Figure 6. Reaction of BG-800, CBG-800-QC1 and CBG-800-PEG- QC1 substrates with purified SNAPf-EGF protein. (A) BG substrates (100 nM) were incubated with SNAPf-EGF protein (15 ng/ ml) in a 96-well plate. The plate was scanned after different time periods of reaction to measure the fluorescence signal. (B) The reaction mixtures of SNAPf-EGF with different BG substrates were resolved on a SDS-PAGE gel and scanned to visualize the labeled protein. doi:10.1371/journal.pone.0034003.g006 Discussion Fluorescence technology has become an indispensable tool for biological and biomedical research. SNAP-tag fluorescence imaging has been used in various applications, including protein- protein interaction [20], hydrogen peroxide detection [21], monitoring zinc flux [22], virus-cell interactions [23] and super- resolution imaging of live cells [24]. SNAPf used in this study is a fast-labeling variant of SNAP-tag with some extra mutations [10]. As a new technology discovered less than a decade ago [12], the SNAP-tag has some advantages over fluorescent proteins. Firstly, NIR SNAP-tag substrates could be synthesized readily from NIR dyes. As a result, NIR fluorescence imaging is greatly facilitated with SNAP-tag. This is in contrast to the efforts needed to engineer NIR fluorescent proteins. Secondly, a variety of fluorescent substrates with different colors could be used to label one single SNAP-tag without a requirement for separate cloning and expression for each color. Once a stable cell line or transgenic animal is established, the color on the SNAP-tag can be easily altered by using a substrate with different Ex/Em spectra. However, changing to a different color with fluorescent proteins entails laborious processes of re-establishing stable cell lines or transgenic animals de novo. Thirdly, the labeling time with SNAP- tag can be controlled easily, allowing for ‘‘pulse-chase’’ experi- ments that require labeling with different probes at different time points. Although SNAP-tag technology has been widely used in cell imaging, much less work has been done to apply this technology in animal imaging. A fusion protein of SNAP-tag with a single-chain NIR Fluorescence Imaging with SNAP-Tag antibody fragment has enabled targeting EGFR-overexpressing tumors. However, the labeling of SNAP-tag by NIR substrates was conducted in vitro in that study [25]. In a recent report, BG-782 was successfully used to label SNAP-tag in vivo. Tumors expressing SNAP-tag fusion proteins were visualized and the half-lives of SNAP-tag fusion proteins were measured in vivo [26]. A NIR fluorescent SNAP-tag substrate BG-800 was synthesized by conjugating IRDye 800CW to BG-NH2. Because BG-800 is cell impermeable, we choose to use the SNAPf-ADRb2 fusion protein, in which ADRb2 directs the localization of SNAPf fusion protein to the cell membrane. BG-800 reacted with SNAPf- ADRb2 in both cell lysate and live cell culture. It was also found that BG-800 produced a higher signal and S/B ratio compared to BG-782 in cell-based assay. The tumors expressing SNAPf- ADRb2 could be visualized by BG-800. The signal was from the specific reaction between SNAPf-ADRb2 and BG-800, as evidenced by gel analysis of protein exacts from the dissected tumors. However the relatively high signal in the skin, presumably due to the non-specific accumulation of BG-800 in this tissue, produced high background. The accumulation of BG-800 in the skin is unlikely caused by IRDye 800CW because little skin signal has been detected with either free dye or various other IRDye 800CW conjugates, including small organic molecules (such as 2- DG), small peptides (such as RGD), large peptides (such as EGF, Affibody) and antibodies [14,15,27,28]. It is also noteworthy that a HaloTag probe containing IRDye 800CW was used to detect HaloTag expressing tumors. No skin accumulation problem was noted in that study [29]. One possible solution to the background issue is to use quenched substrates, which only fluoresce upon reaction with SNAP-tag. The quenched substrates are desirable in cell-based assay because they could eliminate the wash step which is needed for the conventional unquenched substrates [10,30]. More importantly, the quenched substrate will produce minimal background when used in animal imaging, where the clearance of the substrate from the body is more difficult and much slower compared to the cell culture system. Various quenching mechanisms, such as self-quenching, Fo¨ rster resonance energy transfer (FRET), H-dimer formation and photon-induced electron transfer (PeT), have been employed in fluorescence imaging [31,32]. As guanine is known to quench the fluorescence of certain dyes by PeT, various dyes were tested for their quenching efficiency by guanine after conjugation to BG. Several BG substrates were discovered to have a strong (.10-fold) increase in their fluorescence upon covalent labeling of the SNAP-tag [33]. A more general method is based on the FRET principle. A fluorescent dye and a quencher could be linked to the benzyl moiety and the guanine moiety, respectively. In this substrate, the fluorescence of the dye is quenched by the closely-linked quencher. After reaction with SNAP-tag, the guanine-quencher group will separate from the benzyl-dye group, resulting in the restoration of the fluorescence. The quencher was linked to the C-8 or N-9 positions, and the resulting substrates were characterized. Although the substrates were quenched in both situations, the C-8 modification exhibited better reaction kinetics [30,34]. A variety of quenched substrates with different combinations of fluorescent dyes and quenchers linked at C-8 position have been synthesized and tested [10]. However, none of these quenched substrates has Ex/Em spectra in the NIR region. We synthesized the NIR quenched substrate CBG-800-QC1 by conjugating IRDye 800CW and IRDye QC1 at the benzyl group and C-8 position of guanine group, respectively. The reaction speed of this substrate was greatly reduced compared to the unquenched BG-800. This is not surprising given that C-8 PLoS ONE | www.plosone.org 6 March 2012 | Volume 7 | Issue 3 | e34003