High-throughput engineering of ligand-activated splicing ribozyme through domain insertion

Area of Science:

• Molecular Biology
• Synthetic Biology
• RNA Engineering

Background:

• Protein engineering uses domain insertion for ligand-mediated control.
• Systematic application to large, structured RNAs like catalytic introns is not well-established.

Purpose of the Study:

• Investigate engineering ligand-activated splicing ribozymes (LASRs) from group I catalytic introns.
• Map aptamer insertion sites for ligand-dependent control.
• Establish LASRs as a versatile RNA-based control platform.

Main Methods:

• Domain-insertion profiling coupled with high-throughput screening.
• Nucleotide-resolution landscape mapping of aptamer insertion.
• Integration with a genetic recorder for ribosomal RNA information writing.

Main Results:

• Identified specific sites for robust ligand-dependent control in ribozymes.
• Demonstrated LASR functionality across bacteria and fungi.
• Enabled sequencing-based recovery of intracellular chemical signals from microbial consortia.

Conclusions:

• LASRs are a feasible RNA-based inducible control platform.
• LASRs can sense diverse chemical inputs and regulate gene expression.
• LASRs facilitate recording of intracellular information in RNA.