a ribozyme core into a precursor RNA for cis-splicing.
• Design of homologous arms to form the P9.0 duplex for self-splicing.
• Structural optimization of RNA elements (P10, P1-ex) to enhance circularization.
• Analysis of circRNA production, protein expression, and innate immune response.

Main Results:

• The cis-splicing system efficiently produces circRNAs without additional spacer elements.
• Formation of the P9.0 duplex is critical for self-splicing and circularization.
• Structural features adjacent to the ωG position, not just nucleotide composition, dictate recognition of the transesterification site.
• Optimized structural elements significantly improved circularization efficiency.
• Generated circRNAs demonstrated prolonged protein expression and low innate immune activation.

Conclusions:

• The developed cis-splicing system provides an efficient strategy for circRNA generation.
• Understanding RNA structural elements is key to optimizing circRNA synthesis.
• This approach holds promise for advancing circRNA-based therapeutics with improved stability and reduced immunogenicity.