Screening for engineered neomycin riboswitches that control translation initiation.

Area of Science:

• Molecular Biology
• RNA Biology
• Synthetic Biology

Background:

• Riboswitches are natural genetic elements controlling gene expression via small molecule binding.
• Aminoglycosides like neomycin are antibiotics that target bacterial ribosomes.

Purpose of the Study:

• To develop artificial riboswitches responsive to the aminoglycoside neomycin.
• To explore structure-function relationships of engineered neomycin-binding riboswitches.

Main Methods:

• A two-stage strategy combining in vitro selection and genetic screening.
• Structural probing and structure-function analyses were performed.

Main Results:

• Several artificial riboswitches binding neomycin were successfully identified.
• These riboswitches share structural motifs with natural neomycin-binding sites but not sequence homology.
• A composite binding pocket model involving internal and flaplike loops was proposed.

Conclusions:

• The combination of in vitro selection and in vivo screening is effective for identifying functional RNA molecules.
• Engineered riboswitches can mimic natural ligand-binding mechanisms.
• This work provides a foundation for designing novel RNA-based genetic controllers.