Thermozymes: Synthetic RNA thermometers based on ribozyme activity

Area of Science:

• Synthetic biology
• Molecular biology
• RNA therapeutics

Background:

• Synthetic biology enables the creation of novel cellular functions by combining natural biological components.
• Ribozymes, such as the hammerhead ribozyme (HHR), are RNA molecules with catalytic activity, capable of cleaving other RNA molecules.
• RNA thermometers (RNATs) are RNA structures that regulate gene expression in response to temperature changes.

Purpose of the Study:

• To design and construct a novel regulatory RNA device with temperature-controlled gene expression.
• To engineer a synthetic ribozyme system that responds inversely to natural RNA thermometers.
• To explore the biotechnological potential of combining hammerhead ribozymes with RNA thermometers.

Main Methods:

• Fusion of a temperature-responsive RNA hairpin (Salmonella RNAT) to a hammerhead ribozyme (HHR).
• Development of a screening approach to isolate functional temperature-controlled ribozymes (thermozymes).
• In vivo gene expression studies and in vitro RNA structure probing to elucidate the mechanism of action.

Main Results:

• Isolation of two functional thermozymes capable of regulating gene expression based on temperature.
• Demonstration that increased temperatures disrupt the RNA thermometer structure, inhibiting HHR self-cleavage.
• Characterization of a system where gene expression is repressed upon temperature increase, acting oppositely to natural RNATs.

Conclusions:

• Engineered thermozymes exhibit a novel, inverse temperature-dependent gene regulation mechanism.
• The modularity of ribozyme-based systems allows for the creation of sophisticated RNA regulatory devices.
• This work highlights the significant biotechnological potential of synthetic ribozyme-RNA thermometer fusions for gene control applications.