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. 2024 May 1;15(21):8097-8105.
doi: 10.1039/d3sc06957e. eCollection 2024 May 29.

Bioisostere-conjugated fluorescent probes for live-cell protein imaging without non-specific organelle accumulation

Takuya Kamikawa  1 , Akari Hashimoto  2 , Nozomi Yamazaki  2 , Junya Adachi  3 , Ayami Matsushima  3 , Kazuya Kikuchi  2   4 , Yuichiro Hori  3
Affiliations

Bioisostere-conjugated fluorescent probes for live-cell protein imaging without non-specific organelle accumulation

Takuya Kamikawa et al. Chem Sci. .

Abstract

Specific labeling of proteins using membrane-permeable fluorescent probes is a powerful technique for bioimaging. Cationic fluorescent dyes with high fluorescence quantum yield, photostability, and water solubility provide highly useful scaffolds for protein-labeling probes. However, cationic probes generally show undesired accumulation in organelles, which causes a false-positive signal in localization analysis. Herein, we report a design strategy for probes that suppress undesired organelle accumulation using a bioisostere for intracellular protein imaging in living cells. Our design allows the protein labeling probes to possess both membrane permeability and suppress non-specific accumulation and has been shown to use several protein labeling systems, such as PYP-tag and Halo tag systems. We further developed a fluorogenic PYP-tag labeling probe for intracellular proteins and used it to visualize multiple localizations of target proteins in the intracellular system. Our strategy offers a versatile design for undesired accumulation-suppressed probes with cationic dye scaffolds and provides a valuable tool for intracellular protein imaging.

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Conflict of interest statement

There are no conflicts to declare.

Figures

Fig. 1
Fig. 1. Schematic illustration of probe design strategy for intracellular protein imaging using various cationic scaffolds and protein tag labeling system. (a) Cationic probes for protein imaging are cell permeable but show organelle accumulation. (b) Zwitterionic probes with tetrazole as an organelle accumulation suppression group show cell permeability and low background. POI denotes protein of interest.
Fig. 2
Fig. 2. (a) Molecular structures of PYP labeling probes (PCAF-RB, PCAF-RBis, PCAF-SP, PCAF-SPis, and PCAF-SP-s). (b and c) The result of CBB staining and fluorescence images of the SDS-PAGE analysis. (b) PYPWT (2.0 μM) and PYPNQN (2.0 μM) incubated with PCAF-RB (1.0 μM) or PCAF-RBis (1.0 μM) for 30 min. PYPWT (2.0 μM) and PYPNQN (2.0 μM) incubated with PCAF-SP (1.0 μM), PCAF-SPis (1.0 μM), or PCAF-SP-s (1.0 μM) for 20 min. The SDS analysis images were obtained with the excitation at 470 nm. CBB and FL denote Coomassie brilliant blue staining and fluorescence images, respectively. Experiments were performed twice with similar results. (c) Normalized fluorescence spectra of PYP-tag labeling probes (PCAF-SP, PCAF-SPis, and PCAF-SP-s) reacted with/without PYPWT. PCAF-SP, PCAF-SPis, and PCAF-SP-s (5.0 μM) were incubated with/without PYPWT (6.0 μM) for 30 min in the solution of 20 mM HEPES, 150 mM NaCl, and 0.1% DMSO buffered to pH 7.4 at 37 °C. All spectra were obtained with the excitation at 484 nm.
Fig. 3
Fig. 3. Fluorescence and phase contrast-merged images of HEK293T cells expressing HA-PYPNQN-NLS or mock cells (transfected with empty vector) stained with (a) PCAF-RBis (5.0 μM) and (b) PCAF-RB (5.0 μM). The images were measured at 566–685 nm emission with the excitation at 561 nm. (c) HA-PYPWT-NLS or mock cells stained with (c) PCAF-SPis (1.25 μM) or (d) PCAF-SP (1.25 μM). The images were measured at 550–650 nm emission with the excitation at 488 nm. Experiments were performed twice with similar results. Scale bars, 10 μm. FL denotes fluorescence image.
Fig. 4
Fig. 4. (a) Schematic view of GLUT4 multiple localization imaging with PCAF-SP-s and PCAF-RBis. (b and c) Fluorescence and phase contrast-merged images of HeLa cells stably expressing PYPWT-GLUT4 or HeLa cells (non-expressed PYP-tag) stained with (b) PCAF-SP-s (1.5 μM) in the presence of insulin or (c) PCAF-RB (1.25 μM) in the absence of insulin. (d) Multicolor imaging of HeLa cells stably expressing PYPWT-GLUT4 or HeLa cells (non-expressed PYP-tag) stained with PCAF-RBis (1.25 μM) and PCAF-SP-s (1.5 μM) for visualization of multiple localizations of GLUT4. All images were obtained with the excitation at 488 and 561 nm and detection at 500–630 (green) and 635–705 (red) nm emission, respectively. Scale bars, 10 μm. FL and PC denote fluorescence image and phase contrast image, respectively. FL merge denotes FL red image merged with FL green image. Experiments were performed twice with similar results.
Fig. 5
Fig. 5. (a) Molecular structures of Halo tag labeling probe with or without tetrazole. (b and c) Fluorescence and phase contrast-merged images of HEK293T cells expressing HA-Halo-NLS or mock cells (transfected empty vector) stained with (b) HTL-RBis (2.5 μM) and (c) HTL-RB (2.5 μM). The images were measured with 566–685 nm emission and excitation at 561 nm. Scale bars, 10 μm. FL denotes Fluorescence image. Experiments were performed twice with similar results.
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References

    1. Griffin B. A. Adams S. R. Tsien R. Y. Science. 1998;281:269–272. doi: 10.1126/science.281.5374.269. - DOI - PubMed
    1. Adams S. R. Campbell R. E. Gross L. A. Martin B. R. Walkup G. K. Yao Y. Llopis J. Tsien R. Y. J. Am. Chem. Soc. 2002;124:6063–6076. doi: 10.1021/ja017687n. - DOI - PubMed
    1. Chena X. Wu Y. W. Org. Biomol. Chem. 2016;14:5417–5439. doi: 10.1039/C6OB00126B. - DOI - PubMed
    1. Dean K. M. Palmer A. E. Nat. Chem. Biol. 2014;10:512–523. doi: 10.1038/nchembio.1556. - DOI - PMC - PubMed
    1. Tsien R. Y. Annu. Rev. Biochem. 1998;67:509–544. doi: 10.1146/annurev.biochem.67.1.509. - DOI - PubMed