Hybrid Small-Molecule/Protein Fluorescent Probes.

Hybrid small-molecule/protein fluorescent probes are powerful tools for visualizing protein localization and function in living cells. These hybrid probes are constructed by diverse site-specific chemical protein labeling approaches through chemical reactions to exogenous peptide/small protein tags, enzymatic post-translational modifications, bioorthogonal reactions for genetically incorporated unnatural amino acids, and ligand-directed chemical reactions. The hybrid small-molecule/protein fluorescent probes are employed for imaging protein trafficking, conformational changes, and bioanalytes surrounding proteins. In addition, fluorescent hybrid probes facilitate visualization of protein dynamics at the single-molecule level and the defined structure with super-resolution imaging. In this review, we discuss development and the bioimaging applications of fluorescent probes based on small-molecule/protein hybrids.

Rational Design of Hydroxylated Thiazole Orange Photocages for Green Light-Triggered DNA Recombination.

The precise control of DNA recombination enables the cell- or time-dependent regulation of gene expression in studies of gene function. Caged estrogen receptor ligands combined with a Cre-ERT2/loxP system are useful tools for light-triggered DNA recombination. However, the photolysis of most caged compounds requires ultraviolet or blue light, which is toxic and displays low tissue penetration. Although a cyanine-based photo-responsive protecting group (PPG) can release estrogen receptor ligands with longer-wavelength light, its low photolytic efficiency requires long illumination times. We developed a caged estrogen receptor ligand with improved green light-responsive PPGs. The rational modification of Hydroxylated Thiazole Orange (HTO) photocages using electron-donating groups (EDGs), such as dimethoxy (DiMeO)-substituted HTO, resulted in high photolytic efficiency (up to ÏµΦ ≈320 M-1  cm-1 ). Theoretical calculations demonstrated that the enhanced photolytic efficiencies were derived from the increased intramolecular charge transfer by EDGs upon excitation. The efficient uncaging of estrogen receptor ligands enabled the control of gene recombination in a ligand-dependent Cre-ERT2/loxP system in live cells.

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.

Elastic Polymer Coated Nanoparticles with Fast Clearance for 19 F†MR Imaging.

19 F magnetic resonance imaging (MRI) is a powerful molecular imaging technique that enables high-resolution imaging of deep tissues without background signal interference. However, the use of nanoparticles (NPs) as 19 F MRI probes has been limited by the immediate trapping and accumulation of stiff NPs, typically of around 100 nm in size, in the mononuclear phagocyte system, particularly in the liver. To address this issue, elastic nanomaterials have emerged as promising candidates for improving delivery efficacy in vivo. Nevertheless, the impact of elasticity on NP elimination has remained unclear due to the lack of suitable probes for real-time and long-term monitoring. In this study, we present the development of perfluorocarbon-encapsulated polymer NPs as a novel 19 F MRI contrast agent, with the aim of suppressing long-term accumulation. The polymer NPs have high elasticity and exhibit robust sensitivity in 19 F MRI imaging. Importantly, our 19 F MRI data demonstrate a gradual decline in the signal intensity of the polymer NPs after administration, which contrasts starkly with the behavior observed for stiff silica NPs. This innovative polymer-coated NP system represents a groundbreaking nanomaterial that successfully overcomes the challenges associated with long-term accumulation, while enabling tracking of biodistribution over extended periods.

Development of a Versatile Protein Labeling Tool for Live-Cell Imaging Using Fluorescent ß-Lactamase Inhibitors.

To understand the function of protein in live cells, real-time monitoring of protein dynamics and sensing of their surrounding environment are important methods. Fluorescent labeling tools are thus needed that possess fast labeling kinetics, high efficiency, and long-term stability. We developed a versatile chemical protein-labeling tool based on fluorophore-conjugated diazabicyclooctane ß-lactamase inhibitors (BLIs) and wild-type TEM-1 ß-lactamase protein tag. The fluorescent probes efficiently formed a stable carbamoylated complex with ß-lactamase, and the labeled proteins were visualized over a long period of time in live cells. Moreover, use of an α-fluorinated carboxylate ester-based BLI prodrug enabled the probe to permeate cell membranes and stably label intracellular proteins after unexpected spontaneous ester hydrolysis. Lastly, combining the labeling tool with a pH-activatable fluorescent probe allowed visual monitoring of lysosomal protein translocation during autophagy.

Hybrid, dual visible and near-infrared fluorescence emission of (6,5) single-walled carbon nanotubes modified with fluorescein through aryl diazonium salt chemistry.

