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.     

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.     

Metal-ion-dependent folding of a uranyl-specific DNAzyme: insight into function from fluorescence resonance energy transfer studies

Fluorescence resonance energy transfer (FRET) has been used to study the global folding of an uranyl (UO₂²⁺)‐specific 39E DNAzyme in the presence of Mg²⁺, Zn²⁺, Pb²⁺, or UO₂²⁺. At pH 5.5 and physiological ionic strength (100 mM Na⁺), two of the three stems in this DNAzyme folded into a compact structure in the presence of Mg²⁺ or Zn²⁺. However, no folding occurred in the presence of Pb²⁺ or UO₂²⁺; this is analogous to the “lock‐and‐key” catalysis mode first observed in the Pb²⁺‐specific 8–17 DNAzyme. However, Mg²⁺ and Zn²⁺ exert different effects on the 8–17 and 39E DNAzymes. Whereas Mg²⁺ or Zn²⁺‐dependent folding promoted 8–17 DNAzyme activity, the 39E DNAzyme folding induced by Mg²⁺ or Zn²⁺ inhibited UO₂²⁺‐specific activity. Group IIA series of metal ions (Mg²⁺, Ca²⁺, Sr²⁺) also caused global folding of the 39E DNAzyme, for which the apparent binding affinity between these metal ions and the DNAzyme decreases as the ionic radius of the metal ions increases. Because the ionic radius of Sr²⁺ (1.12 Å) is comparable to that of Pb²⁺ (1.20 Å), but contrary to Pb²⁺, Sr²⁺ induces the DNAzyme to fold under identical conditions, ionic size alone cannot account for the unique folding behaviors induced by Pb²⁺ and UO₂²⁺. Under low ionic strength (30 mM Na⁺), all four metal ions (Mg²⁺, Zn²⁺, Pb²⁺, and UO₂²⁺), caused 39E DNAzyme folding, suggesting that metal ions can neutralize the negative charge of DNA‐backbone phosphates in addition to playing specific catalytic roles. Mg²⁺ at low (<2 mM ) concentration promoted UO₂²⁺‐specific activity, whereas Mg²⁺ at high (>2 mM ) concentration inhibited the UO₂²⁺‐specific activity. Therefore, the lock‐and‐key mode of DNAzymes depends on ionic strength, and the 39E DNAzyme is in the lock‐and‐key mode only at ionic strengths of 100 mM or greater.     

A Multi‐component All‐DNA Biosensing System Controlled by a DNAzyme

We report on a programmable all‐DNA biosensing system that centers on the use of a 4‐way junction (4WJ) to transduce a DNAzyme reaction into an amplified signal output. A target acts as a primary input to activate an RNA‐cleaving DNAzyme, which then cleaves an RNA‐containing DNA substrate that is designed to be a component of a 4WJ. The formation of the 4WJ controls the release of a DNA output that becomes an input to initiate catalytic hairpin assembly (CHA), which produces a second DNA output that controls assembly of a split G‐quadruplex as a fluorescence signal generator. The 4WJ can be configured to produce either a turn‐off or turn‐on switch to control the degree of CHA, allowing target concentration to be determined in a quantitative manner. We demonstrate this approach by creating a sensor for E. coli that could detect as low as 50 E. coli cells mL^?1 within 85 min and offers an amplified bacterial detection method that does not require a protein enzyme.     

Molecular Swings as Highly Active Ion Transporters

Ions are transported across membrane mostly via carrier or channel mechanisms. Herein, a unique class of molecular‐machine‐inspired membrane transporters, termed molecular swings is reported that utilize a previously unexplored swing mechanism for promoting ion transport in a highly efficient manner. In particular, the molecular swing, which carries a 15‐crown‐5 unit as the ion‐binding and transporting unit, exhibits extremely high ion‐transport activities with EC₅₀ values of 46 nm (a channel:lipid molar ratio of 1:4800 or 0.021 mol % relative to lipid) and 110 nm for K⁺ and Na⁺ ions, respectively. Remarkably, such ion transport activities remain high in a cholesterol‐rich environment, with EC₅₀ values of 130 (0.045 mol % relative to lipid/cholesterol) and 326 nm for K⁺ and Na⁺ ions, respectively.     

