SNAP-tag Protein Research Articles

Protein proximity ligation probe with wide sensing range resolving dynamics of subcellular ph by super-resolution imaging

Subcellular pH plays a key role to modulate protein structures or specific active sites to impact diverse biological processes. The heterogeneity and high dynamics of intracellular pH require probes that can detect pH in the vicinity of the protein and have a wide pH response range. Here, we reported a protein proximity ligation fluorescent probe for super-resolution imaging of subcellular pH dynamics. Relying on SNAP-tag protein labeling technology, the pH-sensitive fluorophore BG-NDM based on photo-induced electron transfer mechanism was localized to the adjacent positions of the target protein in different subcellular organelles. The equilibrium between the non-fluorescent aggregate and the fluorogenic monomer enabled BG-NDM to have the ability of wash-free super-resolution imaging in live cells. And the wide pH response ranges from pH 6 to pH 12 made the probe have the ability of recognizing pH dynamics. The pH changes around functional proteins during mitophagy, cellular alkalosis and acidosis were revealed. Through long-term super-resolution imaging, we could further record the pH dynamics around mitochondrial proteins COX8A during mitochondrial contacts, fusion and fission at the resolution of a single mitochondria.

A general highly efficient synthesis of biocompatible rhodamine dyes and probes for live-cell multicolor nanoscopy

The development of live-cell fluorescence nanoscopy is powered by the availability of suitable fluorescent probes. Rhodamines are among the best fluorophores for labeling intracellular structures. Isomeric tuning is a powerful method for optimizing the biocompatibility of rhodamine-containing probes without affecting their spectral properties. An efficient synthesis pathway for 4-carboxyrhodamines is still lacking. We present a facile protecting-group-free 4-carboxyrhodamines’ synthesis based on the nucleophilic addition of lithium dicarboxybenzenide to the corresponding xanthone. This approach drastically reduces the number of synthesis steps, expands the achievable structural diversity, increases overall yields and permits gram-scale synthesis of the dyes. We synthesize a wide range of symmetrical and unsymmetrical 4-carboxyrhodamines covering the whole visible spectrum and target them to multiple structures in living cells – microtubules, DNA, actin, mitochondria, lysosomes, Halo-tagged and SNAP-tagged proteins. The enhanced permeability fluorescent probes operate at submicromolar concentrations, allowing high-contrast STED and confocal microscopy of living cells and tissues.

A fluorogenic probe for SNAP-tag protein based on ESPT ratiometric signals

Protein self-labeling tags achieve selective fusion and labeling of target proteins through genetic coding technology, but require exogenous fluorescent probes with fluorogenicity for protein tag binding to have the performance of wash-free fluorescence imaging in live cells. In this paper, we reported a fluorogenic probe 1 capable of ratiometric fluorescence recognition of SNAP-tag proteins. In this probe, the O6-benzylguanine derivative of 3‑hydroxy-1,8-naphthalimide underwent a selective covalent linkage reaction with SNAP-tag protein. The hydroxyl group on the naphthalimide fluorophore formed a hydrogen bond with the functional group near the protein cavity. The excited state proton transfer occurred after illumination, to obtain the ratio fluorescence signal from blue emission to red emission, realizing the wash-free fluorescence imaging of the target proteins.

Magnetic nanoparticles covalently immobilizing epidermal growth factor receptor by SNAP-Tag protein as a platform for drug discovery.

Magnetic nanoparticles (NPs) cloaked with cell membranes expressing high levels of the epidermal growth factor receptor (EGFR) have been used to screen for EGFR-targeting active compounds in traditional Chinese medicine (TCM) formulations. However, previous strategies involved physical immobilization of the biomaterials on the surface of the nanocarrier, resulting in highly unstable platforms since the biological materials could dislodge easily. Chemical bonding of biomaterials to the nanoparticles surface can improve the stability of the biomimetic platforms. In this study, membrane fragments from cells expressing SNAP-Tag-EGFR (ST-EGFR) were immobilized on the surface of magnetic NPs. The ST-EGFR magnetic cell membrane nanoparticles (ST-EGFR/MCMNs) showed greater stability, and higher binding capacity, selectivity adsorption of gefitinib after 7 days compared to the un-immobilized magnetic cell membrane nanoparticles (EGFR/MCMNs). The ST-EGFR/MCMNs were used to screen for the EGFR-targeting active compounds of Zanthoxyli Radix (ZR), and identified toddalolactone and nitidine chloride. The latter significantly inhibited the proliferation of EGFR-overexpressing cancer cells, and was more effective compared to gefitinib. This innovative technology can be used to rapidly screen for active compounds from complex extracts, and aid in drug discovery.