The aryl diazonium salt chemistry offers enhancement of near-infrared (NIR) emission of single-walled carbon nanotubes (SWCNTs), although, the attachment of functional molecules which could bring hybrid properties through the process is underdeveloped. In this work, we utilize aryl diazonium salt of fluorescein to createsp3defects on (6,5) SWCNTs. We study the influence of pH on the grafting process identifying that pH 5-6 is necessary for a successful reaction. The fluorescein-modified (6,5) SWCNTs (F-(6,5) SWCNTs) exhibit red-shiftedE11* emission in the NIR region attributed to luminescentsp3defects, but also visible (Vis) fluorescence at 515 nm from surface-attached fluorescein molecules. The fluorescence in both Vis and NIR regions of F-(6,5) SWCNTs exhibit strong pH-dependency associated with the dissociation of fluorescein molecules with an indication of photoinduced-electron transfer quenching the Vis emission of fluorescein dianion. The F-(6,5) SWCNTs could potentially be used for dual-channel medical imaging as indicated by our preliminary experiments. We hope that our research will encourage new, bold modifications of SWCNTs with functional molecules introducing new, unique hybrid properties.

Optical Manipulation of Subcellular Protein Translocation Using a Photoactivatable Covalent Labeling System.

The photoactivatable chemically induced dimerization (photo-CID) technique for tag-fused proteins is one of the most promising methods for regulating subcellular protein translocations and protein-protein interactions. However, light-induced covalent protein dimerization in living cells has yet to be established, despite its various advantages. Herein, we developed a photoactivatable covalent protein-labeling technology by applying a caged ligand to the BL-tag system, a covalent protein labeling system that uses mutant ß-lactamase. We further developed CBHD, a caged protein dimerizer, using caged BL-tag and HaloTag ligands, and achieved light-induced protein translocation from the cytoplasm to subcellular regions. In addition, this covalent photo-CID system enabled quick protein translocation to a laser-illuminated microregion. These results indicate that the covalent photo-CID system will expand the scope of CID applications in the optical manipulation of cellular functions.

Development of Fluorogenic Probes for Rapid High-Contrast Imaging of Transient Nuclear Localization of Sirtuin†3.

Protein labeling using fluorogenic probes enables the facile visualization of proteins of interest. Herein, we report new fluorogenic probes consisting of a rationally designed coumarin ligand for the live-cell fluorogenic labeling of the photoactive yellow protein (PYP)-tag. On the basis of the photochemical mechanisms of coumarin and the probe-tag interactions, we introduced a hydroxy group into an environment-sensitive coumarin ligand to modulate its spectroscopic properties and increase the labeling reaction rate. The resulting probe had a higher labeling reaction rate constant and a greater fluorescence OFF-ON ratio than any previously developed PYP-tag labeling probe. The probe enabled the fluorogenic labeling of intracellular proteins within minutes. Furthermore, we used our probe to investigate the localization of sirtuin 3 (SIRT3), a mitochondrial deacetylase. Although the nuclear localization of SIRT3 has been controversial, this transient nuclear localization was clearly captured by the rapid, high-contrast imaging enabled by our probe.

Live-Cell Imaging of Protein Degradation Utilizing Designed Protein-Tag Mutant and Fluorescent Probe with Turn-Off Switch.

Protein degradation plays various roles in cellular homeostasis and signal transduction. Real-time monitoring of the degradation process not only contributes to the elucidation of relevant biological phenomena but also offers a powerful tool for drug discoveries targeting protein degradation. Fluorescent protein labeling with a protein tag and a synthetic fluorescent probe is a powerful technique that enables the direct visualization of proteins of interest in living cells. Although a variety of protein tags and their labeling probes have been reported, techniques for the visualization of protein degradation in living cells remain limited. In order to overcome this limitation, we herein employed a PYP-tag labeling probe with a fluorescence turn-off switch that enables the imaging of protein degradation. Furthermore, we performed a structure-based design of a PYP-tag to stabilize a complex formed by the probe and the protein tag for long-term live-cell imaging. We successfully applied this technique to live-cell imaging of the degradation process of Regnase-1 in response to immunostimulation.

Chemical Tools with Fluorescence Switches for Verifying Epigenetic Modifications.