Modulation of DNAzyme Activity via Butanol Dehydration

Understanding the activity of biomolecules in cosolvent systems is important for catalysis, separation and developing biosensors. The majority of previously studied solvents are either phase separated with water or miscible with water. Butanol was recently used to extract water for the conjugation of DNA to gold nanoparticles. In this work, the effect of butanol on the activity of a few RNA-cleaving DNAzymes was studied. A 130-fold improvement in sensitivity for the Na⁺-specific EtNa DNAzyme was observed, and butanol also improved the activity of another Na⁺-specific DNAzyme, NaA43T by a few folds. However, when divalent metal ions were used for both EtNa and 17E DNAzymes, the activity was inhibited. A main driven force for enhanced DNAzyme activity is the concentration effect due to butanol dehydration. This study provides insights into the interplay between DNA, metal ions and organic solvents, and such an understanding might be useful for developing sensitive biosensors.     

Facilitated Transfer of Alkali Metal Ions by a Tetraester Derivative of Thiacalix[4]arene at the Liquid–Liquid Interface

The facilitated transfer of alkali metal ions (Na⁺, K⁺, Rb⁺, and Cs⁺) by 25,26,27,28‐tetraethoxycarbonylmethoxy‐thiacalix[4]arene across the water/1,2‐dichloroethane interface was investigated by cyclic voltammetry. The dependence of the half‐wave transfer potential on the metal and ligand concentrations was used to formulate the stoichiometric ratio and to evaluate the association constants of the complexes formed between ionophore and metal ions. While the facilitated transfer of Li⁺ ion was not observed across the water/1,2‐dichloroethane interface, the facilitated transfers were observed by formation of 1 : 1 (metal:ionophore) complex for Na⁺, K⁺, and Rb⁺ ions except for Cs⁺ ion. In the case of Cs⁺ a 1 : 2 (metal:ionophore) complex was obtained from its special electrochemical response to the variation of ligand concentrations in the organic phase. The logarithms of the complex association constants, for facilitated transfer of Na⁺, K⁺, Rb⁺, and Cs⁺, were estimated as 6.52, 7.75, 7.91 (log β₁°), and 8.36 (log β₂°), respectively.     

Artificial K+ Channels Formed by Pillararene‐Cyclodextrin Hybrid Molecules: Tuning Cation Selectivity and Generating Membrane Potential

A class of artificial K⁺ channels formed by pillararene‐cyclodextrin hybrid molecules have been designed and synthesized. These channels efficiently inserted into lipid bilayers and displayed high selectivity for K⁺ over Na⁺ in fluorescence and electrophysiological experiments. The cation transport selectivity of the artificial channels is tunable by varying the length of the linkers between pillararene and cyclodexrin. The shortest channel showed specific transmembrane transport preference for K⁺ over all alkali metal ions (selective sequence: K⁺ > Cs⁺ > Rb⁺ > Na⁺ > Li⁺), and is rarely observed for artificial K⁺ channels. The high selectivity of this artificial channel for K⁺ over Na⁺ ensures specific transmembrane translocation of K⁺, and generated stable membrane potential across lipid bilayers.     

Facilitated Transfer of Alkali and Alkaline‐Earth Metal Ions by a Calix[4]arene Derivative Across Water/1,2‐Dichloroethane Microinterface: Amperometric Detection of Ca2+

The host–guest complexation reactions between 5,11,17,23‐tetra‐tert‐butyl‐25,27‐diethoxycarbonylmethoxy‐26,28‐dimethoxy calix[4]arene (BDDC4) and alkali and alkaline‐earth metal ions were investigated by facilitated ion transfer processes across water/1,2‐dichloroethane microinterface by using steady‐state cyclic voltammetry and differential pulse voltammetry. The obtained facilitated transfers for Li⁺, Na⁺, K⁺, Rb⁺ and Ca²⁺ were evaluated under the different experimental conditions, at the excess concentrations of metal ions with respect to BDDC4 and vice versa. The association constants having 1 : 1 stoichiometry for Li⁺, Na⁺, K⁺ and Rb⁺ in 1,2‐DCE were determined. Also, we demonstrated that BDDC4 can play an important role for the development of highly selective chemical sensor for Ca²⁺ among alkaline‐metal ions in the concentration range of 0.1–1.0 mM in aqueous solution.     

