Activity Enhancement of G‐Quadruplex/Hemin DNAzyme by Flanking d(CCC)

G‐quadruplex (G4)/hemin DNAzymes have been extensively applied in bioanalysis and molecular devices. However, their catalytic activity is still much lower than that of proteinous enzymes. The G4/hemin DNAzyme activity is correlated with the G4 conformations and the solution conditions. However, little is known about the effect of the flanking sequences on the activity, though they are important parts of G4s. Here, we report sequences containing d(CCC), flanked on both ends of the G4‐core sequences remarkably enhance their DNAzyme activity. By using circular dichroism and UV‐visible spectroscopy, the d(CCC) flanking sequences were demonstrated to improve the hemin binding affinity to G4s instead of increasing the parallel G4 formation, which might explain the enhanced DNAzyme activity. Meanwhile, the increased hemin binding ability promoted the degradation of hemin within the DNAzyme by H₂O₂. Furthermore, the DNAzyme with d(CCC) flanking sequences showed strong tolerance to pH value changes, which makes it more suitable for applications requiring wide pH conditions. The results highlight the influence of the flanking sequences on the DNAzyme activity and provide insightful information for the design of highly active DNAzymes.     

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.     

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.     

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).     

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.     

Enzyme‐Regulated Activation of DNAzyme: A Novel Strategy for a Label‐Free Colorimetric DNA Ligase Assay and Ligase‐Based Biosensing

The DNA nick repair catalyzed by DNA ligase is significant for fundamental life processes, such as the replication, repair, and recombination of nucleic acids. Here, we have employed ligase to regulate DNAzyme activity and developed a homogeneous, colorimetric, label‐free and DNAzyme‐based strategy to detect DNA ligase activity. This novel strategy relies on the ligation‐trigged activation or production of horseradish peroxidase mimicking DNAzyme that catalyzes the generation of a color change signal; this results in a colorimetric assay of DNA ligase activity. Using T4 DNA ligase as a model, we have proposed two approaches to demonstrate the validity of the DNAzyme strategy. The first approach utilizes an allosteric hairpin‐DNAzyme probe specifically responsive to DNA ligation; this approach has a wide detection range from 0.2 to 40 U mL^?1 and a detection limit of 0.2 U mL^?1. Furthermore, the approach was adapted to probe nucleic acid phosphorylation and single nucleotide mismatch. The second approach employs a “split DNA machine” to produce numerous DNAzymes after being reassembled by DNA ligase; this greatly enhances the detection sensitivity by a signal amplification cascade to achieve a detection limit of 0.01 U mL^?1.     

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.     

Characterization of G-quadruplex/hemin peroxidase: substrate specificity and inactivation kinetics

Recently, G-quadruplex/hemin (G4/hemin) complexes have been found to exhibit peroxidase activity, and this feature has been extensively exploited for colorimetric detection of various targets. To further understand and characterize this important DNAzyme, its substrate specificity, inactivation mechanism, and kinetics have been examined by comparison with horseradish peroxidase (HRP). G4/hemin DNAzyme exhibits broader substrate specificity and much higher inactivation rate than HRP because of the exposure of the catalytic hemin center. The inactivation of G4/hemin DNAzyme is mainly attributed to the degradation of hemin by H(2)O(2) rather than the destruction of G4. Both the inactivation rate and catalytic oxidation rate of G4/hemin DNAzyme depend on the concentration of H(2)O(2), which suggests that active intermediates formed by G4/hemin and H(2)O(2) are the branch point of catalysis and inactivation. Reducing substrates greatly inhibit the inactivation of G4/hemin DNAzyme by rapidly reacting with the active intermediates. A possible catalytic and inactivation process of G4/hemin has been proposed. These results imply a potential cause for the hemin-mediated cellular injury and provide insightful information for the future application of G4/hemin DNAzyme.     