Epigenetic DNA and histone modifications alter chromatin conformation and regulate gene expression. A major DNA modification is methylation, which is catalyzed by DNA methyltransferase (Dnmt) and results in gene suppression. Compared to DNA, histones undergo a greater variety of modification types, one of which is the acetylation of lysine. While histone acetyltransferase (HAT) catalyzes acetylation and activates gene expression, histone deacetylase (HDAC) removes the modification and causes gene suppression. As precise regulation of these epigenetic marks on DNA and histones is critical for cellular functions, their dysregulation causes various diseases including cancer, metabolic syndromes, immune diseases, and psychiatric diseases. Therefore, elucidation of the epigenetic phenomena is important not only in the field of biology but also in medical and pharmaceutical sciences. Furthermore, this field is also attracting industrial interest, because small-molecule inhibitors modulate enzymatic activity for epigenetic modification and are used for cancer treatment. Under these circumstances, various methods for detecting epigenetic modifications have been developed. However, most methods require cell lysis, which is not suitable for real-time detection of enzymatic activity. Since fluorescent probes are attractive chemical tools to solve this issue, chemists made considerable efforts to create fluorescent probes for epigenetics. To date, we have particularly focused on HDAC activity and DNA methylation and have developed fluorescent probes for their detection. The first part of this review describes our recent efforts to develop fluorescent probes for detecting HDAC activity. Since the discovery of HDAC activity in the late 1960s, no fluorescent probe has been developed that can detect enzymatic reactions in a simple, one-step procedure despite its biological and medical importance. We designed fluorescent probes to overcome this limitation by devising two different types of fluorescence switching mechanisms, which are based on aggregation-induced emission (AIE) and intramolecular transesterification. Using these probes, we detected HDAC activity simply by mixing the probes and HDAC for the first time. In the second part, a hybrid approach using a protein-labeling system was employed to detect DNA methylation in living cells. So far, live-cell detection of DNA methylation was conducted by imaging the localization of Fluorescent Proteins (FPs) fused to a methylated DNA-binding domain. However, FP lacks a fluorescence switch and emits fluorescence without binding to methylated DNA. We created a hybrid probe that comprises a fluorogen and a protein and enhances fluorescence intensity upon binding to methylated DNA. To create the hybrid probe, we applied our protein labeling system using the PYP-tag that we previously developed. This method successfully visualized methylated DNA in living cells and verified its dynamics during cell division. Both of the above-mentioned fluorescent probes have great potential for use not only in HDAC and DNA methylation but also in other epigenetics-associated modifications. For example, the mechanism of the HDAC probes can be used to detect histone demethylation. The hybrid probe can be converted to a sensor for imaging acetylated or methylated histones. In this review, we mainly describe how we designed the probes using chemical principles and solved the current obstacles with the probe design and discuss the future prospects of these probes.

An Acid-Activatable Fluorescence Probe for Imaging Osteocytic Bone Resorption Activity in Deep Bone Cavities.

A rationally designed pH-activatable fluorescent probe (pHocas-RIS) has been used to measure localised pH levels in osteocytic lacunae in bone tissue. Conjugation of the moderate bone-binding drug risedronate to a pH-activatable BODIPY fluorophore enables the probe to penetrate osteocytic lacunae cavities that are embedded deep within the bone matrix. After injection of pHocas-RIS, any osteocytic lacunae caused by bone-resorbing osteocytes cause the probe to fluoresce in vivo, thus allowing imaging by intravital two-photon excitation microscopy. This pH responsive probe enabled the visualization of the bone mineralizing activities of acid producing osteocytes in real time, thus allowing the study of their central role in remodeling the bone-matrix in healthy and disease states.

Development of an effective protein-labeling system based on smart fluorogenic probes.

Proteins are an important component of living systems and play a crucial role in various physiological functions. Fluorescence imaging of proteins is a powerful tool for monitoring protein dynamics. Fluorescent protein (FP)-based labeling methods are frequently used to monitor the movement and interaction of cellular proteins. However, alternative methods have also been developed that allow the use of synthetic fluorescent probes to target a protein of interest (POI). Synthetic fluorescent probes have various advantages over FP-based labeling methods. They are smaller in size than the fluorescent proteins, offer a wide variety of colors and have improved photochemical properties. There are various chemical recognition-based labeling techniques that can be used for labeling a POI with a synthetic probe. In this review, we focus on the development of protein-labeling systems, particularly the SNAP-tag, BL-tag, and PYP-tag systems, and understanding the fluorescence behavior of the fluorescently labeled target protein in these systems. We also discuss the smart fluorogenic probes for these protein-labeling systems and their applications. The fluorogenic protein labeling will be a useful tool to investigate complex biological phenomena in future work on cell biology.

