A programmable ribozyme for RNA signal transduction.

Area of Science:

• Synthetic Biology
• Molecular Biology
• Biochemistry

Background:

• RNA detection is crucial for various applications, but direct functional transduction of sensed RNA remains a challenge.
• Existing methods lack a generalizable approach to link RNA triggers with diverse non-coding RNA effectors.

Purpose of the Study:

• To engineer a programmable RNA-activated system for direct functional transduction of RNA triggers.
• To develop a versatile platform for RNA sensing, amplification, and effector release.

Main Methods:

• Engineering of a dual-site self-cleaving ribozyme platform (UNlocked by Activating RNA - UNBAR) encoded within a single RNA strand.
• Designing UNBAR ribozymes for single-nucleotide trigger specificity and modularity.
• Demonstrating cell-free and in-cell applications, including microRNA and viral RNA detection, and CRISPR-Cas9 gene editing regulation.

Main Results:

• UNBAR ribozymes exhibit high inactivity without triggers and single-nucleotide specificity.
• The system enables protein-free amplification and trigger-dependent release of non-coding RNA effectors (sgRNA, shRNA, aptamer).
• Functional transduction demonstrated via a cleaved aptamer for direct fluorescence readout and trigger-dependent CRISPR-Cas9 editing in zebrafish and human cells.

Conclusions:

• UNBAR represents a first-in-class RNA-activated system for direct functional transduction.
• This programmable platform holds significant potential for synthetic biology, diagnostics, and gene regulation applications.