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DNA-Specific Biosensors Based on Intramolecular β-Lactamase-Inhibitor Complex Formation
Wouter Engelen1, Maarten Merkx2
1Laboratory of Chemical Biology and Institute for Complex Molecular Systems, Department of Biomedical Engineering, Eindhoven University of Technology, Den Dolech 2, 5612, MB, Eindhoven, The Netherlands.
Methods in Molecular Biology (Clifton, N.J.)
|March 16, 2017
Summary
Researchers developed a modular synthetic protein switch that uses DNA to control enzyme activity. This system can be reversibly turned on and off by specific DNA sequences, enabling sensitive detection of oligonucleotide triggers.
Area of Science:
- Biochemistry and Molecular Biology
- Synthetic Biology
- Biomolecular Engineering
Background:
- Synthetic protein switches offer precise control over biomolecular systems.
- Oligonucleotide-based triggers are valuable for programming molecular interactions.
- Controlling protein complex assembly and disassembly is crucial for biosensing applications.
Purpose of the Study:
- To develop a modular and reversible synthetic protein switch.
- To control the DNA-directed assembly and disassembly of a protein complex.
- To enable sensitive detection of oligonucleotide inputs.
Main Methods:
- Conjugating TEM1-β-lactamase and its inhibitor BLIP to unique handle oligonucleotides.
- Utilizing a complementary ssDNA template strand for noncovalent protein complex assembly.
- Employing an input-oligonucleotide to disrupt the complex via dsDNA helix formation.
- Monitoring enzyme activity using a fluorescent substrate.
Main Results:
- Achieved reversible control over the enzyme-inhibitor complex assembly and disassembly.
- Demonstrated straightforward tuning of DNA recognition sequences and lengths.
- Detected as little as 10 pM target oligonucleotide, showing a distinguishable increase in enzyme activity.
Conclusions:
- The developed synthetic protein switch provides a highly modular approach for oligonucleotide-based sensing.
- This system enables sensitive and reversible control of enzyme activity through DNA triggers.
- The modularity allows for easy adaptation to different oligonucleotide inputs and recognition sequences.
