title: Near-infrared fluorescence imaging of mammalian cells and xenograft tumors with SNAP-tag.
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.
authors.affiliation: LI-COR Biosciences, Lincoln, Nebraska, United States of America. herbert.gong@licor.com
authors.forename: Haibiao
authors.initials: H
authors.lastname: Gong
authors.forename: Joy L
authors.initials: JL
authors.lastname: Kovar
authors.forename: Brenda
authors.initials: B
authors.lastname: Baker
authors.forename: Aihua
authors.initials: A
authors.lastname: Zhang
authors.forename: Lael
authors.initials: L
authors.lastname: Cheung
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Draney
authors.forename: Ivan R
authors.initials: IR
authors.lastname: Corrêa
authors.forename: Ming-Qun
authors.initials: MQ
authors.lastname: Xu
authors.forename: D Michael
authors.initials: DM
authors.lastname: Olive
mesh_terms: D000818:Animals; D002460:Cell Line; D015254:DNA Modification Methylases / Q000737:chemistry / Q000235:genetics; D045643:DNA Repair Enzymes / Q000737:chemistry / Q000235:genetics; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D057054:Molecular Imaging / Q000379:methods*; D009369:Neoplasms / Q000378:metabolism*; D018343:Receptors, Adrenergic, beta-2 / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D059629:Signal-To-Noise Ratio; D019265:Spectroscopy, Near-Infrared / Q000379:methods*; D013194:Staining and Labeling; D014162:Transfection; D014183:Transplantation, Heterologous; D025521:Tumor Suppressor Proteins / Q000737:chemistry / Q000235:genetics
chemical_list: D005456:Fluorescent Dyes; D018343:Receptors, Adrenergic, beta-2; D011993:Recombinant Fusion Proteins; D025521:Tumor Suppressor Proteins; D015254:DNA Modification Methylases; C515491:MGMT protein, human; D045643:DNA Repair Enzymes