title: DNA-Specific Biosensors Based on Intramolecular β-Lactamase-Inhibitor Complex Formation.
abstract: Synthetic protein switches that sequence-specifically respond to oligonucleotide-based input triggers provide valuable tools for the readout of oligonucleotide-based biomolecular systems and networks. Here, we discuss a highly modular approach to reversibly control the DNA-directed assembly and disassembly of a complex between TEM1-β-lactamase and its inhibitor protein BLIP. By conjugating each protein to a unique handle oligonucleotide, the enzyme-inhibitor pair is noncovalently assembled upon the addition of a complementary ssDNA template strand, resulting in inhibition of enzyme activity. Hybridization of an input-oligonucleotide that is complementary to a target recognition sequence in the ssDNA template strand results in the formation of a rigid dsDNA helix that mechanically disrupts the enzyme-inhibitor complex, hereby restoring enzyme activity. Following this noncovalent approach allowed straightforward tuning of the ssDNA template recognition sequence and target oligonucleotide lengths with only a single set of oligonucleotide-functionalized enzyme and inhibitor domains. Using a fluorescent substrate, as little as 10 pM target oligonucleotide resulted in a distinguishable increase in enzyme activity.
authors.affiliation: Laboratory 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.
authors.forename: Wouter
authors.initials: W
authors.lastname: Engelen
authors.affiliation: Laboratory 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. M.Merkx@tue.nl.
authors.forename: Maarten
authors.initials: M
authors.lastname: Merkx
mesh_terms: D015374:Biosensing Techniques / Q000379:methods; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000378:metabolism*; D009693:Nucleic Acid Hybridization / Q000502:physiology; D009841:Oligonucleotides / Q000378:metabolism; D011487:Protein Conformation; D001618:beta-Lactamases / Q000378:metabolism*
chemical_list: D004277:DNA, Single-Stranded; D004791:Enzyme Inhibitors; D009841:Oligonucleotides; D001618:beta-Lactamases
keywords: Biosensor; DNA detection; Protein-DNA conjugation; Reporter enzyme; Synthetic biology; β-lactamase