Highly efficient expression of circular RNA aptamers in cells using autocatalytic transcripts

Area of Science:

• Molecular Biology
• Biotechnology
• Synthetic Biology

Background:

• RNA aptamers are valuable tools for cellular research but suffer from low expression and degradation in mammalian cells.
• Existing methods limit the utility of RNA aptamers for in vivo applications.
• Genetic encoding of RNA aptamers offers potential for cellular manipulation but requires robust expression systems.

Purpose of the Study:

• To develop a novel expression system for enhanced RNA aptamer stability and expression in mammalian cells.
• To overcome limitations of low expression and rapid degradation of RNA aptamers.
• To improve the functionality of RNA aptamers as cellular probes and therapeutic agents.

Main Methods:

• Developed the Tornado (Twister-optimized RNA for durable overexpression) expression system.
• Utilized Twister ribozymes for rapid RNA circularization.
• Leveraged endogenous RNA ligase RtcB for ligation of RNA termini.
• Engineered protein-binding aptamers and a fluorescent biosensor for S-adenosyl methionine (SAM).

Main Results:

• Tornado system achieved rapid RNA circularization, significantly increasing RNA aptamer stability and expression levels.
• Circularized aptamers demonstrated potent inhibition of cellular signaling pathways.
• An RNA-based SAM biosensor showed enhanced sensitivity for detecting intracellular SAM dynamics.
• The system enabled functional expression of RNA aptamers previously limited by stability issues.

Conclusions:

• The Tornado expression system markedly enhances the utility of RNA-based approaches in mammalian cells.
• RNA circularization via Tornado improves the stability and efficacy of RNA aptamers.
• This system provides a powerful tool for advancing RNA therapeutics and diagnostics.