Hydrogen Bond-Enhanced Nanoaggregation and Antisolvatochromic Fluorescence for Protein-Recognition by Si-Coumarins.

Silicon-substituted coumarin (SiC) was established as a substantial family of both intramolecular and intermolecular hydrogen bond (H-bond) enhanced fluorescent probes for sensitively tracking proteins in vivo through the assemble and disassemble of its nanoaggregates. The intramolecular H-bond in SiC has led to significant aggregation, antisolvatochromism, and strong fluorescence with bathochromically shifted spectra into far-red or near-infrared (NIR) regions in polar, protic environments. Without further furnishing with organic linkers, the compact skeleton of SiC bearing H-bond has ensured sensitively and selectively sensing the targeting proteins with the protic reaction pockets through efficient disassemble of the aggregates. In the existence of strong intermolecular H-bonds with the target protein pocket, SiC resolved as high as >250-fold fluorescence enhancement. Selectively tracking proteins, including human serum albumin, human carbonic anhydrase (hCAII), avidin, SNAP-tag protein, and translocator protein, has confirmed SiC a versatile skeleton for sensitively monitoring proteins in complicated biological systems.

Photo-SNAP-tag, a Light-Regulated Chemical Labeling System

Methods that allow labeling and tracking of proteins have been instrumental for understanding their function. Traditional methods for labeling proteins include fusion to fluorescent proteins or self-labeling chemical tagging systems such as SNAP-tag or Halo-tag. These latter approaches allow bright fluorophores or other chemical moieties to be attached to a protein of interest through a small fusion tag. In this work, we sought to improve the versatility of self-labeling chemical-tagging systems by regulating their activity with light. We used light-inducible dimerizers to reconstitute a split SNAP-tag (modified human O6-alkylguanine-DNA-alkyltransferase, hAGT) protein, allowing tight light-dependent control of chemical labeling. In addition, we generated a small split SNAP-tag fragment that can efficiently self-assemble with its complement fragment, allowing high labeling efficacy with a small tag. We envision these tools will extend the versatility and utility of the SNAP-tag chemical system for protein labeling applications.

Modulation of emission and singlet oxygen photosensitisation in live cells utilising bioorthogonal phosphorogenic probes and protein tag technology.

Herein we report a strategy to utilise the bioorthogonal reactivity and phosphorogenic property of iridium(iii) polypyridine nitrone complexes and SNAP-tag protein for the modulation of emission and singlet oxygen (1O2) photosensitisation in live cells.

Fluorogenic Protein Labeling Probes to Study the Morphological Interplay between PreLamin and Mature Lamin

Although many protein labeling probes have been developed to elucidate the trafficking and turnover processes of cell surface proteins, real-time tracking of intracellular proteins remains a challenging task. Herein, we describe a new design to construct a cell-permeable, photostable, and far-red fluorescent turn-on probe to enable no-wash, organelle-specific, and long-term visualization of intracellular SNAP-tagged proteins in living cells. When the probe was used in dual-color pulse chase labeling experiments to differentiate between preLamin and mature Lamin, our results reveal that the shape of mature Lamin can be altered by the newly synthesized preLamin and that this alteration is progressive, cumulative, and due to a concentration-dependent dominant-negative effect of preLamin. We believe that this probe can also be applied to other intracellular proteins whose cellular localization and synthesis changes dynamically in response to external stimuli.

Two-Color 810 nm STED Nanoscopy of Living Cells with Endogenous SNAP-Tagged Fusion Proteins.

A 810 nm STED nanoscopy setup and an appropriate combination of two fluorescent dyes (Si-rhodamine 680SiR and carbopyronine 610CP) have been developed for near-IR live-cell super-resolution imaging. Vimentin endogenously tagged using the CRISPR/Cas9 approach with the SNAP tag, together with a noncovalent tubulin label, provided reliable and cell-to-cell reproducible dual-color confocal and STED imaging of the cytoskeleton in living cells.

Covalent Protein Labeling and Improved Single-Molecule Optical Properties of Aqueous CdSe/CdS Quantum Dots.

Semiconductor quantum dots (QDs) have proven to be superior probes for single-molecule imaging compared to organic or genetically encoded fluorophores, but they are limited by difficulties in protein targeting, their larger size, and on-off blinking. Here, we report compact aqueous CdSe/CdS QDs with significantly improved bioconjugation efficiency and superior single-molecule optical properties. We have synthesized covalent protein labeling ligands (i.e., SNAP tags) that are optimized for nanoparticle use, and QDs functionalized with these ligands label SNAP-tagged proteins ∼10-fold more efficiently than existing SNAP ligands. Single-molecule analysis of these QDs shows 99% of time spent in the fluorescent on-state, ∼4-fold higher quantum efficiency than standard CdSe/ZnS QDs, and 350 million photons detected before photobleaching. Bright signals of these QDs enable us to track the stepping movement of a kinesin motor in vitro, and the improved labeling efficiency enables tracking of single kinesins in live cells.