Fine Control of the Redox Reactivity of a Nonheme Iron(III)–Peroxo Complex by Binding Redox‐Inactive Metal Ions

Redox‐inactive metal ions are one of the most important co‐factors involved in dioxygen activation and formation reactions by metalloenzymes. In this study, we have shown that the logarithm of the rate constants of electron‐transfer and C−H bond activation reactions by nonheme iron(III)–peroxo complexes binding redox‐inactive metal ions, [(TMC)Fe^III(O₂)]⁺‐M^{n +} (M^{n +}=Sc³⁺, Y³⁺, Lu³⁺, and La³⁺), increases linearly with the increase of the Lewis acidity of the redox‐inactive metal ions (ΔE ), which is determined from the g_zz values of EPR spectra of O₂^.−‐M^{n +} complexes. In contrast, the logarithm of the rate constants of the [(TMC)Fe^III(O₂)]⁺‐M^{n +} complexes in nucleophilic reactions with aldehydes decreases linearly as the ΔE value increases. Thus, the Lewis acidity of the redox‐inactive metal ions bound to the mononuclear nonheme iron(III)–peroxo complex modulates the reactivity of the [(TMC)Fe^III(O₂)]⁺‐M^{n +} complexes in electron‐transfer, electrophilic, and nucleophilic reactions.     

Ion‐Responsive Hemin–G‐Quadruplexes for Switchable DNAzyme and Enzyme Functions

Programmed nucleic acid sequences undergo K⁺ ion‐induced self‐assembly into G‐quadruplexes and separation of the supramolecular structures by the elimination of K⁺ ions by crown ether or cryptand ion‐receptors. This process allows the switchable formation and dissociation of the respective G‐quadruplexes. The different G‐quadruplex structures bind hemin, and the resulting hemin–G‐quadruplex structures reveal horseradish peroxidase DNAzyme catalytic activities. The following K⁺ ion/receptor switchable systems are described: 1) The K⁺‐induced self‐assembly of the Mg²⁺‐dependent DNAzyme subunits into a catalytic nanostructure using the assembly of G‐quadruplexes as bridging unit. 2) The K⁺‐induced stabilization of the anti‐thrombin G‐quadruplex nanostructure that inhibits the hydrolytic functions of thrombin. 3) The K⁺‐induced opening of DNA tweezers through the stabilization of G‐quadruplexes on the “tweezers’ arms" and the release of a strand bridging the tweezers into a closed structure. In all of the systems reversible, switchable, functions are demonstrated. For all systems two different signals are used to follow the switchable functions (fluorescence and the catalytic functions of the derived hemin–G‐quadruplex DNAzyme).     

G-quadruplex-hemin DNAzyme-amplified colorimetric detection of Ag ion

A G-quadruplex-hemin DNAzyme-amplified Ag⁺-sensing method was developed based on the ability of Ag⁺ to stabilize C-C mismatches by forming C-Ag⁺-C base pairs. In this method, only one unlabelled oligonucleotide strand was used. In the absence of Ag⁺, the oligonucleotide strand formed an intramolecular duplex. The G-rich sequence in the oligonucleotide was partially caged in this duplex structure and cannot fold into the G-quadruplex structure. The addition of Ag⁺ promoted the formation of another intramolecular duplex in which C-C mismatches were stabilized by C-Ag⁺-C base pairs, leading to the release of the G-rich sequence which can fold into a G-quadruplex capable to bind hemin to form a catalytically active G-quadruplex-hemin DNAzyme. As a result, a UV-vis absorbance increasing was observed in the H₂O₂-ABTS (2,2′-azinobis(3-ethylbenzothiozoline)-6-sulfonic acid) reaction system. This “turn-on” process allowed the detection of aqueous Ag⁺ at concentrations as low as 6.3 nM using a simple colorimetric technique, showing a high selectivity over a range of other metal ions.     

