ABCB1 Can Actively Pump-out the Background-Free Tame Fluorescent Probe CO-1 From Live Cells.

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.

Uptake mechanisms of cell-internalizing nucleic acid aptamers for applications as pharmacological agents.

Nucleic acid aptamers, also regarded as chemical antibodies, show potential as targeted therapeutic and delivery agents since they possess unique advantages over antibodies. Generated by an iterative selection and amplification process from oligonucleotide libraries using cultured cells, the aptamers bind to their target molecules expressed on the cell surface. Excitingly, most aptamers also demonstrate a cell-internalizing property in native living cells, allowing them to directly enter the cells via endocytosis depending on the target. In this review, we discuss selection methods in generating cell-internalizing aptamers via a cell-based selection process, along with their challenges and optimization strategies. We highlight the cellular uptake routes adopted by the aptamers and also their intracellular fate after the uptake, to give an overview of their mechanism of action for applications as promising pharmacological agents.

Azide-based bioorthogonal chemistry: Reactions and its advances in cellular and biomolecular imaging.

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.

Advances in the design of cell-permeable fluorescent probes for applications in live cell imaging.

Small fluorescent molecules have been an important tool for fluorescence-based imaging, thanks to their technical simplicity, sensitivity and structural flexibility. However, probing specific intracellular systems with a small fluorescent molecule is not an easy task owing to the intricate nature of the cells. Ideal imaging probes should be highly permeable and be without background noise to allow undisrupted observations. These probes will unquestionably be more advantageous for interrogating intracellular architectures and dynamics without compromising the cellular integrity, and could therefore be applied for applications in the native cellular environment. This review will highlight the advances in design strategies for cell-permeable fluorescent probes through a diversity-oriented fluorescence library approach and rational design via computational-aided predictive models. Here, we discuss a series of cell-permeable probe applications for imaging specific cell types and intracellular biomolecules, as well as the cellular environment.

A palette of background-free tame fluorescent probes for intracellular multi-color labelling in live cells.

A multi-color labelling technique for visualizing multiple intracellular apparatuses in their native environment using small fluorescent probes remains challenging. This approach requires both orthogonal and biocompatible coupling reactions in heterogeneous biological systems with minimum fluorescence background noise. Here, we present a palette of BODIPY probes containing azide and cyclooctyne moieties for copper-free click chemistry in living cells. The probes, referred to as 'tame probes', are highly permeable and specific in nature, leaving no background noise in cells. Such probes, which are rationally designed through optimized lipophilicity, water solubility and charged van der Waals surface area, allow us to demonstrate rapid and efficient concurrent multi-labelling of intracellular target components. We show that these probes are capable of not only labelling organelles and engineered proteins, but also showing the intracellular glycoconjugates' dynamics, through the use of metabolic oligosaccharide engineering technology in various cell types. The results demonstrated in this study thus provide flexibility for multi-spectral labelling strategies in native systems in a high spatiotemporal manner.

Development of background-free tame fluorescent probes for intracellular live cell imaging.

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.

A highly selective fluorescent probe for direct detection and isolation of mouse embryonic stem cells.

Stem cell research has gathered immense attention in the past decade due to the remarkable ability of stem cells for self-renewal and tissue-specific differentiation. Despite having numerous advancements in stem cell isolation and manipulation techniques, there is a need for highly reliable probes for the specific detection of live stem cells. Herein we developed a new fluorescence probe (CDy9) with high selectivity for mouse embryonic stem cells. CDy9 allows the detection and isolation of intact stem cells with marginal impact on their function and capabilities.

Designing cyclic peptide inhibitor of dengue virus NS3-NS2B protease by using molecular docking approach.

Peptides are preferred for designing inhibitors because of their high activity and specificity. Seven cyclopentapeptide inhibitors were designed in this study against dengue virus type 2 (DEN-2) NS3-NS2B protease: CKRRC, CGRRC, CRGRC, CRTRC, CTRRC, CKRKC and CRRKC. Docking analysis was performed to study the enzyme-inhibitor binding interactions. The free energy binding and estimated Ki values for all the inhibitors were found to be small (within micromolar range), indicating that the inhibitors bind considerably well to the binding site. The results showed that the cyclopentapeptide CKRKC was the best peptide inhibitor candidate with estimated free binding energy of -8.39 kcal/mol and Ki of 0.707 µM when compared to the standard inhibitor Bz-Nle-Lys-Arg-Arg-H that has been experimentally tested and shown to exhibit Ki value of 5.8 µM. Several modes of weak interactions were observed between the cyclopentapeptide CKRKC and the active site of DEN-2 NS3-NS2B protease. Thus, the cyclopentapeptide is proposed as a potential inhibitor to the NS3-NS2B protease activities of DEN-2. While these preliminary results are promising, further experimental investigation is necessary to validate the results.