Binding characteristics of galectin-3 fusion proteins.

Galectin-3 modulates cell adhesion and signaling events by specific binding and cross-linking galactoside containing carbohydrate ligands. Proteolytic cleavage by metalloproteinases yields in vivo N-terminally truncated galectin-3 still bearing the carbohydrate recognition domain. Truncated galectin-3 has been demonstrated to act in vivo as a negative inhibitor of galectin-3 due to higher affinity for carbohydrate ligands. We here present our studies on a series of 12 human galectin-3 protein constructs. Truncated galectin-3 (∆1-62 and ∆1-116) and fusions with SNAP-tag and/or yellow fluorescent protein (YFP) display altered binding efficiencies (ratio of maximum binding signal and apparent affinity constant Kd) to asialofetuin (ASF) in solid-phase enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) binding assays. Galectin-3(Δ1-62) and full-length (native) galectin-3 have highest affinity to ASF in ELISA and SPR experiments, respectively, whereas galectin-3(Δ1-116) shows only weak binding. We demonstrate here for the first time that SNAP-tag and YFP fusions of galectin-3 and truncated galectin-3 proteins improve binding efficiencies to ASF. SNAP-tagged galectin-3, galectin-3(Δ1-62) and galectin-3(Δ1-116) are found with significant (3- to 6-fold) higher binding efficiencies in SPR when compared with native galectin-3. Fusion of truncated galectin-3 with YFP renders binding properties similar to native galectin-3, whereas in combination with SNAP-tag improved binding characteristics are obtained. Our results emphasize the importance of the N-terminal domain of human galectin-3 for ligand binding. Most importantly, in combination with fusion proteins suitable for the design of diagnostic and therapeutic tools binding properties can be beneficially tuned. The resulting novel protein tools may be advantageous for potential galectin-3 directed applications in tumor diagnostics and therapy.

Development and applications of a near-infrared dye-benzylguanine conjugate to specifically label SNAP-tagged proteins.

Near-infrared (NIR) fluorescent probes are advantageous over visible ones, for they can avoid the interference from the short-wavelength background emission in biological systems. However, there are a very limited number of NIR probes that can specifically label target proteins in living cells. In this work, a series of long-wavelength dyes (N-NIR, S-NIR, and K-NIR) analogous to the novel Changsha NIR family are synthesized conveniently through a new approach that is different from the previously reported one. These three dyes have similar conjugation structures but exhibit tunable photophysical properties. N-NIR and S-NIR have large extinction coefficients over 100 000, and high fluorescence quantum yields. Although NIR absorption and emission of K-NIR are inferior to the former two, it emits in a much longer wavelength region. And all the three dyes can easily pass through the cell membranes to obtain the high-resolution NIR fluorescence images. Furthermore, N-NIR is chosen as the NIR fluorophore to develop a protein-labeling reagent PYBG-D, since it demonstrates the highest fluorescence quantum yield of up to 0.4 (in methanol). PYBG-D is efficiently synthesized through Sonogashira coupling between bromo-substituted N-NIR and alkyne-substituted benzylguanine (PYBG). The conjugate PYBG-D proves to be a specific and efficient label for O6-alkylguanine-DNA alkyltransferase (SNAP-tag) that fused to target proteins in living cells, which contributes to high resolution NIR fluorescence images under a laser confocal microscope.

Specifically and wash-free labeling of SNAP-tag fused proteins with a hybrid sensor to monitor local micro-viscosity

Viscosity, as one of the major factors of intracellular microenvironment, influences the function of proteins. To detect local micro-viscosity of a protein, it is a precondition to apply a viscosity sensor for specifically target to proteins. However, all the reported small-molecule probes are just suitable for sensing/imaging of macro-viscosity in biological fluids of entire cells or organelles. To this end, we developed a hybrid sensor BDP-V BG by connecting a viscosity-sensitive boron-dipyrromethene (BODIPY) molecular rotor (BDP-V) to O6-benzylguanine (BG) for specific detection of local micro-viscosity of SNAP-tag fused proteins. We measured and calculated the reaction efficiency between the sensor and SNAP-tag protein in vitro to confirm the high labeling specificity. We also found that the labeling reaction results in a 53-fold fluorescence enhancement for the rotor, which qualifies it as a wash-free sensor with ignorable background fluorescence. The high sensitivity of protein labeled sensor (BDP-V-SNAP) to the changes of local viscosity was evaluated by detecting the enhancement of fluorescence lifetimes. Further, with the sensor BDP-V BG, we achieved high specific labeling of cells expressing two SNAP-tag fused proteins (nuclear histone H2B and mitochondrial COX8A). Two-photon excited fluorescence lifetime imaging revealed that, the micro-viscosities nearby the SNAP-tag fused two proteins are distinct. The different changes of local micro-viscosity of SNAP-tag fused histone protein in apoptosis induced by three nucleus-targeted drugs were also characterized for the first time.

