Protein-responsive ribozyme switches in eukaryotic cells

Area of Science:

• Synthetic biology
• Molecular biology
• Biochemistry

Background:

• Genetic devices are crucial for synthetic biology, enabling targeted cellular responses.
• Ribozyme-based devices offer a platform for sensing and regulating gene expression based on intracellular conditions.

Purpose of the Study:

• To develop novel ribozyme-based genetic devices for detecting intracellular protein concentrations.
• To enable both gene activation (ON) and repression (OFF) in response to protein ligands.
• To validate device performance in eukaryotic hosts like yeast and mammalian cells.

Main Methods:

• Design and construction of ribozyme switches responsive to protein ligands.
• In vitro characterization pipeline for prescreening device designs using magnesium concentration gradients.
• In vivo testing of gene-regulatory activities in yeast and mammalian cells.
• Ligand localization studies to determine cellular compartment activity (nucleus/cytoplasm).

Main Results:

• Developed functional ribozyme devices for gene regulation in yeast and mammalian cells.
• Demonstrated both gene-ON and gene-OFF responses to protein ligands.
• Correlated in vitro cleavage activity with in vivo gene-regulatory function across different magnesium concentrations.
• Confirmed ribozyme switch activity in both nuclear and cytoplasmic cellular compartments.

Conclusions:

• Ribozyme-based devices are effective tools for sensing protein concentrations and regulating gene expression in eukaryotic cells.
• The developed in vitro characterization pipeline aids in efficient screening of synthetic gene-regulatory devices.
• These findings advance the application of ribozyme switches for detecting protein biomarkers and engineering cellular functions.