SCOTfluors: Small, Conjugatable, Orthogonal, and Tunable Fluorophores for In†Vivo Imaging of Cell Metabolism.

The transport and trafficking of metabolites are critical for the correct functioning of live cells. However, in situ metabolic imaging studies are hampered by the lack of fluorescent chemical structures that allow direct monitoring of small metabolites under physiological conditions with high spatial and temporal resolution. Herein, we describe SCOTfluors as novel small-sized multi-colored fluorophores for real-time tracking of essential metabolites in live cells and in vivo and for the acquisition of metabolic profiles from human cancer cells of variable origin.

Synthetic-Molecule/Protein Hybrid Probe with Fluorogenic Switch for Live-Cell Imaging of DNA Methylation.

Hybrid probes consisting of synthetic molecules and proteins are powerful tools for detecting biological molecules and signals in living cells. To date, most targets of the hybrid probes have been limited to pH and small analytes. Although biomacromolecules are essential to the physiological function of cells, the hybrid-probe-based approach has been scarcely employed for live-cell detection of biomacromolecules. Here, we developed a hybrid probe with a chemical switch for live-cell imaging of methylated DNA, an important macromolecule in the repression of gene expression. Using a protein labeling technique, we created a hybrid probe containing a DNA-binding fluorogen and a methylated-DNA-binding domain. The hybrid probe enhanced fluorescence intensity upon binding to methylated DNA and successfully monitored methylated DNA during mitosis. The hybrid probe offers notable advantages absent from probes based on small molecules or fluorescent proteins and is useful for live-cell analyses of epigenetic phenomena and diseases related to DNA methylation.

In vivo visualisation of different modes of action of biological DMARDs inhibiting osteoclastic bone resorption.

OBJECTIVES: Osteoclasts play critical roles in inflammatory bone destruction. Precursor cell migration, cell differentiation, and functional cell activation are all in play. Biological disease-modifying antirheumatic drugs (DMARDs) have been shown to significantly inhibit both bone erosion as well as synovitis, although how such agents reduce osteoclastic bone destructionin vivo has not been fully explained. Here, we used an intravital time-lapse imaging technique to directly visualise mature osteoclasts and their precursors, and explored how different biological DMARDs acted in vivo. METHODS: Lipopolysaccharide (LPS) was injected into the calvarial periosteum of fluorescent reporter mice to induce inflammatory bone destruction. Time-lapse imaging was performed via intravital multiphoton microscopy 5 days after LPS injection. Biological DMARDs, including monoclonal antibodies (mAbs) against the interleukin (IL) 6 receptor (IL-6R) and tumour necrosis factor α (TNFα), or cytotoxic T-lymphocyte-associated protein 4 (CTLA4)-Ig, were intraperitoneally administered at the time of LPS injection. We determined CD80/86 expression levels in mature osteoclasts and their precursors by flow cytometry, quantitative PCR and immunohistochemistry. RESULTS: Of the biologicals tested, anti-IL-6R and anti-TNFα mAbs affected mature osteoclasts and switched bone-resorbing osteoclasts to non-resorbing cells. CTLA4-Ig had no action on mature osteoclasts but mobilised osteoclast precursors, eliminating their firm attachment to bone surfaces. In agreement with these results, CD80/86 (the target molecules of CTLA4-Ig) were prominently expressed only in osteoclast precursor cells, being suppressed during osteoclast maturation. CONCLUSIONS: Intravital imaging revealed that various biological DMARDs acted at specific therapeutic time points during osteoclastic bone destruction, with different efficacies. These results enable us to grasp the real modes of action of drugs, optimising the usage of drug regimens.

Highly Sensitive Detection of Caspase-3/7 Activity in Living Mice Using Enzyme-Responsive 19F MRI Nanoprobes.