Highly Selective Artificial Potassium Ion Channels Constructed from Pore‐Containing Helical Oligomers

Potassium ion channels specifically transport K⁺ ions over Na⁺ ions across a cell membrane. A queue of four binding sites in the K⁺ channel pore plays significant roles during highly selective conduction. A kind of aromatic helical oligomer was synthesized that can selectively bind K⁺ over Na⁺. By aromatic stacking of helical oligomers, a type of artificial K⁺ channels with contiguous K⁺ binding sites was constructed. Such artificial channels exhibited exceptionally high K⁺/Na⁺ selectivity ratios during transmembrane ion conduction.     

Molecular Design of Calixarene 5. Syntheses and Cation Selectivities of Novel Schiff's Base p‐tert‐Butylcalix[4]arenes

Five novel Schiff's bases p‐tert‐butylcalix[4]arenes have been synthesized in high yields by the reaction of 1,3‐distally disubstituted p‐tert‐butylcalix[4]arene amine (1) with the corresponding aromatic aldehydes, and their cation binding abilities and selectivities with alkali and heavy metal ions have been evaluated by solvent extraction of aqueous metal picrates to show the highest Ag⁺ extractability for Schiff's base p‐tert‐butylcalix[4]arene (6) and the best Na⁺/Li⁺ and Ag⁺/Ti⁺ selectivities for Schiff's base p‐tert‐butylcalix[4]arene (4 and 2) over any other calix[4]arene derivatives, respectively.     

Intelligent demethylase-driven DNAzyme sensor for highly reliable metal-ion imaging in living cells

The accurate intracellular imaging of metal ions requires an exquisite site-specific activation of metal-ion sensors, for which the pervasive epigenetic regulation strategy can serve as an ideal alternative thanks to its orthogonal control feature and endogenous cell/tissue-specific expression pattern. Herein, a simple yet versatile demethylation strategy was proposed for on-site repairing-to-activating the metal-ion-targeting DNAzyme and for achieving the accurate site-specific imaging of metal ions in live cells. This endogenous epigenetic demethylation-regulating DNAzyme system was prepared by modifying the DNAzyme with an m⁶A methylation group that incapacitates the DNAzyme probe, thus eliminating possible off-site signal leakage, while the cell-specific demethylase-mediated removal of methylation modification could efficiently restore the initial catalytic DNAzyme for sensing metal ions, thus allowing a high-contrast bioimaging in live cells. This epigenetic repair-to-activate DNAzyme strategy may facilitate the robust visualization of disease-specific biomarkers for in-depth exploration of their biological functions.

A simple yet versatile demethylation strategy is proposed for an on-site repairing-to-activating metal-ion-targeting DNAzyme and for achieving the highly reliable site-specific imaging of metal ions in live cells.     

Detection of Metal Ions (Cu2+, Hg2+) and Cocaine by Using Ligation DNAzyme Machinery

