DNAzyme-Mediated Genetically Encoded Sensors for Ratiometric Imaging of Metal Ions in Living Cells

Genetically encoded fluorescent proteins (FPs) have been used for metal ion detection. However, their applications are restricted to a limited number of metal ions owing to the lack of available metal-binding proteins or peptides that can be fused to FPs and the difficulty in transforming the binding of metal ions into a change of fluorescent signal. We report herein the use of Mg²⁺-specific 10–23 or Zn²⁺-specific 8–17 RNA-cleaving DNAzymes to regulate the expression of FPs as a new class of ratiometric fluorescent sensors for metal ions. Specifically, we demonstrate the use of DNAzymes to suppress the expression of Clover2, a variant of the green FP (GFP), by cleaving the mRNA of Clover2, while the expression of Ruby2, a mutant of the red FP (RFP), is not affected. The Mg²⁺ or Zn²⁺ in HeLa cells can be detected using both confocal imaging and flow cytometry. Since a wide variety of metal-specific DNAzymes can be obtained, this method can likely be applied to imaging many other metal ions, expanding the range of the current genetically encoded fluorescent protein-based sensors.     

Genetically Encoded Ratiometric RNA‐Based Sensors for Quantitative Imaging of Small Molecules in Living Cells

Precisely determining the intracellular concentrations of metabolites and signaling molecules is critical in studying cell biology. Fluorogenic RNA‐based sensors have emerged to detect various targets in living cells. However, it is still challenging to apply these genetically encoded sensors to quantify the cellular concentrations and distributions of targets. Herein, using a pair of orthogonal fluorogenic RNA aptamers, DNB and Broccoli, we engineered a modular sensor system to apply the DNB‐to‐Broccoli fluorescence ratio to quantify the cell‐to‐cell variations of target concentrations. These ratiometric sensors can be broadly applied for live‐cell imaging and quantification of metabolites, signaling molecules, and other synthetic compounds.     

Genetically Encoded Activators of Small Molecules for Imaging and Drug Delivery

Chemical biologists have developed many tools based on genetically encoded macromolecules and small, synthetic compounds. The two different approaches are extremely useful, but they have inherent limitations. In this Minireview, we highlight examples of strategies that combine both concepts to tackle challenging problems in chemical biology. We discuss applications in imaging, with a focus on super‐resolved techniques, and in probe and drug delivery. We propose future directions in this field, hoping to inspire chemical biologists to develop new combinations of synthetic and genetically encoded probes.     

Ratiometric Single‐Nanoparticle Oxygen Sensors for Biological Imaging

It makes sense : Conjugated polymer nanoparticles doped with a platinum porphyrin dye exhibit bright phosphorescence that is highly sensitive to the concentration of molecular oxygen. The small size, extraordinary brightness, excellent sensitivity, and ratiometric emission, together with the demonstration of single‐particle sensing and cellular uptake, indicate the potential of the nanoparticle sensors for quantitative mapping of local molecular oxygen concentration.

     

Photocaged DNAzymes as a General Method for Sensing Metal Ions in Living Cells

DNAzymes, which are sequences of DNA with catalytic activity, have been demonstrated as a potential platform for sensing a wide range of metal ions. Despite their significant promise, cellular sensing using DNAzymes has however been difficult, mainly because of the “always‐on” mode of first‐generation DNAzyme sensors. To overcome this limitation, a photoactivatable (or photocaged) DNAzyme was designed and synthesized, and its application in sensing Zn^II in living cells was demonstrated. In this design, the adenosine ribonucleotide at the scissile position of the 8–17 DNAzyme was replaced by 2′‐O‐nitrobenzyl adenosine, rendering the DNAzyme inactive and thus allowing its delivery into cells intact, protected from nonspecific degradation within cells. Irradiation at 365 nm restored DNAzyme activity, thus allowing the temporal control over the sensing activity of the DNAzyme for metal ions. The same strategy was also applied to the GR‐5 DNAzyme for the detection of Pb^II, thus demonstrating the possible scope of the method.     

Near‐Infrared Photothermally Activated DNAzyme–Gold Nanoshells for Imaging Metal Ions in Living Cells

DNAzymes have enjoyed success as metal ion sensors outside cells. Their susceptibility to metal‐dependent cleavage during delivery into cells has limited their intracellular applications. To overcome this limitation, a near‐infrared (NIR) photothermal activation method is presented for controlling DNAzyme activity in living cells. The system consists of a three‐stranded DNAzyme precursor (TSDP), the hybridization of which prevents the DNAzyme from being active. After conjugating the TSDP onto gold nanoshells and upon NIR illumination, the increased temperature dehybridizes the TSDP to release the active DNAzyme, which then carries out metal‐ion‐dependent cleavage, resulting in releasing the cleaved product containing a fluorophore. Using this construct, detecting Zn²⁺ in living HeLa cells is demonstrated. This method has expanded the DNAzyme versatility for detecting metal ions in biological systems under NIR light that exhibits lower phototoxicity and higher tissue penetration ability.     

