Generation of precise and accurate engineered circRNAs using enzymatic ligation

Area of Science:

• Biotechnology
• Molecular Biology
• RNA Therapeutics

Background:

• Linear messenger RNA (mRNA) therapeutics show promise but are limited by in vivo degradation.
• Engineered circular RNAs (circRNAs) offer greater stability and are valuable for therapeutics and functional studies.
• Precise and homogenous circRNA production is crucial for accurate biological interpretation.

Purpose of the Study:

• To develop and optimize methods for generating precise, high-fidelity engineered circRNAs.
• To overcome limitations of linear RNA degradation in therapeutic applications.
• To provide a reliable method for producing homogenous circRNAs for research.

Main Methods:

• Enzymatic ligation of linear RNAs was used instead of autocatalytic splicing to minimize extraneous nucleotides.
• A permuted DNA transcription template with internal guanosines and a modified reverse primer was designed.
• GMP-primed in vitro transcription, T4 RNA ligase 2 circularization, and urea-PAGE gel extraction were optimized.

Main Results:

• The optimized method successfully produced precise linear precursor RNAs with correct ends.
• The combination of GMP-primed transcription, T4 RNA ligase 2, and urea-PAGE yielded the highest fidelity circRNAs.
• This workflow minimizes sequence and structural integrity issues in circRNA production.

Conclusions:

• The developed methods enable the production of precise and homogenous engineered circRNAs.
• These findings advance the potential of circRNAs as stable therapeutic agents.
• The optimized protocol supports accurate functional studies using engineered circRNAs.