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

New England Biolabs, Inc. 240 County Road, Ipswich, MA 01938, USA.
ChemBioChem (Impact Factor: 3.09). 09/2011; 12(14):2217-26. DOI: 10.1002/cbic.201100173
Source: PubMed


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 O(6)-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 SNAP(f), which displays up to a tenfold increase in its reactivity towards benzylguanine substrates. The presented data demonstrate that the combination of SNAP(f) 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.

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Available from: Luo Sun
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    • "We then compared the labeling dynamics between the FKBP-AP21967 and SNAP-tag approaches in living cells. As shown in Fig. 1F, the t 1/2 (time required to label half of the cell surface proteins) was much smaller for the FKBP- AP21967 method (13 s) than for the SNAP-Surface 488 method (72 s), which is consistent with a previously published report (Sun et al., 2011). These results indicate that labeling with the FKBP-AP21967 method is much more rapid than SNAP-tag labeling and that the fluorescence background of the FKBP-AP21967 method is extremely low. "

    Full-text · Article · Aug 2014 · Protein & Cell
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    • "To get around this problem, one interesting alternative consists in using a substrate conjugated to both a fluorophore and a quencher. The probe becomes highly fluorescent only upon reacting with the SLP (Sun et al., 2011). "
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    ABSTRACT: The concept of oligomerization of G protein-coupled receptor (GPCR) opens new perspectives regarding physiological function regulation. The capacity of one GPCR to modify its binding and coupling properties by interacting with a second one can be at the origin of regulations unsuspected two decades ago. Although the concept is interesting, its validation at a physiological level is challenging and probably explains why receptor oligomerization is still controversial. Demonstrating direct interactions between two proteins is not trivial since few techniques present a spatial resolution allowing this precision. Resonance energy transfer (RET) strategies are actually the most convenient ones. During the last two decades, bioluminescent resonance energy transfer and time-resolved fluorescence resonance energy transfer (TR-FRET) have been widely used since they exhibit high signal-to-noise ratio. Most of the experiments based on GPCR labeling have been performed in cell lines and it has been shown that all GPCRs have the propensity to form homo- or hetero-oligomers. However, whether these data can be extrapolated to GPCRs expressed in native tissues and explain receptor functioning in real life, remains an open question. Native tissues impose different constraints since GPCR sequences cannot be modified. Recently, a fluorescent ligand-based GPCR labeling strategy combined to a TR-FRET approach has been successfully used to prove the existence of GPCR oligomerization in native tissues. Although the RET-based strategies are generally quite simple to implement, precautions have to be taken before concluding to the absence or the existence of specific interactions between receptors. For example, one should exclude the possibility of collision of receptors diffusing throughout the membrane leading to a specific FRET signal. The advantages and the limits of different approaches will be reviewed and the consequent perspectives discussed.
    Full-text · Article · Jul 2012 · Frontiers in Endocrinology
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    • "SNAPf is a fast-labeling variant of SNAP-tag, which is derived from the human DNA repair protein O6-alkylguanine-DNA-alkyltransferase (AGT) [10]. It reacts specifically and rapidly with benzylguanine (BG) derivatives, leading to covalent labeling of the SNAPf with a variety of functional moieties, such as fluorescent dyes, biotin and solid surfaces. "
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    ABSTRACT: Fluorescence in the near-infrared (NIR) spectral region is suitable for in vivo imaging due to its reduced background and high penetration capability compared to visible fluorescence. SNAP(f) is a fast-labeling variant of SNAP-tag that reacts with a fluorescent dye-conjugated benzylguanine (BG) substrate, leading to covalent attachment of the fluorescent dye to the SNAP(f). This property makes SNAP(f) a valuable tool for fluorescence imaging. The NIR fluorescent substrate BG-800, a conjugate between BG and IRDye 800CW, was synthesized and characterized in this study. HEK293, MDA-MB-231 and SK-OV-3 cells stably expressing SNAP(f)-Beta-2 adrenergic receptor (SNAP(f)-ADRβ2) fusion protein were created. The ADRβ2 portion of the protein directs the localization of the protein to the cell membrane. The expression of SNAP(f)-ADRβ2 in the stable cell lines was confirmed by the reaction between BG-800 substrate and cell lysates. Microscopic examination confirmed that SNAP(f)-ADRβ2 was localized on the cell membrane. The signal intensity of the labeled cells was dependent on the BG-800 concentration. In vivo imaging study showed that BG-800 could be used to visualize xenograph tumors expressing SNAP(f)-ADRβ2. However, the background signal was relatively high, which may be a reflection of non-specific accumulation of BG-800 in the skin. To address the background issue, quenched substrates that only fluoresce upon reaction with SNAP-tag were synthesized and characterized. Although the fluorescence was successfully quenched, in vivo imaging with the quenched substrate CBG-800-PEG-QC1 failed to visualize the SNAP(f)-ADRβ2 expressing tumor, possibly due to the reduced reaction rate. Further improvement is needed to apply this system for in vivo imaging.
    Full-text · Article · Mar 2012 · PLoS ONE
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