The Cu²⁺‐dependent ligation DNAzyme is implemented as a biocatalyst for the colorimetric or chemiluminescence detection of Cu²⁺ ions, Hg²⁺ ions, or cocaine. These sensing platforms are based on the structural tailoring of the sequence of the Cu²⁺‐dependent ligation DNAzyme for specific analytes. The tethering of a subunit of the hemin/G‐quadruplex DNAzyme to the ligation DNAzyme sequence, and the incorporation of an imidazole‐functionalized nucleic‐acid sequence, which acts as a co‐substrate for the ligation DNAzyme that is tethered to the complementary hemin/G‐quadruplex subunit. In the presence of different analytes, Cu²⁺ ions, Hg²⁺ ions, or cocaine, the pretailored Cu²⁺‐dependent ligation DNAzyme sequence stimulates the respective ligation process by combining the imidazole‐functionalized co‐substrate with the ligation DNAzyme sequence. These reactions lead to the self‐assembly of stable hemin/G‐quadruplex DNAzyme nanostructures that enable the colorimetric analysis of the substrate through the DNAzyme‐catalyzed oxidation of 2,2′‐azinobis(3‐ethylbenzothiazoline‐6‐sulfonic acid), ABTS^2?, by H₂O₂ into the colored product ABTS^.?, or the chemiluminescence detection of the substrate through the DNAzyme‐catalyzed oxidation of luminol by H₂O₂. The detection limits for the sensing of Cu²⁺ ions, Hg²⁺ ions, and cocaine correspond to 1 nM , 10 nM and 2.5 μM , respectively. These different sensing platforms also reveal impressive selectivities.     

A Multi-component All-DNA Biosensing System Controlled by a DNAzyme

We report on a programmable all-DNA biosensing system that centers on the use of a 4-way junction (4WJ) to transduce a DNAzyme reaction into an amplified signal output. A target acts as a primary input to activate an RNA-cleaving DNAzyme, which then cleaves an RNA-containing DNA substrate that is designed to be a component of a 4WJ. The formation of the 4WJ controls the release of a DNA output that becomes an input to initiate catalytic hairpin assembly (CHA), which produces a second DNA output that controls assembly of a split G-quadruplex as a fluorescence signal generator. The 4WJ can be configured to produce either a turn-off or turn-on switch to control the degree of CHA, allowing target concentration to be determined in a quantitative manner. We demonstrate this approach by creating a sensor for E. coli that could detect as low as 50 E. coli cells mL⁻¹ within 85 min and offers an amplified bacterial detection method that does not require a protein enzyme.     

Colorimetric detection of sodium ion in serum based on the G-quadruplex conformation related DNAzyme activity

There has been a big challenge in developing the Na⁺ sensor that can be practically used in the physiological system with the interference of large amounts of K⁺. In this research, a novel Na⁺ sensor has been designed based on the G-quadruplex-conformation related DNAzyme activity. The sensor exhibits high selectivity and sensitivity with the detection limit of 0.6 μM, which enables the sensor to be practically used in determination of the Na⁺ level in serum. The research not only provides a simple Na⁺ sensor but also opens a new way for developing the detection technology of Na⁺.     

Competitive binding exchange between alkali metal ions (K+, Rb+, and Cs+) and Na+ ions bound to the dimeric quadruplex [d(G4T4G4)]2: a 23Na and 1H NMR study

A comparative study of the competitive cation exchange between the alkali metal ions K⁺, Rb⁺, and Cs⁺ and the Na⁺ ions bound to the dimeric quadruplex [d(G₄T₄G₄)]₂ was performed in aqueous solution by a combined use of the ²³Na and ¹H NMR spectroscopy. The titration data confirm the different binding affinities of these ions for the G‐quadruplex and, in particular, major differences in the behavior of Cs⁺ as compared to the other ions were found. Accordingly, Cs⁺ competes with Na⁺ only for the binding sites at the quadruplex surface (primarily phosphate groups), while K⁺ and Rb⁺ are also able to replace sodium ions located inside the quadruplex. Furthermore, the ¹H NMR results relative to the CsCl titration evidence a close approach of Cs⁺ ions to the phosphate groups in the narrow groove of [d(G₄T₄G₄)]₂. Based on a three‐site exchange model, the ²³Na NMR relaxation data lead to an estimate of the relative binding affinity of Cs⁺ versus Na⁺ for the quadruplex surface of 0.5 at 298 K. Comparing this value to those reported in the literature for the surface of the G‐quadruplex formed by 5′‐guanosinemonophosphate and for the surface of double‐helical DNA suggests that topology factors may have an important influence on the cation affinity for the phosphate groups on DNA. Copyright © 2009 John Wiley & Sons, Ltd.     

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.