NicOPURE: nickless RNA circularization and one-step purification with engineered group II introns and cyclizing UTRs

Area of Science:

• Molecular Biology
• Biochemistry
• RNA Therapeutics

Background:

• Circular RNAs (circRNAs) are increasingly recognized for their biological roles and therapeutic potential.
• Current methods for circRNA synthesis often rely on group 1 introns, requiring high temperatures and leading to nicked RNA byproducts.
• Complex purification steps are necessary for preparing circRNAs for downstream applications.

Purpose of the Study:

• To enhance the efficiency and simplicity of circular RNA production.
• To develop a method for generating therapeutic circular messenger RNAs with reduced nicks.
• To streamline the purification process for engineered circRNAs.

Main Methods:

• Strategic design of structural elements and sequence features in group 2 introns for improved RNA circularization.
• Incorporation of split streptavidin aptamer tags into the enzymatic sites of group 2 introns.
• In vitro circularization reactions performed at reduced temperatures.

Main Results:

• Group 2 introns demonstrated enhanced efficacy in RNA circularization compared to group 1 introns.
• The novel approach resulted in significantly fewer nicked RNA byproducts.
• The integrated aptamer tags enabled a one-step purification of the circularized RNA.
• The developed system is straightforward, efficient, and scalable for generating therapeutic circRNAs.

Conclusions:

• Engineered group 2 introns offer a more efficient and gentler method for circRNA synthesis.
• The nick-free, one-step purification system simplifies the production of therapeutic circular messenger RNAs.
• This innovative approach has significant implications for advancing RNA-based therapeutics.