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).     

A G‐Quadruplex/Hemin Complex with Switchable Peroxidase Activity by DNA Hybridization

A hemin‐binding DNA G‐quadruplex (also known as a hemin aptamer or DNAzyme) has been previously reported to be able to enhance the peroxidase activity of hemin. In this work, we described a DNAzyme structure that had an effector‐recognizing part appearing as a single stranded DNA linkage flanked by two split G‐quadruplex halves. Hybridization of the single stranded part in the enzyme with a perfectly matched DNA strand (effector) formed a rigid DNA duplex between the two G‐quadruplex halves and thus efficiently suppressed the enzymatic activity of the G‐quadruplex/hemin complex, while the mismatched effector strand was not able to regulate the peroxidase activity effectively. With 2,2′‐azinobis(3‐ethylbenzthiazoline)‐6‐sulfonic acid (ABTS) as an oxidizable substrate, we were able to characterize the formation of the re‐engineered G‐quadruplex/hemin complex and verify its switchable peroxidase activity. Our results show that the split G‐quadruplex is an especially useful module to design low‐cost and label‐free sensors toward various biologically or environmentally interesting targets.     

Light‐Induced Reversible Reconfiguration of DNA‐Based Constitutional Dynamic Networks: Application to Switchable Catalysis

The light‐induced reversible and cyclic reconfiguration of constitutional dynamic networks, consisting of supramolecular nucleic acid structures as constituents and a photoisomerizable trans/cis‐azobenzene‐functionalized nucleic acid as the trigger is demonstrated. In addition, the cyclic photochemical reconfiguration of the constitutional dynamic networks guides the switchable on/off operation of an emerging hemin/G‐quadruplex DNAzyme.     

Rational Design of an “OFF–ON” Phosphorescent Chemodosimeter Based on an Iridium(III) Complex and Its Application for Time‐Resolved Luminescent Detection and Bioimaging of Cysteine and Homocysteine

Biothiols, such as cysteine (Cys) and homocysteine (Hcy), play very crucial roles in biological systems. Abnormal levels of these biothiols are often associated with many types of diseases. Therefore, the detection of Cys (or Hcy) is of great importance. In this work, we have synthesized an excellent “OFF‐ON” phosphorescent chemodosimeter 1 for sensing Cys and Hcy with high selectivity and naked‐eye detection based on an Ir^III complex containing a 2,4‐dinitrobenzenesulfonyl (DNBS) group within its ligand. The “OFF‐ON” phosphorescent response can be assigned to the electron‐transfer process from Ir^III center and C^N ligands to the DNBS group as the strong electron‐acceptor, which can quench the phosphorescence of probe 1 completely. The DNBS group can be cleaved by thiols of Cys or Hcy, and both the ³M LCT and ³LC states are responsible for the excited‐state properties of the reaction product of probe 1 and Cys (or Hcy). Thus, the phosphorescence is switched on. Based on these results, a general principle for designing “OFF‐ON” phosphorescent chemodosimeters based on heavy‐metal complexes has been provided. Importantly, utilizing the long emission‐lifetime of phosphorescence signal, the time‐resolved luminescent assay of 1 in sensing Cys was realized successfully, which can eliminate the interference from the short‐lived background fluorescence and improve the signal‐to‐noise ratio. As far as we know, this is the first report about the time‐resolved luminescent detection of biothiols. Finally, probe 1 has been used successfully for bioimaging the changes of Cys/Hcy concentration in living cells.     

DNA Switches: From Principles to Applications

The base sequence of nucleic acid encodes structural and functional properties into the biopolymer. Structural information includes the formation of duplexes, G‐quadruplexes, i‐motif, and cooperatively stabilized assemblies. Functional information encoded in the base sequence involves the strand‐displacement process, the recognition properties by aptamers, and the catalytic functions of DNAzymes. This Review addresses the implementation of the information encoded in nucleic acids to develop DNA switches. A DNA switch is a supramolecular nucleic acid assembly that undergoes cyclic, switchable, transitions between two distinct states in the presence of appropriate triggers and counter triggers, such as pH value, metal ions/ligands, photonic and electrical stimuli. Applications of switchable DNA systems to tailor switchable DNA hydrogels, for the controlled drug‐release and for the activation of switchable enzyme cascades, are described, and future perspectives of the systems are addressed.     

