Near-Infrared Fluorescence Imaging of Mammalian Cells and Xenograft Tumors with SNAP-Tag Haibiao Gong1*, Joy L. Kovar1, Brenda Baker2, Aihua Zhang2, Lael Cheung 1 , Daniel R. Draney1, Ivan R. Correˆ a, Jr.2, Ming-Qun Xu 2 , D. Michael Olive1 1 LI-COR Biosciences, Lincoln, Nebraska, United States of America, 2 New England Biolabs, Ipswich, Massachusetts, United States of America 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. SNAPf 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 SNAPf. This property makes SNAPf 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 SNAPf-Beta-2 adrenergic receptor (SNAPf-ADRb2) fusion protein were created. The ADRb2 portion of the protein directs the localization of the protein to the cell membrane. The expression of SNAPf-ADRb2 in the stable cell lines was confirmed by the reaction between BG-800 substrate and cell lysates. Microscopic examination confirmed that SNAPf-ADRb2 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 SNAPf-ADRb2. 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 SNAPf-ADRb2 expressing tumor, possibly due to the reduced reaction rate. Further improvement is needed to apply this system for in vivo imaging. Citation: Gong H, Kovar JL, Baker B, Zhang A, Cheung L, et al. (2012) Near-Infrared Fluorescence Imaging of Mammalian Cells and Xenograft Tumors with SNAP- Tag. PLoS ONE 7(3): e34003. doi:10.1371/journal.pone.0034003 Editor: Juri G. Gelovani, University of Texas, M.D. Anderson Cancer Center, United States of America Received November 9, 2011; Accepted February 22, 2012; Published March 30, 2012 Copyright: 2012 Gong et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors have no support or funding to report. Competing Interests: The authors are employees at LI-COR Biosciences (HG, JLK, LC, DRD and DMO) and New England Biolabs (BB, AZ, IRC and M-QX). This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials. IRDye(r) 800CW and SNAP(r)-tag are proprietary materials of LI-COR Biosciences and New England Biolabs, respectively. * E-mail: herbert.gong@licor.com Introduction Fluorescence has been extensively used in biological research to visualize molecular and cellular events. Its application ranges from visualizing targeting molecules in single cells to imaging physio- logical and pathological alterations in whole animals [1,2]. Its high sensitivity and stability, and simplicity of multiplexing offer advantages over other imaging methods in many applications. The most commonly used fluorophores include organic dyes, fluorescent proteins and quantum dots [1]. Each class of fluorophores has its own advantages and limitations. For example, fluorescent proteins can be easily expressed in cells and whole organisms. On the other hand, fluorescent organic dyes are more suitable for conjugation to other molecules, such as nucleic acids and proteins. It is of great interest to develop fluorophores with excitation (Ex) and emission (Em) maxima in the near-infrared (NIR) region (700–900 nm). With fluorescence in the NIR region, cells, buffers and plastic materials used in assays have reduced background. As a result, NIR fluorescence imaging offers higher sensitivity and better signal-to-background (S/B) ratio compared to visible spectra. More importantly, due to the reduced light absorption and scattering of NIR light in animal tissues, and the low tissue autofluorescence in the NIR region, NIR fluorescence is well- suited for in vivo animal imaging [2,3,4]. Significant efforts have been made to shift the spectra of the fluorescent proteins to longer wavelengths [5,6,7,8]. The most red-shifted fluorescent proteins are bacteriophytochrome-based near-infrared fluorescent proteins IFP1.4 [8] and iRFP [9]. However, the Ex/Em peaks of IFP1.4 (Ex/Em: 684/708 nm) and iRFP (Ex/Em: 690/713 nm) are still significantly lower compared to those of NIR fluorescent dyes such as IRDye 800CW (Ex/Em: 774/789 nm). 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. The fusion of SNAPf to a protein of interest yields a tagged protein capable of forming a covalent linkage to fluorescent dyes [11,12]. The NIR fluorescent dye IRDye 800CW has been conjugated to a variety of molecules for different applications. Examples include labeled antibodies for Western, In-Cell-Western, and labeled 2-deoxyglucose, RGD peptide and target-specific peptides for animal imaging [13,14,15]. An epidermal growth factor receptor (EGFR)-specific Affibody molecule labeled with IRDye 800CW has been successfully used in cell-based plate assays, PLoS ONE | www.plosone.org 1 March 2012 | Volume 7 | Issue 3 | e34003