A ligand-dependent hammerhead ribozyme switch for controlling mammalian gene expression

Area of Science:

• Synthetic Biology
• Molecular Biology
• Gene Regulation

Background:

• External control of gene expression is crucial for life sciences research and applications.
• Existing methods in mammalian cells often require ligand-sensing transcription factors, necessitating stable cell line generation.
• Direct RNA-ligand interactions offer an alternative for gene expression control, with engineered RNA switches showing promise.

Purpose of the Study:

• To engineer and validate an artificial RNA switch for controlling gene expression in mammalian cells.
• To demonstrate ligand-dependent regulation of gene expression via mRNA self-cleavage.
• To develop a system that bypasses the need for exogenous transcription factors.

Main Methods:

• Adaptation and optimization of a bacterial aptazyme switch for mammalian systems.
• Engineering of artificial start codons and the linker sequence between aptamer and ribozyme.
• Utilizing a theophylline-inducible hammerhead ribozyme for mRNA self-cleavage and gene expression 'off-switching'.

Main Results:

• Successfully implemented a theophylline-inducible RNA switch in mammalian cells.
• Demonstrated ligand-dependent triggering of mRNA self-cleavage, leading to gene expression repression.
• Optimized components of the RNA switch for efficient function in mammalian systems.

Conclusions:

• An artificial aptazyme functions as an effective RNA-based gene expression switch in mammalian cells.
• This theophylline-inducible system provides external control over transgenic expression without requiring transcription factors.
• The developed RNA switch represents a versatile tool for gene regulation in diverse mammalian applications.