Persistent Fluorescence Switching of a Probe Using a Photochromic Quencher with High Photostability Assisted by Protein-Surface Modification

Photoswitchable fluorescent molecules (PSFMs) are widely applicable in the life sciences for super-resolution imaging. Owing to the large and hydrophobic molecular structures of PSFMs that may aggregate in a biological medium, the development of synthetic PSFMs with persistent reversible photoswitching is challenging. Here, we established a protein-surface-assisted photoswitching strategy that allows for persistent reversible fluorescence photoswitching of a PSFM in an aqueous solution. As a first step, we applied the photochromic chromophore furylfulgimide (FF) as a photoswitchable fluorescence quencher and developed a Förster resonance energy transfer-based PSFM, named FF-TMR. Most importantly, the protein-surface modification strategy allows FF-TMR to exhibit persistent reversible photoswitching performance in an aqueous solution. In fixed cells, the fluorescence intensity of FF-TMR bound to antitubulin antibody was repetitively modulated. The protein-surface-assisted photoswitching strategy will be a useful platform to broaden the utility of functionalized synthetic chromophores enabling persistent fluorescence switching that inherits their high resistance to light irradiation

Simple and Real-Time Colorimetric Assay for Glycosidases Activity Using Functionalized Gold Nanoparticles and Its Application for Inhibitor Screening

The development of real-time assays for enzymes has been receiving a great deal of attention in biomedical research recently. Self-immolative elimination is the spontaneous and irreversible disassembly of a multicomponent construct into its constituent fragments through a cascade of elimination processes, in response to external stimuli. Here, we report a simple and real-time colorimetric assay for glycosidases (β-galactosidase and β-glucosidase). Self-immolative elimination was utilized to release amines to give rise to aggregation and color change by electrostatic attraction after cleavage of the trigger by enzymes displayed on functionalized gold nanoparticles (Gal-Lip-AuNPs and Glc-Lip-AuNPs, where AuNPs denotes gold nanoparticles). The detection limits for β-galactosidase and β-glucosidase were as low as 9.2 and 22.3 nM at 20 min, and they improved slightly over time. Thus, glycosidase activity was detected successfully in real time, and this technique could be used for glycosidase inhibitor screening, based on real-time colorimetric variation

Development of a Fluorogenic Probe Based on a DNA Staining Dye for Continuous Monitoring of the Histone Deacetylase Reaction

We designed a simple, rapid, and continuous method for the detection of the activity of histone deacetylases (HDACs), which are key enzymes involved in epigenetic gene regulation, using a DNA-based fluorogenic probe. We designed and synthesized a fluorogenic probe, BOXTO-GK­(Ac)­G, which is a DNA staining dye–peptide conjugate containing an acetylated lysine. The DNA-dependent fluorescence of BOXTO-GK­(Ac)­G was greatly enhanced upon deacetylation of the acetylated lysine moiety, owing to the increased DNA binding ability of the probe. The HDAC reaction was detected through a simple procedure that combined this probe with DNA. Our detection system monitored the enzymatic reaction in real time and could be applied to the inhibition assay. These findings demonstrated that our system might be a useful tool for the analysis of HDAC function and for the evaluation of the inhibitor potencies of drug candidates that target HDACs

Covalent Protein Labeling Based on Noncatalytic β-Lactamase and a Designed FRET Substrate

Covalent Protein Labeling Based on Noncatalytic β-Lactamase and a Designed FRET Substrat

Anion Sensor-Based Ratiometric Peptide Probe for Protein Kinase Activity

A new fluorescent sensor consisting of CdII-cylcen appended aminocoumarin and a substrate peptide for protein kinase A (PKA) has been designed. Upon phosphorylation by PKA, the metal complex moiety binds to a phosphorylated residue, which in turn displaces the coumarin fluorophore, and this event results in ratiometric change of excitation spectrum in neutral aqueous solution

Lanthanide-Based Protease Activity Sensors for Time-Resolved Fluorescence Measurements

Lanthanide-Based Protease Activity Sensors for Time-Resolved Fluorescence Measurement

Design and Synthesis of Coumarin-Based Zn²⁺ Probes for Ratiometric Fluorescence Imaging

