Engineering protein and DNA tools for creating DNA-dependent protein switches

Area of Science:

• Biotechnology
• Molecular Biology
• Synthetic Biology

Background:

• Switchable proteins alter conformation (inactive to active) in response to stimuli like ligands, pH, temperature, or light.
• CRISPR Cas nucleases are DNA-activated protein switches, inspiring the engineering of other enzymes for sequence-specific control.
• Recent advances enable the integration of DNA-binding control into diverse enzymatic functions.

Purpose of the Study:

• To highlight engineered protein switches for creating advanced biosensors.
• To demonstrate methods for detecting pathogen and other genomic sequences using engineered biosensors.
• To showcase the potential of protein and DNA engineering in developing novel diagnostic tools.

Main Methods:

• Utilizing alternate frame folding to convert proteins into ligand-activated switches by fusing input and output domains.
• Employing GCN4 as a DNA recognition domain and nanoluciferase as a luminescent reporter for DNA-binding-induced color changes.
• Implementing DNA engineering protocols for de novo designed hairpins and modified aptamers to enable arbitrary sequence and small molecule/protein activation.

Main Results:

• Demonstrated the successful engineering of switchable proteins activated by DNA binding.
• Illustrated a method for creating sequence-specific biosensors using protein and DNA engineering.
• Showcased the versatility of the approach with a GCN4-nanoluciferase system and DNA tools.

Conclusions:

• Engineered protein switches offer a powerful platform for developing sequence-specific biosensors.
• The described methodologies can be broadly applied to control protein function via DNA binding.
• This approach holds significant potential for pathogen detection and other genomic sequence-based diagnostics.