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heteroquenching) conjugated to the peptide biosensor, can be employed to
reduce basal fluorescence of the biosensor, although this does not necessarily
ensure that fluorescence will be completely quenched 142 ( Fig. 6.13B ).
3.5.2 Light activation
Another means of optimizing the signal-to-noise ratio and increasing the
sensitivity of the response involves generating light-activatable probes that
can be selectively activated in response to UV irradiation. Light-activatable
probes are generated through molecular caging, by masking the main func-
tion with a photolabile group, which will be selectively released upon illu-
mination. This provides spatial and temporal control over the activation of
the probe, thereby allowing dampening or silencing any nonspecific fluores-
cence and reducing nonspecific noise attributable to basal fluorescence prior
to activation 143 ( Fig. 6.13C ). The first caged fluorescent reporter of intra-
cellular enzymatic activity was a light-activatable variant of the NBD-PKC
probe, engineered through incorporation of a single photolytically sensitive
cage on the phosphorylatable serine group of the peptide, which precluded
A Dye quenching thanks to synthetic quencher
P
Quencher
Phosphorylation
Peptide substrate
B Homodimer quenching—autoquenching
P
Phosphorylation
Peptide substrate
C Caging of the phosphorylatable residue
Photoactivation
and phosphorylation
P
Peptide substrate
Figure 6.13 Quenching and photoactivation strategies for peptide biosensors. (A) Incor-
poration of a synthetic quencher into the peptide backbone. (B) Intramolecular
homoquenching between two fluorophores conjugated to the peptide biosensor.
(C) Selective photoactivation of caged compounds by UV irradiation. 110,127,133
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