Multifunctional G‐Quadruplex Aptamers and Their Application to Protein Detection

Two significant G‐quadruplex aptamers named AGRO100 and T30695 are identified as multifunctional aptamers that can bind the protein ligands nucleolin or HIV‐1 integrase and hemin. Besides their strong binding to target proteins, both AGRO100 and T30695 exhibit high hemin‐binding affinities comparable to that of the known aptamer (termed PS2M) selected by the in vitro evolution process. Most importantly, their corresponding hemin–DNA complexes reveal excellent peroxidase‐like activities, higher than that of the reported hemin–PS2M DNAzyme. This enables these multifunctional aptamers to be applied to the sensitive detection of proteins, which is demonstrated by applying AGRO100 to the chemiluminescence detection of nucleolin expressed at the surface of HeLa cells. Based on the specific AGRO100–nucleolin interaction, the surface‐expressed nucleolin of HeLa cells is labeled in situ with the hemin–AGRO100 DNAzyme, and then determined in the luminol–H₂O₂ system. Through this approach, the sensitive detection of total nucleolin expressed at the surface of about 6000 HeLa cells is accomplished. Our results suggest that exploiting new functions of existing aptamers will help to extend their potential applications in the biochemical field.     

Inducement and stabilization of G-quadruplex DNA by a thiophene-containing dinuclear ruthenium(II) complex

G-quadruplex structures are attractive targets for the development of anticancer drugs, as their formation in human telomere could impair telomerase activity, thus inducing apoptosis in cancer cells. In this work, a thiophene-containing dinuclear ruthenium(II) complex, [Ru₂(bpy)₄(H₂bipt)]⁴⁺ {bpy = 2,2′-bipyridine, H₂bipt = 2,5-bis[1,10]phenanthrolin[4,5-f]-(imidazol-2-yl)thiophene}, was prepared and the interaction between the complex and human telomeric DNA oligomers 5′-G₃(T₂AG₃)₃-3′ (HTG21) has been investigated by UV-Vis, fluorescence and circular dichroism (CD) spectroscopy, fluorescence resonance energy transfer (FRET) melting assay, polymerase chain reaction (PCR) stop assay, fluorescent intercalator displacement (FID) titrations, Job plot and color reaction studies. The results indicate that the complex can well induce and stabilize the formation of antiparallel G-quadruplex of telomeric DNA in the presence or absence of metal cations, and the ΔT_m value of the G-quadruplex DNA treated with the complex was obtained to be 12.8 °C even at levels of 50-fold molar of duplex DNA (calf-thymus DNA), suggesting that the complex exhibits higher G-quadruplex DNA selectivity over duplex DNA. The complex shows high interaction ability with G-quadruplex DNA at (1.17 ± 0.12) × 10⁷ M^?1 binding affinity using a 2:1 [complex]/[quadruplex] binding mode ratio. A novel visual method has been developed here for making a distinction between G-quadruplex DNA and duplex DNA by our ruthenium complex binding hemin to form the hemin-G-quadruplex DNAzyme.     

Switchable Enzyme/DNAzyme Cascades by the Reconfiguration of DNA Nanostructures

Mimicking cellular transformations and signal transduction pathways by means of biocatalytic cascades proceeding in organized media is a scientific challenge. We describe two DNA machines that enable the “ON/OFF” switchable activation and deactivation of three‐component biocatalytic cascades. One system consists of a reconfigurable DNA tweezers‐type structure, whereas in the second system the catalytic cascade proceeds on a switchable DNA clamp scaffold. The three‐component catalytic cascades consist of β‐galactosidase (β‐Gal), glucose oxidase (GOx), and the K⁺‐ion‐stabilized hemin‐G‐quadruplex horseradish peroxidase (HRP)‐mimicking DNAzyme. The hemin‐G‐quadruplex‐bridged closed structure of the tweezers or clamp allows the biocatalytic cascades to operate (switched “ON′′), whereas separation of the hemin‐G‐quadruplex by means of 18‐crown‐6‐ether opens the tweezers/clamp structures, thus blocking the catalytic cascade (switched ”OFF“). This study is complemented by two‐component, switchable biocatalytic cascades composed of GOx and hemin‐G‐quadruplex assembled on hairpin‐bridged DNA tweezers or clamp nanostructures.     

