179
Viktor Stein (ed.), Synthetic Protein Switches: Methods and Protocols, Methods in Molecular Biology, vol. 1596,
DOI 10.1007/978-1-4939-6940-1_12, © Springer Science+Business Media LLC 2017
Chapter 12
DNA-Specific Biosensors Based on Intramolecular
β-Lactamase-Inhibitor Complex Formation
Wouter Engelen and Maarten Merkx
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.
Key words β-lactamase, Reporter enzyme, DNA detection, Synthetic biology, Protein-DNA conjugation,
Biosensor
1 Introduction
Synthetic protein switches are extensively used in synthetic biology,
molecular imaging, and molecular diagnostics to study biological
processes both in vitro and in vivo by responding to the presence
of a specific input molecule. Ideally, molecular recognition (input)
and signal generating functions (output) are part of separate
domains in these switches, as such a modular organization allows
straightforward exchange of input or output function [1]. A successful design approach that ensures efficient translation of input
activation to output modulation, is to design switches that are able
to adopt two conformational states; one in which two output
domains can interact intramolecularly and one where their interaction
is prohibited. The dynamic response of such a protein switch is
Viktor Stein (ed.), Synthetic Protein Switches: Methods and Protocols, Methods in Molecular Biology, vol. 1596,
DOI 10.1007/978-1-4939-6940-1_12, © Springer Science+Business Media LLC 2017
Chapter 12
DNA-Specific Biosensors Based on Intramolecular
β-Lactamase-Inhibitor Complex Formation
Wouter Engelen and Maarten Merkx
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.
Key words β-lactamase, Reporter enzyme, DNA detection, Synthetic biology, Protein-DNA conjugation,
Biosensor
1 Introduction
Synthetic protein switches are extensively used in synthetic biology,
molecular imaging, and molecular diagnostics to study biological
processes both in vitro and in vivo by responding to the presence
of a specific input molecule. Ideally, molecular recognition (input)
and signal generating functions (output) are part of separate
domains in these switches, as such a modular organization allows
straightforward exchange of input or output function [1]. A successful design approach that ensures efficient translation of input
activation to output modulation, is to design switches that are able
to adopt two conformational states; one in which two output
domains can interact intramolecularly and one where their interaction
is prohibited. The dynamic response of such a protein switch is