A cis-acting ligase ribozyme generates circular RNA in vitro for ectopic protein functioning.

Area of Science:

• Molecular Biology
• RNA Therapeutics
• Biochemistry

Background:

• Delivering synthetic RNA for ectopic protein expression is a novel therapeutic strategy.
• Covalently joining linear RNA head-to-tail can enhance its functional longevity.

Purpose of the Study:

• To generate circular RNA (circRNA) in vitro using a cis-acting ligase ribozyme (RzL) for ectopic protein expression.
• To investigate the translational efficiency of circRNA and its potential for antiviral applications.

Main Methods:

• RNA circularization was achieved using a ligase ribozyme (RzL).
• circRNA integrity was confirmed by 5' phosphate masking, RNase R resistance, and RT-PCR sequencing.
• Translational competence was assessed using internal ribosome entry sites (IRES).

Main Results:

• Successful in vitro generation of circular RNA (circRNA) was confirmed through multiple methods.
• A single IRES element enabled efficient circRNA translation, while two IRES elements in cis hindered it.
• The translated circRNA demonstrated potent antiviral activity, particularly when expressing Cas13 ribonuclease.

Conclusions:

• In vitro generated circRNA is a viable platform for enhanced protein expression.
• circRNA exhibits significant potential as an antiviral therapeutic agent, especially when engineered to express specific nucleases like Cas13.