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

Abstract

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.

The periciliary ring in polarized epithelial cells is a hot spot for delivery of the apical protein gp135

“…The tag binds covalently to O 6 -benzylguanine (BG), resulting in the irreversible transfer of the substituted benzyl group to the reactive thiol within the SNAP tag. BG derivatives bearing fluorophores, biotin, or other groups can be used to label a SNAP-tagged protein (Farr et al, 2009;Maurel et al, 2010;Sun et al, 2011;Lukinavičius et al, 2013).…”

Analysis of Protein Turnover by Quantitative SNAP‐Based Pulse‐Chase Imaging

“…Labeling and washing steps require approximately 1 hour rendering this method inappropriate to assess protein dynamics at short timescales (seconds to minutes), as pulse labeled proteins will have reached their steady state equilibrium before imaging can determine their dynamics. However, improvements are currently being made to both the SNAP-enzyme and the fluorescent substrates thereof, which would in principle allow labeling steps of 5 minutes without the need for any washes (see below and Sun et al, 2011).…”

“…These so called 'dark-dyes' are either quenched by guanine itself (Stöhr et al, 2010), or by a side-group fused the guanine moiety of benzylguanine (Komatsu et al, 2011;Sun et al, 2011). These dark-dyes provide a number of advantages over traditional fluorescent SNAP-substrates, most importantly leading to highly reduced (unspecific) background fluorescence.…”

“…Tagging of two different proteins by SNAP and CLIP allows for simultaneous labeling of two different proteins in different colors (Gautier et al, 2008;Prendergast et al, 2011). More recently, variants of SNAP and CLIP named SNAPf and CLIPf have been developed that present faster reaction kinetics (Pellett et al, 2011;Sun et al, 2011). We evaluated SNAPf and CLIPf performance in vivo by sideby-side comparison with SNAP and CLIP, using the intracellular protein CENP-A as a labeling target (data not shown and Figure 7A).…”

Fluorescent labeling and modification of proteins

“…Protein tags such as Halo [38], SNAP [58], and CLIP [22] are self-labeling enzymes that covalently link to fluorescently labeled substrates. These tags are highly specific and many commercial kits are available making the process relatively easy.…”