Ruthenium Complex “Light Switches” that are Selective for Different G‐Quadruplex Structures

Recognition and regulation of G‐quadruplex nucleic acid structures is an important goal for the development of chemical tools and medicinal agents. The addition of a bromo‐substituent to the dipyridylphenazine (dppz) ligands in the photophysical “light switch”, [Ru(bpy)₂dppz]²⁺, and the photochemical “light switch”, [Ru(bpy)₂dmdppz]²⁺, creates compounds with increased selectivity for an intermolecular parallel G‐quadruplex and the mixed‐hybrid G‐quadruplex, respectively. When [Ru(bpy)₂dppz‐Br]²⁺ and [Ru(bpy)₂dmdppz‐Br]²⁺ are incubated with the G‐quadruplexes, they have a stabilizing effect on the DNA structures. Activation of [Ru(bpy)₂dmdppz‐Br]²⁺ with light results in covalent adduct formation with the DNA. These complexes demonstrate that subtle chemical modifications of Ru^II complexes can alter G‐quadruplex selectivity, and could be useful for the rational design of in vivo G‐quadruplex probes.     

Dual Amplified Electrochemical Immunosensor for Hepatitis B Virus Surface Antigen Detection Using Hemin/G‐Quadruplex Immobilized onto Fe3O4‐AuNPs or (Hemin‐Amino‐rGO‐Au) Nanohybrid

A sensitive electrochemical immunosensor for Hepatitis B virus surface antigen (HBsAg) detection was fabricated based on hemin/G‐quadruplex interlaced onto Fe₃O₄‐AuNPs or hemin ‐amino‐reduced graphene oxide nanocomposite (H‐amino‐rGO‐Au). G‐quadruplex DNAzyme, which is composed of hemin and guanine‐rich nucleic acid, is an effective signal amplified tool for its outstanding peroxidase activity and Fe₃O₄‐AuNPs or (H‐amino‐rGO‐Au) nanocomposites with quasi‐enzyme activity provide appropriate support for the immobilization of hemin/G‐quadruplex. The target protein was sandwiched between the primary antibody immobilized on the GO and secondary antibody immobilized on the Fe₃O₄‐AuNPs or (H‐amino‐rGO‐Au) nanocomposites and glutaraldehyde was used as linking agent for the immobilization of primary antibody on the surface of GO. Both Fe₃O₄‐AuNPs and H‐amino‐rGO‐Au nanocomposite and also hemin/G‐quadruplex can cooperate the electrocatalytic reduction of H₂O₂ in the presence of methylene blue as mediator. The proposed immunosensor has a wide linear dynamic range of 0.1 pg/ml to 300 pg/ml with a detection limit of 60 fg/ml when Fe₃O₄‐AuNPs was used for immobilization of hemin/G‐quadruplex, while the dynamic range and DL were 0. 1–1000 pg/mL and 10 fg/mL, respectively in the presence of H‐amino‐rGO‐ Au nanocomposite as platform for immobilizing of hemin/G‐quadruplex. The proposed immunosensor was also used for analysis of HBsAg in spiked human serum samples with satisfactory results.     

Multitasking Water‐Soluble Synthetic G‐Quartets: From Preferential RNA–Quadruplex Interaction to Biocatalytic Activity

Natural G‐quartets, a cyclic and coplanar array of four guanine residues held together through a Watson–Crick/Hoogsteen hydrogen‐bond network, have received recently much attention due to their involvement in G‐quadruplex DNA, an alternative higher‐order DNA structure strongly suspected to play important roles in key cellular events. Besides this, synthetic G‐quartets (SQ), which artificially mimic native G‐quartets, have also been widely studied for their involvement in nanotechnological applications (i.e., nanowires, artificial ion channels, etc.). In contrast, intramolecular synthetic G‐quartets (iSQ), also named template‐assembled synthetic G‐quartets (TASQ), have been more sparingly investigated, despite a technological potential just as interesting. Herein, we report on a particular iSQ named ^PNADOTASQ, which demonstrates very interesting properties in terms of DNA and RNA interaction (notably its selective recognition of quadruplexes according to a bioinspired process) and catalytic activities, through its ability to perform peroxidase‐like hemin‐mediated oxidations either in an autonomous fashion (i.e., as pre‐catalyst for TASQzyme reactions) or in conjunction with quadruplex DNA (i.e., as enhancing agents for DNAzyme processes). These results provide a solid scientific basis for TASQ to be used as multitasking tools for bionanotechnological applications.     