The physiological roles of free Zn2+ have attracted great attention. To clarify those roles, there has been a need for ratiometric fluorescent Zn2+ probes for practical use. We report the rational design and synthesis of a series of ratiometric fluorescent Zn2+ probes. The structures of the probes are based on the 7-hydroxycoumarin structure. We focused on the relationship between the electron-donating ability of the 7-hydroxy group and the excitation spectra of 7-hydroxycoumarins, and exploited that relationship in the design of the ratiometric probes; as a result, most of the synthesized probes showed ratiometric Zn2+-sensing properties. Then, we designed and synthesized ratiometric Zn2+ probes that can be excited with visible light, by choosing adequate substituents on coumarin dyes. Since one of the probes could permeate living cell membranes, we introduced the probe to living RAW264 cells and observed the intracellular Zn2+ concentration via ratiometric fluorescence microscopy. As a result, the ratio value of the probe changed quickly in response to intracellular Zn2+ concentration

Multicolor Protein Labeling in Living Cells Using Mutant β-Lactamase-Tag Technology

Protein labeling techniques using small molecule probes have become important as practical alternatives to the use of fluorescent proteins (FPs) in live cell imaging. These labeling techniques can be applied to more sophisticated fluorescence imaging studies such as pulse-chase imaging. Previously, we reported a novel protein labeling system based on the combination of a mutant β-lactamase (BL-tag) with coumarin-derivatized probes and its application to specific protein labeling on cell membranes. In this paper, we demonstrated the broad applicability of our BL-tag technology to live cell imaging by the development of a series of fluorescence labeling probes for this technology, and the examination of the functions of target proteins. These new probes have a fluorescein or rhodamine chromophore, each of which provides enhanced photophysical properties relative to coumarins for the purpose of cellular imaging. These probes were used to specifically label the BL-tag protein and could be used with other small molecule fluorescent probes. Simultaneous labeling using our new probes with another protein labeling technology was found to be effective. In addition, it was also confirmed that this technology has a low interference with respect to the functions of target proteins in comparison to GFP. Highly specific and fast covalent labeling properties of this labeling technology is expected to provide robust tools for investigating protein functions in living cells, and future applications can be improved by combining the BL-tag technology with conventional imaging techniques. The combination of probe synthesis and molecular biology techniques provides the advantages of both techniques and can enable the design of experiments that cannot currently be performed using existing tools

Improvement and Biological Applications of Fluorescent Probes for Zinc, ZnAFs

The development and cellular applications of novel fluorescent probes for Zn2+, ZnAF-1F, and ZnAF-2F are described. Fluorescein is used as a fluorophore of ZnAFs, because its excitation and emission wavelengths are in the visible range, which minimizes cell damage and autofluorescence by excitation light. N,N-Bis(2-pyridylmethyl)ethylenediamine, used as an acceptor for Zn2+, is attached directly to the benzoic acid moiety of fluorescein, resulting in very low quantum yields of 0.004 for ZnAF-1F and 0.006 for ZnAF-2F under physiological conditions (pH 7.4) due to the photoinduced electron-transfer mechanism. Upon the addition of Zn2+, the fluorescence intensity is quickly increased up to 69-fold for ZnAF-1F and 60-fold for ZnAF-2F. Apparent dissociation constants (Kd) are in the nanomolar range, which affords sufficient sensitivity for biological applications. ZnAFs do not fluoresce in the presence of other biologically important cations such as Ca2+ and Mg2+, and are insensitive to change of pH. The complexes with Zn2+ of previously developed ZnAFs, ZnAF-1, and ZnAF-2 decrease in fluorescence intensity below pH 7.0 owing to protonation of the phenolic hydroxyl group of fluorescein, whose pKa value is 6.2. On the other hand, the Zn2+ complexes of ZnAF-1F and ZnAF-2F emit stable fluorescence around neutral and slightly acidic conditions because the pKa values are shifted to 4.9 by substitution of electron-withdrawing fluorine at the ortho position of the phenolic hydroxyl group. For application to living cells, the diacetyl derivative of ZnAF-2F, ZnAF-2F DA, was synthesized. ZnAF-2F DA can permeate through the cell membrane, and is hydrolyzed by esterase in the cytosol to yield ZnAF-2F, which is retained in the cells. Using ZnAF-2F DA, we could measure the changes of intracellular Zn2+ in cultured cells and hippocampal slices