105
concentrations of analyte, and is calculated as the ratio between the
upper and the lower plateaus. The concentration of analyte that
causes half the maximum signal change is defined as the c 50 . The c 50
for a certain analyte can be tuned by altering the affinity of the
intramolecular ligand for the binding protein. The analyte response
range can thus be altered to fall within physiological concentrations of the analyte.
Some common design principles can be followed to construct a
SNIFIT or a LUCID for a specific analyte. Over the years, we have
gathered experience from our design of biosensors from analytes
ranging from sulfonamides and neurotransmitters to cancer therapeutics such as methotrexate. Here, we give an overview of how to
design these biosensors, followed by an overview of their practical
applications.
A binding protein is required to display sufficient affinity and specificity for the analyte of interest. Since SNIFITs and LUCIDs are
based on competition with another ligand, the dissociation constant of the binding protein for the analyte of interest must be
lower compared to the desired response range.
Another important factor when choosing the binding protein
is the availability of structural information, ideally in complex with
a potential intramolecular ligand. This is not only crucial for the
geometrical optimization of the sensor, but also for the choice of
derivatization points of the ligand. In this regard, it can be advantageous if the binding protein is small, monomeric, and stable. Yet,
if no suitable natural binding protein is available for the analyte of
interest, computational methods can be used to design a protein
with tailor-engineered binding properties [20].
The synthetic component of SNIFITs and LUCIDs consists of
three parts: (1) a BG group that serves as the attachment site for
SNAP-tag, (2) a fluorophore as RET acceptor, and (3) an intramolecular ligand (Fig. 4). These three parts are connected by oligoethylene glycol (EG) linkers that must be sufficiently long not to
affect the binding of the intramolecular ligand and the closing of
the sensor. We routinely use an (EG) 11 linker between the BG
group and the fluorophore, and an (EG) 2 linker between the fluorophore and the intramolecular ligand.
Three main points must be considered when designing the
intramolecular ligand of the sensor: (1) its affinity must be strong
enough for the sensor to be predominantly closed in the absence
of analyte, (2) its affinity determines the sensor’s analyte response
range, and (3) its dissociation kinetics determine the response
kinetics of the sensor.
1.2 The Rational
Design of SNIFIT
and LUCID Biosensors
1.2.1 The Binding
Protein
1.2.2 The
Intramolecular Tether
SNIFITS and LUCIDs: Semi-Synthetic Modular Biosensors
concentrations of analyte, and is calculated as the ratio between the
upper and the lower plateaus. The concentration of analyte that
causes half the maximum signal change is defined as the c 50 . The c 50
for a certain analyte can be tuned by altering the affinity of the
intramolecular ligand for the binding protein. The analyte response
range can thus be altered to fall within physiological concentrations of the analyte.
Some common design principles can be followed to construct a
SNIFIT or a LUCID for a specific analyte. Over the years, we have
gathered experience from our design of biosensors from analytes
ranging from sulfonamides and neurotransmitters to cancer therapeutics such as methotrexate. Here, we give an overview of how to
design these biosensors, followed by an overview of their practical
applications.
A binding protein is required to display sufficient affinity and specificity for the analyte of interest. Since SNIFITs and LUCIDs are
based on competition with another ligand, the dissociation constant of the binding protein for the analyte of interest must be
lower compared to the desired response range.
Another important factor when choosing the binding protein
is the availability of structural information, ideally in complex with
a potential intramolecular ligand. This is not only crucial for the
geometrical optimization of the sensor, but also for the choice of
derivatization points of the ligand. In this regard, it can be advantageous if the binding protein is small, monomeric, and stable. Yet,
if no suitable natural binding protein is available for the analyte of
interest, computational methods can be used to design a protein
with tailor-engineered binding properties [20].
The synthetic component of SNIFITs and LUCIDs consists of
three parts: (1) a BG group that serves as the attachment site for
SNAP-tag, (2) a fluorophore as RET acceptor, and (3) an intramolecular ligand (Fig. 4). These three parts are connected by oligoethylene glycol (EG) linkers that must be sufficiently long not to
affect the binding of the intramolecular ligand and the closing of
the sensor. We routinely use an (EG) 11 linker between the BG
group and the fluorophore, and an (EG) 2 linker between the fluorophore and the intramolecular ligand.
Three main points must be considered when designing the
intramolecular ligand of the sensor: (1) its affinity must be strong
enough for the sensor to be predominantly closed in the absence
of analyte, (2) its affinity determines the sensor’s analyte response
range, and (3) its dissociation kinetics determine the response
kinetics of the sensor.
1.2 The Rational
Design of SNIFIT
and LUCID Biosensors
1.2.1 The Binding
Protein
1.2.2 The
Intramolecular Tether
SNIFITS and LUCIDs: Semi-Synthetic Modular Biosensors