Fluorescence labelling of an intracellular biomolecule in native living cells is a powerful strategy to achieve in-depth understanding of the biomolecule's roles and functions. Besides being nontoxic and specific, desirable labelling probes should be highly cell permeable without nonspecific interactions with other cellular components to warrant high signal-to-noise ratio. While it is critical, rational design for such probes is tricky. Here we report the first predictive model for cell permeable background-free probe development through optimized lipophilicity, water solubility and charged van der Waals surface area. The model was developed by utilizing high-throughput screening in combination with cheminformatics. We demonstrate its reliability by developing CO-1 and AzG-1, a cyclooctyne- and azide-containing BODIPY probe, respectively, which specifically label intracellular target organelles and engineered proteins with minimum background. The results provide an efficient strategy for development of background-free probes, referred to as ‘tame' probes, and novel tools for live cell intracellular imaging.
Advances in the design strategy of cell-permeable small fluorescent probes are discussed. Their applications in imaging specific cell types and intracellular bioanalytes, as well as the cellular environment in live conditions, are presented.
Cell labelling using a small fluorescent probe is an important technique in biomedical sciences. We previously developed a biocompatible and membrane‐permeable probe, CO‐1, which has low nonspecific binding affinity towards nontarget molecules. Although this background‐free tame probe has been utilized for labelling of various intracellular biomolecules in live cells, the probes’ backgroung‐free staining mechanism was not fully understood. Here, we propose that Gating‐Oriented Live‐cell Distinction (GOLD) mechanism occurs when ABCB1 transporter removes unbound CO‐1 molecules from mammalian cells and, in a minor role, DIRC2 pumps CO‐1 out from lysosomes. We also showed that solute carrier transporters were not involved in carrying CO‐1 inside of cells. The role of reporters in assisting the probes’ influx‐efflux was analyzed by the combination of CRISPR library sceenings and inhibitors test. In summary, tame probe CO‐1 cellular staining occurs in a dual mechanism where the probe moves freely through the cells membrane, but its washable property can be directly related to the action of ABCB1 transporter.
Nucleic acid aptamers, also regarded as chemical antibodies, manifest potentials for targeted therapeutic and delivery agents since they possess unique advantages over antibodies. Generated by an iterative in vitro selection...
Since the term “bioorthogonal” was first demonstrated in 2003, new tools for bioorthogonal chemistry have been rapidly developed. Bioorthogonal chemistry has now been widely utilized for applications in imaging various biomolecules, such as proteins, glycoconjugates, nucleic acids, and lipids. Contrasting the chemical reactions or synthesis that are typically executed in vitro with organic solvents, bioorthogonal reactions can occur inside cells under physiological conditions. Functional groups or chemical reporters for bioorthogonal chemistry are highly selective and will not perturb the native functions of biological systems. Advances in azide-based bioorthogonal chemical reporters make it possible to perform chemical reactions in living systems for wide-ranging applications. This review discusses the milestones of azide-based bioorthogonal reactions, from Staudinger ligation and copper(I)-catalyzed azide-alkyne cycloaddition to strain-promoted azide-alkyne cycloaddition. The development of bioorthogonal reporters and their capability of being built into biomolecules in vivo have been extensively applied in cellular imaging. We focus on strategies used for metabolic incorporation of chemically tagged molecular building blocks (e.g., amino acids, carbohydrates, nucleotides, and lipids) into cells via cellular machinery systems. With the aid of exogenous bioorthogonally compatible small fluorescent probes, we can selectively visualize intracellular architectures, such as protein, glycans, nucleic acids, and lipids, with high specificity to help in answering complex biological problems.
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