Imaging Endogenous Metal Ions in Living Cells Using a DNAzyme–Catalytic Hairpin Assembly Probe

DNAzymes are a promising platform for metal ion detection, and a few DNAzyme‐based sensors have been reported to detect metal ions inside cells. However, these methods required an influx of metal ions to increase their concentrations for detection. To address this major issue, the design of a catalytic hairpin assembly (CHA) reaction to amplify the signal from photocaged Na⁺‐specific DNAzyme to detect endogenous Na⁺ inside cells is reported. Upon light activation and in the presence of Na⁺, the NaA43 DNAzyme cleaves its substrate strand and releases a product strand, which becomes an initiator that trigger the subsequent CHA amplification reaction. This strategy allows detection of endogenous Na⁺ inside cells, which has been demonstrated by both fluorescent imaging of individual cells and flow cytometry of the whole cell population. This method can be generally applied to detect other endogenous metal ions and thus contribute to deeper understanding of the role of metal ions in biological systems.     

Activity‐Based Genetically Encoded Fluorescent and Luminescent Probes for Detecting Formaldehyde in Living Cells

Formaldehyde (FA) is endogenously produced in living systems through a variety of biological processes and has been implicated in many pathological conditions. Detection tools for biological FA are therefore of great interest. Reported here are novel activity‐based genetically encoded fluorescent and luminescent probes for detecting FA in aqueous solutions and living mammalian cells. A FA‐reactive lysine analogue, PrAK, was site‐specifically incorporated into the essential lysine sites of enhanced green fluorescent protein (EGFP) and firefly luciferase (fLuc) to afford fluorescent and luminescent FA probes, respectively. FA selectively reacts with PrAK residues on EGFP and fLuc through a 2‐aza‐Cope rearrangement, resulting in fluorescence and luminescence turn‐on responses, respectively, to FA selectively over potentially interfering reactive species in aqueous buffer. Moreover, the genetically encoded probes are capable of visualizing FA at physiologically relevant levels in living mammalian cells by fluorescence and luminescence imaging, demonstrating their potential as new tools to explore FA biology.     

Ratiometric and Near‐Infrared Molecular Probes for the Detection and Imaging of Zinc Ions

The detection and imaging of Zn²⁺ in biological samples are of paramount interest owing to the role of this cation in physiological functions. This is possible only with molecular probes that specifically bind to Zn²⁺ and result in changes in emission properties. A “turn‐on” emission or shift in the emission color upon binding to Zn²⁺ should be ideal for in vivo imaging. In this context, ratiometric and near‐IR probes are of particular interest. Therefore, in the area of chemosensors or molecular probes, the design of fluorophores that allow ratiometric sensing or imaging in the near‐IR region is attracting the attention of chemists. The purpose of this Focus Review is to highlight recent developments in this area and stress the importance of further research for future applications.     

Oligonucleotide‐Based Luminescent Detection of Metal Ions

Metal ions are prevalent in biological systems and are critically involved in essential life processes. However, excess concentrations of metals can pose a serious danger to living organisms. Oligonucleotides represent a versatile sensing platform for the detection of various molecular entities including metal ions. This review summarizes the recent advances in the development of oligonucleotide‐based luminescent detection methods for metal ions.     

DNAzyme‐Based Probes for Telomerase Detection in Early‐Stage Cancer Diagnosis

Human telomerase is a polymerase enzyme that adds tandem repeats of DNA (TTAGGG) in the telomeric region to the ends of chromosomes. Since telomerase can be detected in immortalized, but not normal, somatic cells, it has been considered a selective target for cancer chemotherapy. Here, we describe a DNAzyme‐based probe to detect the presence of telomerase in cell lysates. Telomerase elongates the primer site on the probe. Subsequent addition of the Pb^II cofactor activates the DNAzyme, which cleaves the elongated fragment at the RNA site, releasing the probe for repetitive cycling and signal amplification. The cleaved fragment is detected by a reporter molecular beacon. Enzymatic amplification with rapid turnover allows detection of telomerase in the range of 0.1 to 1 μg cell lysate, with a fivefold increase in signal level for cancer cells over normal cells. This probe design can provide a simple, yet rapid and sensitive, measurement of telomerase activity.     

Two‐Photon Fluorescent Probes for Metal Ions

Two‐photon microscopy (TPM) has become an indispensible tool in biology and medicine owing to the capability of imaging the intact tissue for a long period of time. To make it a versatile tool in biology, a variety of two‐photon probes for specific applications are needed. In this context, many research groups are developing two‐photon probes for various applications. In this Focus Review, we summarize recent results on model studies and selected examples of two‐photon probes that can detect intracellular free metal ions in live cells and tissues to provide a guideline for the design of useful two‐photon probes for various in vivo imaging applications.     

A Novel Type of Crown Ether‐Containing Metal Ions Optical Sensors Based on Polymer‐Stabilized Cholesteric Liquid Crystalline Films

For the first time, the films based on polymer‐stabilized cholesteric composites containing crown ether fragments with the optical properties sensitive to the complexation with potassium and barium ions were obtained. The complexation with these ions leads to blue spectral shift of the selective light reflection of planar cholesteric texture of composite films. Peculiarities of spectral changes and kinetics of selective light reflection shift were studied. The proposed approach can be used for the creation of the effective and selective sensor materials for different ions or groups of ions.     