A pseudo triple-enzyme electrochemical aptasensor based on the amplification of Pt–Pd nanowires and hemin/G-quadruplex

Our present work aimed at developing a pseudo triple-enzyme cascade electrocatalytic electrochemical aptasensor for determination of thrombin with the amplification of alcohol dehydrogenase (ADH)-Pt–Pd nanowires bionanocomposite and hemin/G-quadruplex structure that simultaneously acted as NADH oxidase and HRP-mimicking DNAzyme. With the addition of ethanol to the electrolyte, the ADH immobilized on the Pt–Pd nanowires catalyzed ethanol to acetaldehyde accompanied by NAD⁺ being converted to NADH. Then the hemin/G-quadruplex firstly served as NADH oxidase, converting the produced NADH to NAD⁺ with the concomitant local formation of high concentration of H₂O₂. Subsequently, the hemin/G-quadruplex acted as HRP-mimicking DNAzyme, bioelectrocatalyzing the produced H₂O₂. At the same time, the Pt–Pd nanowires employed in our strategy not only provided a large surface area for immobilizing thrombin binding aptamer (TBA) and ADH, but also served as HRP-mimicking DNAzyme which rapidly bioelectrocatalyzed the reduction of the produced H₂O₂. Thus, such a pseudo triple-enzyme cascade electrochemical aptasensor could greatly promote the electron transfer of hemin and resulted in the dramatic enhancement of electrochemical signal. As a result, a wide dynamic concentration linear range from 0.2 pM to 20 nM with a low detection limit of 0.067 pM for thrombin (TB) determination was obtained. The excellent performance indicated that our strategy was a promising way for ultrasensitive assays in electrochemical aptasensors.     

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.     

Ultrathin Covalently Bound Organic Layers on Mica: Formation of Atomically Flat Biofunctionalizable Surfaces

Mica is the substrate of choice for microscopic visualization of a wide variety of intricate nanostructures. Unfortunately, the lack of a facile strategy for its modification has prevented the on‐mica assembly of nanostructures. Herein, we disclose a convenient catechol‐based linker that enables various surface‐bound metal‐free click reactions, and an easy modification of mica with DNA nanostructures and a horseradish peroxidase mimicking hemin/G‐quadruplex DNAzyme.     

Peroxidase activity-structure relationship of the intermolecular four-stranded G-quadruplex-hemin complexes and their application in Hg ion detection

The peroxidase activities of the complexes of hemin and intermolecular four-stranded G-quadruplexes formed by short-stranded X_nG_mX_p sequences (X = A, T or C), especially T_nG_mT_p sequences, were compared. The results, combining with those of circular dichroism (CD) spectra and acid-base transition study for DNA-hemin complexes, provide some important information about DNAzymes based on G-quadruplex-hemin complexes, such as the formation of a G-quadruplex structure is an important factor for determining whether a DNA sequence can enhance the catalytic activity of hemin; both intramolecular parallel G-quadruplexes and intermolecular four-stranded parallel G-quadruplexes can enhance the catalytic activity of hemin; the addition of T nucleotides to the 5′-end of a G-tract confers corresponding G-quadruplex greatly enhanced catalytic activity, whereas the addition of T nucleotides to the 3′-end of the G-tract has little effect; the high catalytic activity of hemin in the presence of some short-stranded G-rich sequences may be a result of the reduction of the acidity of the bound hemin cofactor. These studies provide more information for the DNA-hemin peroxidase model system, may help to elucidate the structure-function relationship of peroxidase enzymes and to develop novel, highly efficient peroxidase-liking DNAzymes. As a sequence of such an investigation, a new Hg²⁺ detection method was developed.     

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.     

Stabilization of Human Telomeric G‐Quadruplex and Inhibition of Telomerase Activity by Propeller‐Shaped Trinuclear PtII Complexes