Binding of a Telomestatin Derivative Changes the Mechanical Anisotropy of a Human Telomeric G‐Quadruplex

Mechanical anisotropy is an essential property for biomolecules to assume structural and functional roles in mechanobiology. However, there is insufficient information on the mechanical anisotropy of ligand–biomolecule complexes. Herein, we investigated the mechanical property of individual human telomeric G‐quadruplexes bound to telomestatin, using optical tweezers. Stacking of the ligand to the G‐tetrad planes changes the conformation of the G‐quadruplex, which resembles a balloon squeezed in certain directions. Such a squeezed balloon effect strengthens the G‐tetrad planes, but dislocates and weakens the loops in the G‐quadruplex upon ligand binding. These dynamic interactions indicate that the binding between the ligand and G‐quadruplex follows the induced‐fit model. We anticipate that the altered mechanical anisotropy of the ligand–G‐quadruplex complex can add additional level of regulations on the motor enzymes that process DNA or RNA molecules.     

A Solution‐Processable Donor–Acceptor Compound Containing Boron(III) Centers for Small‐Molecule‐Based High‐Performance Ternary Electronic Memory Devices

A novel small‐molecule boron(III)‐containing donor–acceptor compound has been synthesized and employed in the fabrication of solution‐processable electronic resistive memory devices. High ternary memory performances with low turn‐on (V_Th1=2.0 V) and distinct threshold voltages (V_Th2=3.3 V), small reading bias (1.0 V), and long retention time (>10⁴ seconds) with a large ON/OFF ratio of each state (current ratio of “OFF”, “ON1”, and “ON2”=1:10³:10⁶) have been demonstrated, suggestive of its potential application in high‐density data storage. The present design strategy provides new insight in the future design of memory devices with multi‐level transition states.     

Impedimetric DNA‐Based Biosensor for Silver Ions Detection with Hemin/G‐Quadruplex Nanowire as Enhancer

A DNA‐based biosensor was reported for detection of silver ions (Ag⁺) by electrochemical impedance spectroscopy (EIS) with [Fe(CN)₆]^4?/3? as redox probe and hybridization chain reaction (HCR) induced hemin/G‐quadruplex nanowire as enhanced label. In the present of target Ag⁺, Ag⁺ interacted with cytosine‐cytosine (C? C) mismatch to form the stable C? Ag⁺? C complex with the aim of immobilizing the primer DNA on electrode, which thus triggered the HCR to form inert hemin/G‐quadruplex nanowire with an amplified EIS signal. As a result, the DNA biosensor showed a high sensitivity with the concentration range spanning from 0.1 nM to 100 µM and a detection limit of 0.05 nM.     

A Metalloregulated Four‐State Nanoswitch Controls Two‐Step Sequential Catalysis in an Eleven‐Component System

The nanomechanical switch 1 with its three orthogonal binding motifs—the zinc(II) porphyrin, azaterpyridine, and shielded phenanthroline binding station—is quantitatively and reversibly toggled back and forth between four different switching states by means of addition and removal of appropriate metal‐ion inputs. Two of the four switching stages are able to initiate catalytic transformations (ON1, ON2), while the two others shut down any reaction (OFF1, OFF2). Thus, in a cyclic four‐state switching process the sequential transformation A + B + C → AB + C → ABC can be controlled, which proceeds stepwise along the switching states OFF1→ON1 (click reaction: A + B → AB )→OFF2→ON2 (Michael addition: AB + C → ABC )→OFF1. Two consecutive cycles of the sequential catalysis were realized without loss in activity in a reaction system with eleven different components.     