Chelating Rotaxane Ligands as Fluorescent Sensors for Metal Ions

Although metal‐ion‐binding interlocked molecules have been under intense investigation for over three decades, their application as scaffolds for the development of sensors for metal ions remains underexplored. In this work, we demonstrate the potential of simple rotaxanes as metal‐ion‐responsive ligand scaffolds through the development of a proof‐of‐concept selective sensor for Zn²⁺.     

Activity-Based Genetically Encoded Fluorescent and Luminescent Probes for Detecting Formaldehyde in Living Cells

Formaldehyde (FA) is endogenously produced in living systems through a variety of biological processes and has been implicated in many pathological conditions. Detection tools for biological FA are therefore of great interest. Reported here are novel activity-based genetically encoded fluorescent and luminescent probes for detecting FA in aqueous solutions and living mammalian cells. A FA-reactive lysine analogue, PrAK, was site-specifically incorporated into the essential lysine sites of enhanced green fluorescent protein (EGFP) and firefly luciferase (fLuc) to afford fluorescent and luminescent FA probes, respectively. FA selectively reacts with PrAK residues on EGFP and fLuc through a 2-aza-Cope rearrangement, resulting in fluorescence and luminescence turn-on responses, respectively, to FA selectively over potentially interfering reactive species in aqueous buffer. Moreover, the genetically encoded probes are capable of visualizing FA at physiologically relevant levels in living mammalian cells by fluorescence and luminescence imaging, demonstrating their potential as new tools to explore FA biology.     

A Naphthalimide-Based Fluorescent Probe for the Detection and Imaging of Mercury Ions in Living Cells

The selective and efficient monitoring of mercury (Hg²⁺) contamination found in the environment and ecosystem has been carried out. Thus, a new 1,8-naphthalimide-based fluorescent probe NADP for the detection of Hg²⁺ based on a fluorescence enhancement strategy has been designed and synthesized. The NADP probe can detect Hg²⁺ with high selectivity and sensitivity and a low detection limit of 13 nm . The detection mechanism was based on a Hg²⁺-triggered deprotection reaction, resulting in a dramatic change in fluorescence from colorless to green at physiological pH. Most importantly, biological investigation has shown that the NADP probe can be successfully applied to the monitoring of Hg²⁺ in living cells and zebrafish with low cytotoxicity.     

Enzyme‐Mediated Endogenous and Bioorthogonal Control of a DNAzyme Fluorescent Sensor for Imaging Metal Ions in Living Cells

Bioorthogonal control of metal‐ion sensors for imaging metal ions in living cells is important for understanding the distribution and fluctuation of metal ions. Reported here is the endogenous and bioorthogonal activation of a DNAzyme fluorescent sensor containing an 18‐base pair recognition site of a homing endonuclease (I‐SceI), which is found by chance only once in 7×10¹⁰ bp of genomic sequences, and can thus form a near bioorthogonal pair with I‐SceI for DNAzyme activation with minimal effect on living cells. Once I‐SceI is expressed inside cells, it cleaves at the recognition site, allowing the DNAzyme to adopt its active conformation. The activated DNAzyme sensor is then able to specifically catalyze cleavage of a substrate strand in the presence of Mg²⁺ to release the fluorophore‐labeled DNA fragment and produce a fluorescent turn‐on signal for Mg²⁺. Thus I‐SceI bioorthogonally activates the 10–23 DNAzyme for imaging of Mg²⁺ in HeLa cells.     

A PEGylated Fluorescent Turn‐On Sensor for Detecting Fluoride Ions in Totally Aqueous Media and Its Imaging in Live Cells

Owing to the considerable significance of fluoride anions for health and environmental issues, it is of great importance to develop methods that can rapidly, sensitively and selectively detect the fluoride anion in aqueous media and biological samples. Herein, we demonstrate a robust fluorescent turn‐on sensor for detecting the fluoride ion in a totally aqueous solution. In this study, a biocompatible hydrophilic polymer poly(ethylene glycol) (PEG) is incorporated into the sensing system to ensure water solubility and to enhance biocompatibility. tert‐Butyldiphenylsilyl (TBDPS) groups were then covalently introduced onto the fluorescein moiety, which effectively quenched the fluorescence of the sensor. Upon addition of fluoride ion, the selective fluoride‐mediated cleavage of the Si? O bond leads to the recovery of the fluorescein moiety, resulting in a dramatic increase in fluorescence intensity under visible light excitation. The sensor is responsive and highly selective for the fluoride anion over other common anions; it also exhibits a very low detection limit of 19 ppb. In addition, this sensor is operative in some real samples such as running water, urine, and serum and can accurately detect fluoride ions in these samples. The cytotoxicity of the sensor was determined to be Grade I toxicity according to United States Pharmacopoeia and ISO 10993‐5, suggesting the very low cytotoxicity of the sensor. Moreover, it was found that the senor could be readily internalized by both HeLa and L929 cells and the sensor could be utilized to track fluoride level changes inside the cells.