PCanPIE: A group I intron platform for efficient circRNA synthesis at ambient temperatures

Area of Science:

• Molecular Biology
• RNA Therapeutics
• Biochemistry

Background:

• Ribozyme-based permuted intron-exon (PIE) systems facilitate protein-independent circular RNA (circRNA) production.
• Existing PIE platforms necessitate high temperatures, which can lead to RNA degradation and limit applications.

Purpose of the Study:

• To introduce and characterize a novel PIE platform, PCanPIE, utilizing the Candida albicans mitochondrial large subunit (C.a.mtLSU) group I intron.
• To demonstrate efficient circRNA synthesis under near-physiological conditions using PCanPIE.

Main Methods:

• The C.a.mtLSU group I intron was engineered as a PIE system (PCanPIE) with permutation sites at peripheral stems P5, P6b, and P8.
• Circularization efficiency was assessed under near-physiological conditions (25 °C, 6 mM MgCl₂).
• Kinetic analysis and optimization of spacer sequences were performed to evaluate splicing rates and payload capacity.

Main Results:

• All three evaluated permutation sites (P5, P6b, P8) supported circRNA synthesis at 25 °C, eliminating the need for high-temperature incubation.
• The P6b permutation site achieved the highest circularization efficiency, with 95% precursor splicing to circRNA.
• PCanPIE successfully circularized long RNAs (up to 1,657 nt), including structured and repetitive sequences, after optimization of flanking sequences.

Conclusions:

• PCanPIE represents the first application of the C.a.mtLSU group I intron as a PIE platform for circRNA synthesis.
• This system functions efficiently under near-physiological conditions, offering a more stable and versatile alternative to existing high-temperature PIE methods.
• PCanPIE expands the toolkit for circRNA production, enabling the synthesis of diverse and complex RNA molecules.