Highly sensitive imaging of enzymatic activities in the deep tissues of living mammals provides useful information about their biological functions and for developing new drugs; however, such imaging is challenging. 19F magnetic resonance imaging (MRI) is suitable for noninvasive visualization of enzymatic activities without endogenous background signals. Although various enzyme-responsive 19F MRI probes have been developed, most cannot be used for in vivo imaging because of their low sensitivity. Recently, we developed unique nanoparticles, called FLAMEs, that are composed of a liquid perfluorocarbon core and a robust silica shell, and demonstrated their outstanding sensitivity in vivo. Here, we report a highly functionalized nanoprobe, FLAME-DEVD 2, with an OFF/ON 19F MRI switch for detecting caspase-3/7 activity based on the paramagnetic relaxation enhancement effect. To improve the cleavage efficiency of peptides by caspase-3, we designed a novel Gd3+ complex-conjugated peptide, DEVD X ( X = 1, 2), which is a substrate peptide sequence tandemly repeated X times, and demonstrated that DEVD 2 showed faster cleavage kinetics than DEVD 1. By incorporating this novel concept into a signal activation strategy, FLAME-DEVD 2 showed a high 19F MRI signal enhancement rate in response to caspase-3 activity. After intravenous injection of FLAME-DEVD 2 and an apoptosis-inducing reagent, caspase-3/7 activity in the spleen of a living mouse was successfully imaged by 19F MRI. This imaging platform shows great potential for highly sensitive detection of enzymatic activities in vivo.

Fluorogenic probes reveal a role of GLUT4 N-glycosylation in intracellular trafficking.

Glucose transporter 4 (GLUT4) is an N-glycosylated protein that maintains glucose homeostasis by regulating the protein translocation. To date, it has been unclear whether the N-glycan of GLUT4 contributes to its intracellular trafficking. Here, to clarify the role of the N-glycan, we developed fluorogenic probes that label cytoplasmic and plasma-membrane proteins for multicolor imaging of GLUT4 translocation. One of the probes, which is cell impermeant, selectively detected exocytosed GLUT4. Using this probe, we verified the 'log' of the trafficking, in which N-glycan-deficient GLUT4 was transiently translocated to the cell membrane upon insulin stimulation and was rapidly internalized without retention on the cell membrane. The results strongly suggest that the N-glycan functions in the retention of GLUT4 on the cell membrane. This study showed the utility of the fluorogenic probes and indicated that this imaging tool will be applicable for research on various membrane proteins that show dynamic changes in localization.

Real-time intravital imaging of pH variation associated with osteoclast activity.

Intravital imaging by two-photon excitation microscopy (TPEM) has been widely used to visualize cell functions. However, small molecular probes (SMPs), commonly used for cell imaging, cannot be simply applied to intravital imaging because of the challenge of delivering them into target tissues, as well as their undesirable physicochemical properties for TPEM imaging. Here, we designed and developed a functional SMP with an active-targeting moiety, higher photostability, and a fluorescence switch and then imaged target cell activity by injecting the SMP into living mice. The combination of the rationally designed SMP with a fluorescent protein as a reporter of cell localization enabled quantitation of osteoclast activity and time-lapse imaging of its in vivo function associated with changes in cell deformation and membrane fluctuations. Real-time imaging revealed heterogenic behaviors of osteoclasts in vivo and provided insights into the mechanism of bone resorption.

Live-Cell Imaging of DNA Methylation Based on Synthetic-Molecule/Protein Hybrid Probe.

The epigenetic modification of DNA involves the conversion of cytosine to 5-methylcytosine, also known as DNA methylation. DNA methylation is important in modulating gene expression and thus, regulating genome and cellular functions. Recent studies have shown that aberrations in DNA methylation are associated with various epigenetic disorders or diseases including cancer. This stimulates great interest in the development of methods that can detect and visualize DNA methylation. For instance, fluorescent proteins (FPs) in conjugation with methyl-CpG-binding domain (MBD) have been employed for live-cell imaging of DNA methylation. However, the FP-based approach showed fluorescence signals for both the DNA-bound and -unbound states and thus differentiation between these states is difficult. Synthetic-molecule/protein hybrid probes can provide an alternative to overcome this restriction. In this article, we discuss the synthetic-molecule/protein hybrid probe that we developed recently for live-cell imaging of DNA methylation, which exhibited fluorescence enhancement only after binding to methylated DNA.

[Development of fluorescent probes for bone imaging in vivo ~Fluorescent probes for intravital imaging of osteoclast activity~.]

Fluorescent molecules are widely used as a tool to directly visualize target biomolecules in vivo. Fluorescent probes have the advantage that desired function can be rendered based on rational design. For bone-imaging fluorescent probes in vivo, they should be delivered to bone tissue upon administration. Recently, a fluorescent probe for detecting osteoclast activity was developed. The fluorescent probe has acid-sensitive fluorescence property, specific delivery to bone tissue, and durability against laser irradiation, which enabled real-time intravital imaging of bone-resorbing osteoclasts for a long period of time.