Fluorescent Probe Encapsulated in SNAP-Tag Protein Cavity To Eliminate Nonspecific Fluorescence and Increase Detection Sensitivity.

Despite the promising improvements made recently on fluorescence probes for the detection of enzymes and reactive small molecules, two fundamental problems remain: weaker fluorescence of many dyes in aqueous buffers and strong nonspecific signals in samples containing high protein levels. In this paper, we introduce a novel fluorescent probe encapsulated in protein cavity (FPEPC) concept as demonstrated by SNAP-tag protein and three environment-sensitive fluorescence probes to overcome these two problems. The probes were constructed by following the current probe design for enzymes and reactive small molecules but with an additional benzylguanine moiety for selective SNAP-tag conjugation. The SNAP-tag conjugated probes achieved quantitative nitroreductase and hydrogen sulfide detection in blood plasma, whereas analyte concentrations were overestimated up to 700-fold when bare fluorescent probes were employed for detection. Furthermore, detection sensitivity was increased dramatically, as our probes displayed 390-fold fluorescence enhancement upon SNAP-tag conjugation, in stark contrast to the weak fluorescence of the free probes in aqueous solutions. Compared with the conventional approaches where fluorescent probes are encapsulated into polymers and nanoparticles, our simple and general approach successfully overcame many key issues such as dye leakage, long preparation steps, inconsistent dye-host ratios, difficulty in constructing in situ in a complex medium, and limited application to detect only small metabolites.

A Monoclonal Antibody That Discriminates Between SNAP-Tagged and CLIP-Tagged Proteins.

SNAP-tag technology allows recombinant proteins to be covalently labeled to O(6)-benzylguanine (BG)-modified substrates with 1:1 stoichiometry. By attaching according fluorophores, this method is ideally suited for in vitro and in vivo imaging, as well as protein interaction analyses. Fluorophores modified with BG react with the SNAP-tag, whereas those modified with O(2)-benzylcytosine (BC) conjugate to a more recent derivative known as the CLIP-tag. The orthogonal substrate specificity of the SNAP- and CLIP-tags extends the range of applications by allowing double labeling. We previously developed a monoclonal antibody (mAb) that recognizes both tags. In this study, we describe a new mAb, which is specific for the SNAP-tag alone. Therefore, this mAb allows discrimination between SNAP- and CLIP-tags within a broad range of immunological methods, including enzyme-linked immunosorbent assays, western blotting, flow cytometry, and immunohistochemistry.

Evaluation of Fluorophores to Label SNAP-Tag Fused Proteins for Multicolor Single-Molecule Tracking Microscopy in Live Cells

Single-molecule tracking has become a widely used technique for studying protein dynamics and their organization in the complex environment of the cell. In particular, the spatiotemporal distribution of membrane receptors is an active field of study due to its putative role in the regulation of signal transduction. The SNAP-tag is an intrinsically monovalent and highly specific genetic tag for attaching a fluorescent label to a protein of interest. Little information is currently available on the choice of optimal fluorescent dyes for single-molecule microscopy utilizing the SNAP-tag labeling system. We surveyed 6 green and 16 red excitable dyes for their suitability in single-molecule microscopy of SNAP-tag fusion proteins in live cells. We determined the nonspecific binding levels and photostability of these dye conjugates when bound to a SNAP-tag fused membrane protein in live cells. We found that only a limited subset of the dyes tested is suitable for single-molecule tracking microscopy. The results show that a careful choice of the dye to conjugate to the SNAP-substrate to label SNAP-tag fusion proteins is very important, as many dyes suffer from either rapid photobleaching or high nonspecific staining. These characteristics appear to be unpredictable, which motivated the need to perform the systematic survey presented here. We have developed a protocol for evaluating the best dyes, and for the conditions that we evaluated, we find that Dy 549 and CF 640 are the best choices tested for single-molecule tracking. Using an optimal dye pair, we also demonstrate the possibility of dual-color single-molecule imaging of SNAP-tag fusion proteins. This survey provides an overview of the photophysical and imaging properties of a range of SNAP-tag fluorescent substrates, enabling the selection of optimal dyes and conditions for single-molecule imaging of SNAP-tagged fusion proteins in eukaryotic cell lines.