Molecular Logic Gates and Switches Based on 1,3,4‐Oxadiazoles Triggered by Metal Ions

Organic molecular devices for information processing applications are highly useful building blocks for constructing molecular‐level machines. The development of “intelligent” molecules capable of performing logic operations would enable molecular‐level devices and machines to be created. We designed a series of 2,5‐diaryl‐1,3,4‐oxadiazoles bearing a 2‐(para‐substituted)phenyl and a 5‐(o‐pyridyl) group (substituent X=NMe₂, OEt, Me, H, and Cl; 1 a – e ) that form a bidentate chelating environment for metal ions. These compounds showed fluorescence response profiles varying in both emission intensity and wavelength toward the tested metal ions Ni²⁺, Cu²⁺, Zn²⁺, Cd²⁺, Hg²⁺, and Pb²⁺ and the responses were dependent on the substituent X, with those of 1 d being the most substantial. The 1,3,4‐oxadiazole O or N atom and pyridine N atom were identified as metal‐chelating sites. The fluorescence responses of 1 d upon metal chelation were employed for developing truth tables for OR, NOR, INHIBIT, and EnNOR logic gates as well as “ON‐OFF‐ON” and “OFF‐ON‐OFF” fluorescent switches in a single 1,3,4‐oxadiazole molecular system.     

G‐Quadruplex‐Linked Supersandwich DNA Structure for Electrochemical Amplified Detection of Thrombin

In this paper, a novel strategy of electrochemical amplified detection of thrombin based on G‐quadruplex‐linked supersandwich structure was described. In the presence of K⁺ and hemin, the original hairpin DNA sequence activated an autonomous cross‐opening process to build up hemin/G‐quadruplex structure and can hybridize to form supersandwich structure containing multiple signal labels. With the addition of thrombin, it conjugated with its aptamer, leading to a remarkably descended signal. The supersandwich‐amplified electrochemical sensor system was highly sensitive in the concentration range from 10^?6 to 10^?10 M with a detection limit of 10 pM and also demonstrated excellent selectivity. The amplifying supersandwich structure with multiple labels can be implemented as a versatile sensing platform for analyzing other DNA in the presence of the appropriate probe.     

Metal‐Ion‐Triggered Exonuclease III Activity for the Construction of DNA Colorimetric Logic Gates

The logic system is obtained by using a series of double‐stranded (ds) DNA templates with mismatched base pairs (T–T or C–C) and ion‐modulated exonuclease III (Exo III) activity, in which the Exo III cofactors, Hg²⁺ and Ag⁺ ions, are used as inputs for the activation of the respective scission of Exo III based on the formation of T–Hg²⁺–T or C–Ag⁺–C base pairs. Additionally, two kinds of signal probes are utilized to transduce the logic operations. One is the two split G‐rich DNA strands that are used to design the OR, AND, INHIBIT, and XOR gates, whereas the other is the self‐assembled split G‐quadruplex structure to construct NOR, NAND, IMPLICATION, and XNOR operations based on DNA hybridization and strand displacement. In the presence of hemin, the split G‐quadruplex biocatalyzes the formation of a colored product, which is an output signal for the different logic gates. Thus, we have constructed a complete set of colorimetric DNA logic gates based on the Exo III and split G‐quadruplex for the first time. In addition, we are able to effortlessly recognize the logic output signals by the naked eye and their simplicity and cost‐effective design is the most apparent feature for the logic gates developed in this work.     

Enantioconvergent Biocatalytic Redox Isomerization

Alcohol dehydrogenases can act as powerful catalysts in the preparation of optically pure γ‐hydroxy‐δ‐lactones by means of an enantioconvergent dynamic redox isomerization of readily available Achmatowicz‐type pyranones. Imitating the traditionally metal‐mediated “borrowing hydrogen” approach to shuffle hydrides across molecular architectures and interconvert functional groups, this chemoinspired and purely biocatalytic interpretation effectively expands the enzymatic toolbox and provides new opportunities in the assembly of multienzyme cascades and tailor‐made cellular factories.     

