Chapter title |
DNA-Specific Biosensors Based on Intramolecular β-Lactamase-Inhibitor Complex Formation
|
---|---|
Chapter number | 12 |
Book title |
Synthetic Protein Switches
|
Published in |
Methods in molecular biology, March 2017
|
DOI | 10.1007/978-1-4939-6940-1_12 |
Pubmed ID | |
Book ISBNs |
978-1-4939-6938-8, 978-1-4939-6940-1
|
Authors |
Engelen, Wouter, Merkx, Maarten, Wouter Engelen, Maarten Merkx |
Editors |
Viktor Stein |
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. |
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