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Review
. 2020 Jul 16;21(14):1935-1946.
doi: 10.1002/cbic.202000037. Epub 2020 Apr 2.

Visualizing and Manipulating Biological Processes by Using HaloTag and SNAP-Tag Technologies

Conner A Hoelzel  1 , Xin Zhang  1
Affiliations
Review

Visualizing and Manipulating Biological Processes by Using HaloTag and SNAP-Tag Technologies

Conner A Hoelzel et al. Chembiochem. .

Abstract

Visualizing and manipulating the behavior of proteins is crucial to understanding the physiology of the cell. Methods of biorthogonal protein labeling are important tools to attain this goal. In this review, we discuss advances in probe technology specific for self-labeling protein tags, focusing mainly on the application of HaloTag and SNAP-tag systems. We describe the latest developments in small-molecule probes that enable fluorogenic (no wash) imaging and super-resolution fluorescence microscopy. In addition, we cover several methodologies that enable the perturbation or manipulation of protein behavior and function towards the control of biological pathways. Thus, current technical advances in the HaloTag and SNAP-tag systems means that they are becoming powerful tools to enable the visualization and manipulation of biological processes, providing invaluable scientific insights that are difficult to obtain by traditional methodologies. As the multiplex of self-labeling protein tag systems continues to be developed and expanded, the utility of these protein tags will allow researchers to address previously inaccessible questions at the forefront of biology.

Keywords: HaloTag; SNAP-tag; bio-orthogonal chemistry; chemical biology; molecular probes.

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Figures

Figure 1.
Figure 1.
Chemical mechanisms of chemical labelling for A) HaloTag and B) SNAP-tag.
Figure 2.
Figure 2.
Structures of commonly employed A) coumarin, B) triarylmethane, and C) cyanine based fluorophores for general labelling of HaloTag and SNAP-tag fused proteins in fluorescence-based microscopy. R1 indicates common positions for coupling chloroalkane and O6-benzyl guanine ligands for HaloTag and SNAP-tag ligands. D) Spectral coverage of commonly employed fluorescent probes. Excitation (top value) and emission (bottom value) maxima for the included fluorophores.
Figure 3.
Figure 3.
No-wash labelling strategies for HaloTag and SNAP-tag. A) Release of a PET or FRET based quencher upon conjugation. B) Fluorogenic rhodamines, lactone ring opening upon conjugation. C) Embedding of environmentally-sensitive fluorophores into the substrate binding tunnel.
Figure 4.
Figure 4.
Structures of A) PET and FRET, B) Si-rhodamine, C) molecular rotor, or D) solvatochromic based fluorogenic, wash-free labelling probes for self-labelling protein tags and their corresponding fold-change upon binding to SNAP-tag or HaloTag in vitro.
Figure 5.
Figure 5.
Mechanisms of viscosity and polarity dependent excited state non-radiative decay in donor-acceptor type chromophores. A) Twisted intramolecular charge transfer. B) Solvent-mediated external conversion.
Figure 6.
Figure 6.
The A) AgHalo and B) AggTag methods to monitor protein conformational dynamics and proteome stress onset.
Figure 7.
Figure 7.
A) Immobilization strategy for Halo- and SNAP-Tag fused proteins of interest and B) downstream applications following immobilization.
Figure 8.
Figure 8.
HaloPROTACs strategy for induced proteasomal degradation of HaloTag fused POIs.
Figure 9.
Figure 9.
A) T-REX and B) CID strategies of optochemical control and perturbation of protein systems.
Scheme 1.
Scheme 1.
Equilibrium of 2-carboxyphenyl xanthene-based chromophores between open quinoid and closed lactone forms.
Scheme 2.
Scheme 2.
Photoredox mechanism of rhodamine photoswitching. (T1 = triplet state, β-ME = β-mercaptoethanol)
Scheme 3.
Scheme 3.
Nucleophilic mechanism of cyanine photo-switching.
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