The destabilization of hydrogen bonds in an external E-field for improved switch performance

The effect of an electric field on a recently proposed molecular switch based on a quinone analogue was investigated using next-generation quantum theory of atoms in molecules (QTAIM) methodology. The reversal of a homogenous external electric field was demonstrated to improve the “OFF” functioning of the switch. This was achieved by destabilization of the H atom participating in the tautomerization process along the hydrogen bond that defines the switch. The “ON” functioning of the switch, from the position of the tautomerization barrier, is also improved by the reversal of the homogenous external electric field: this result was previously inaccessible. The “ON” and “OFF” functioning of the switch was visualized in terms of the response of the most preferred directions of motion of the electronic charge density to the applied external field. All measures from QTAIM and the stress tensor provide consistent results for the factors affecting the “ON” and “OFF” switch performance. Our analysis therefore demonstrates use for future design of molecular electronic devices. © 2019 Wiley Periodicals, Inc.     

A Thermophilic Tetramolecular G‐Quadruplex/Hemin DNAzyme

The quadruplex‐based DNAzyme system is one of the most useful artificial enzymes or catalysts; their unique properties make them reliable alternatives to proteins for performing catalytic transformation. The first prototype of a thermally stable DNAzyme system is presented. This thermophilic DNAzyme is capable of oxidizing substrates at high temperatures (up to 95 °C) and long reaction times (up to 18 h at 75 °C). The catalytic activity of the DNAzymes were investigated with the standard peroxidase‐mimicking oxidation of 2,2′‐azino‐bis(3‐ethylbenzothiozoline‐6‐sulfonic acid) (ABTS) by H₂O₂. The step‐by‐step design of this unique heat‐activated G‐quadruplex/hemin catalyst, including the modification of adenines at both ends of G‐tracts, the choice of cation, and its concentration for DNAzyme stabilization, is described. This work investigates thoroughly the molecular basis of these catalytic properties and provides an example of an industrially relevant application.     

Switchable Amplification of Vibrational Circular Dichroism as a Probe of Local Chiral Structure

A new method to detect the vibrational circular dichroism (VCD) of a localized part of a chiral molecular system is reported. A local VCD amplifier was implemented, and the distance dependence of the amplification was investigated in a series of peptides. The results indicate a characteristic distance of 2.0±0.3 bonds, which suggests that the amplification is a localized phenomenon. The amplifier can be covalently coupled to a specific part of a molecule, and can be switched ON and OFF electrochemically. By subtracting the VCD spectra obtained when the amplifier is in the ON and OFF states, the VCD of the local environment of the amplifier can be separated from the total VCD spectrum. Switchable local VCD amplification thus makes it possible to “zoom in” on a specific part of a chiral molecule.     

Template‐Assembled Synthetic G‐Quadruplex (TASQ): A Useful System for Investigating the Interactions of Ligands with Constrained Quadruplex Topologies

A new biomolecular device for investigating the interactions of ligands with constrained DNA quadruplex topologies, using surface plasmon resonance (SPR), is reported. Biomolecular systems containing an intermolecular‐like G‐quadruplex motif 1 (parallel G‐quadruplex conformation), an intramolecular G‐quadruplex 2 , and a duplex DNA 3 have been designed and developed. The method is based on the concept of template‐assembled synthetic G‐quadruplex (TASQ), whereby quadruplex DNA structures are assembled on a template that allows precise control of the parallel G‐quadruplex conformation. Various known G‐quadruplex ligands have been used to investigate the affinities of ligands for intermolecular 1 and intramolecular 2 DNA quadruplexes. As anticipated, ligands displaying a π‐stacking binding mode showed a higher binding affinity for intermolecular‐like G‐quadruplexes 1 , whereas ligands with other binding modes (groove and/or loop binding) showed no significant difference in their binding affinities for the two quadruplexes 1 or 2 . In addition, the present method has also provided information about the selectivity of ligands for G‐quadruplex DNA over the duplex DNA. A numerical parameter, termed the G‐quadruplex binding mode index (G4‐BMI), has been introduced to express the difference in the affinities of ligands for intermolecular G‐quadruplex 1 against intramolecular G‐quadruplex 2 . The G‐quadruplex binding mode index (G4‐BMI) of a ligand is defined as follows: G4‐BMI=K_D^intra/K_D^inter, where K_D^intra is the dissociation constant for intramolecular G‐quadruplex 2 and K_D^inter is the dissociation constant for intermolecular G‐quadruplex 1 . In summary, the present work has demonstrated that the use of parallel‐constrained quadruplex topology provides more precise information about the binding modes of ligands.