Visualization of phagosomal hydrogen peroxide production by a novel fluorescent probe that is localized via SNAP-tag labeling.

Hydrogen peroxide (H2O2), a member of reactive oxygen species (ROS), plays diverse physiological roles including host defense and cellular signal transduction. During ingestion of invading microorganisms, professional phagocytes such as macrophages release H2O2 specifically into the phagosome to direct toxic ROS toward engulfed microbes. Although H2O2 is considered to exert discrete effects in living systems depending on location of its production, accumulation, and consumption, there have been limitations of techniques for probing this oxygen metabolite with high molecular specificity at the subcellular resolution. Here we describe the development of an O(6)-benzylguanine derivative of 5-(4-nitrobenzoyl)carbonylfluorescein (NBzF-BG), a novel H2O2-specific fluorescent probe; NBzF-BG is covalently and selectively conjugated with the SNAP-tag protein, leading to formation of the fluorophore-protein conjugate (SNAP-NBzF). SNAP-NBzF rapidly reacts with H2O2 and thereby shows a 9-fold enhancement in fluorescence. When SNAP-tag is expressed in HEK293T cells and RAW264.7 macrophages as a protein C-terminally fused to the transmembrane domain of platelet-derived growth factor receptor (PDGFR), the tag is presented on the outside of the plasma membrane; conjugation of NBzF-BG with the cell surface SNAP-tag enables detection of H2O2 added exogenously. We also demonstrate molecular imaging of H2O2 that is endogenously produced in phagosomes of macrophages ingesting IgG-coated latex beads. Thus, NBzF-BG, combined with the SNAP-tag technology, should be useful as a tool to measure local production of H2O2 in living cells.

Fluorescence amplified detection of proteases by the catalytic activation of a semisynthetic sensor

A general approach to mimic the sensing scheme of allosteric enzymes is developed. Through the covalent labeling of a sulfonamide inhibitor to the enzyme HCAII via SNAP-tag protein, the enzyme is rendered inactive. Catalytic activation is triggered only when a protease is present to cleave the recognized peptide sequence.

Development of a split SNAP-tag protein complementation assay for visualization of protein–protein interactions in living cells

A split SNAP-tag protein complementation assay was developed for visualization of protein-protein interactions in living cells. Split SNAP-tagμs, fragments of divided SNAP-tag between amino acid residues 91 and 92, were fused to proteins that can interact with each other. After incubation with a fluorescent SNAP-tag substrate, cells that expressed split SNAP-tag fusion proteins generated fluorescent signals when these proteins interacted. Moreover, by combination with the split CLIP-tag (SNAP-tag mutant), simultaneous labeling was achieved. This split SNAP-tag labeling method should be a useful tool for visualization of protein-protein interaction processes.

Development of SNAP‐Tag Fluorogenic Probes for Wash‐Free Fluorescence Imaging

The ability to specifically attach chemical probes to individual proteins represents a powerful approach to the study and manipulation of protein function in living cells. It provides a simple, robust and versatile approach to the imaging of fusion proteins in a wide range of experimental settings. However, a potential drawback of detection using chemical probes is the fluorescence background from unreacted or nonspecifically bound probes. In this report we present the design and application of novel fluorogenic probes for labeling SNAP-tag fusion proteins in living cells. SNAP-tag is an engineered variant of the human repair protein O6-alkylguanine-DNA alkyltransferase (hAGT) that covalently reacts with benzylguanine derivatives. Reporter groups attached to the benzyl moiety become covalently attached to the SNAP tag while the guanine acts as a leaving group. Incorporation of a quencher on the guanine group ensures that the benzylguanine probe becomes highly fluorescent only upon labeling of the SNAP-tag protein. We describe the use of intramolecularly quenched probes for wash-free labeling of cell surface-localized epidermal growth factor receptor (EGFR) fused to SNAP-tag and for direct quantification of SNAP-tagged β-tubulin in cell lysates. In addition, we have characterized a fast-labeling variant of SNAP-tag, termed SNAPf, which displays up to a tenfold increase in its reactivity towards benzylguanine substrates. The presented data demonstrate that the combination of SNAPf and the fluorogenic substrates greatly reduces the background fluorescence for labeling and imaging applications. This approach enables highly sensitive spatiotemporal investigation of protein dynamics in living cells.