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Tewoderos M. Ayele et al.
limitations for broad applicability due to high background signal, potential
arsenic toxicity, and elevated photobleaching properties of the fluorescent
ligands (Marks & Nolan, 2006; Stroffekova, Proenza, & Beam, 2001).
This inspired the development of small-molecule labeling systems that
instead take advantage of enzymatic recognition to drive selectivity.
These approaches include the SNAP/Clip-tag (Gautier et al., 2008), LAP
(Ferna´ndez-Sua´rez et al., 2007), Halo-tag (Los et al., 2008), and coiled-coil
tag (Reinhardt, Lotze, Mo€rl, Beck-Sickinger, & Seitz, 2015). The advan-
tages of these methods include substantially improved control over the
intensity and timing of fluorescent labeling and the ability to harness
the diverse palette of small-molecule fluorophores. However, covalent pro-
tein labeling techniques that target specific amino acids or short peptide
sequences still lack high labeling selectivity in complex biological systems
where other proteins or molecules are likely to have similar reactive groups.
For example, protein labeling systems that rely on cysteine residue func-
tionalization lose their selectivity in environments that contain high concen-
trations of glutathione or other biological molecules that have nucleophilic
thiol groups. Additionally, these techniques still rely on intrinsically fluores-
cent molecules, which produce high background signal and hence require
extensive washing steps to remove unreacted dye from the cytosol.
In order to address these challenges, several next-generation fluorophore
molecules have been developed that are conditionally fluorescent and
undergo a dramatic increase in emission when particular biochemical or
physical conditions are met (Bruchez, 2015). One class of these probes
are fluorogenic molecules, which can adopt somewhat similar planar archi-
tectures compared to traditional organic fluorophores yet display the impor-
tant inclusion of a freely rotating bond, which prevents the molecule
from spending a significant amount of time in the planar conformation.
This results in low fluorescence in solution, with significant enhancement
of signal upon rotational restriction imparted by temperature or viscosity
changes. Alternatively, this signal enhancement can be achieved through
binding to a nucleic acid strand or protein if the binding mode restricts
the fluorogenic molecule in a planar conformation. In practice, visualization
of POIs has been achieved by combining a fluorogenic small molecule and a
“fluorogen activating protein” (FAP) that has been engineered to tightly
bind to the dye molecule and restrict its overall rotational movement.
Similar to previous genetic tagging methods, the POI can then be visualized
by in-frame genetic fusion with the FAP domain (Fig. 1D), generating high
signal-to-background ratio without the requirement of washing away
unbound fluorogen (Chen et al., 2014; Telmer et al., 2015).