Two novel propeller‐shaped, trigeminal‐ligand‐containing, flexible trinuclear Pt^II complexes, {[Pt(dien)]₃(ptp)}(NO₃)₆ ( 1 ) and {[Pt(dpa)]₃(ptp)}(NO₃)₆ ( 2 ) (dien: diethylenetriamine; dpa: bis‐(2‐pyridylmethyl)amine; ptp: 6′‐(pyridin‐3‐yl)‐3,2′:4′,3′′‐terpyridine), have been designed and synthesized, and their interactions with G‐quadruplex (G4) sequences are characterized. A combination of biophysical and biochemical assays reveals that both Pt^II complexes exhibit higher affinity for human telomeric (hTel) and c‐myc promoter G4 sequences than duplex DNA. Complex 1 binds and stabilizes hTel G4 sequence more effectively than complex 2 . Both complexes are found to induce and stabilize either antiparallel or parallel conformation of G4 structures. Molecular docking studies indicate that complex 1 binds into the large groove of the antiparallel hTel G4 structure (PDB ID: 143D) and complex 2 stacks onto the exposed G‐quartet of the parallel hTel G4 structure (PDB ID: 1KF1). Telomeric repeat amplification protocol assays demonstrate that both complexes are good telomerase inhibitors, with IC₅₀ values of (16.0±0.4) μM and (4.20±0.25) μM for 1 and 2 , respectively. Collectively, the results suggest that these propeller‐shaped flexible trinuclear Pt^II complexes are effective and selective G4 binders and good telomerase inhibitors. This work provides valuable information for the interaction between multinuclear metal complexes with G4 DNA.     

Synthetic multivalent DNAzymes for enhanced hydrogen peroxide catalysis and sensitive colorimetric glucose detection

A peroxidase-mimic DNAzyme is a G-quadruplex (G₄) DNA–hemin complex, in which the G₄-DNA resembles an apoenzyme, and hemin is the cofactor for hydrogen peroxide (H₂O₂) catalysis. Twenty-one-mer CatG4 is a well-proven G₄-DNA as well as a hemin-binding aptamer for constituting a DNAzyme. This work studied if a multivalent DNAzyme with accelerated catalysis could be constructed using a multimeric CatG4 with hemin. We compared CatG4 monomer, dimer, trimer, and tetramer, which were prepared by custom oligo synthesis, for G₄ structure formation. According to circular dichroism (CD) analysis, we found that a CatG4 multimer exhibited more active G₄ conformation than the sum effect of equal-number CatG4 monomers. However, the DNAzyme kinetics was not improved monotonically along with the subunit number of a multimeric CatG4. It was the trivalent DNAzyme, trimeric CatG4:hemin, resulting in the rapidest H₂O₂ catalysis instead of a tetravalent one. We discovered that the trivalent DNAzyme’s highest catalytic rate was correlated to its most stable hemin-binding G₄ structure, evidenced by CD melting temperature analysis. Finally, a trivalent DNAzyme-based colorimetric glucose assay with a detection limit as low as 10 μM was demonstrated, and this assay did not need adenosine 5′-tri-phosphate disodium salt hydrate (ATP) as a DNAzyme boosting agent.     

Adenosine Triphosphate Sensing by Electrocatalysis with DNAzyme

Electrocatalysis of redox enzymes shows wide application for biosensing. DNAzymes exhibiting specific catalytic activities have aroused great interest recently. However, there are few studies on the electrocatalysis between DNAzyme and electron mediator. In this paper, based on the electrocatalysis of methylene blue (MB) and horseradish peroxidase mimicking DNAzyme (HRP‐DNAzyme), an amplified electrochemical biosensor for the detection of adenosine triphosphate (ATP) was designed. In the present system, by means of the ATP‐aptamer interaction, two guanine‐rich DNA sequences, one of which was labeled with MB at the 5′ end, were assembled on the gold electrode. In the presence of K⁺ and hemin, the guanine‐rich DNA sequences transferred to HRP‐DNAzyme. The conformational change of the structure resulted in the approaching of MB and HRP‐DNAzyme which made the electrocatalytic process between MB and HRP‐DNAzyme possible. We used cyclic voltammetry and electrochemical impedance spectroscopy to study the electrocatalytic process. The system was therefore utilized for amplified detection of ATP without imposing any new constraints to the platform which showed satisfactory result.     

Design of a High-Sensitivity Dimeric G-Quadruplex/Hemin DNAzyme Biosensor for Norovirus Detection

G-quadruplexes can bind with hemin to form peroxidase-like DNAzymes that are widely used in the design of biosensors. However, the catalytic activity of G-quadruplex/hemin DNAzyme is relatively low compared with natural peroxidase, which hampers its sensitivity and, thus, its application in the detection of nucleic acids. In this study, we developed a high-sensitivity biosensor targeting norovirus nucleic acids through rationally introducing a dimeric G-quadruplex structure into the DNAzyme. In this strategy, two separate molecular beacons each having a G-quadruplex-forming sequence embedded in the stem structure are brought together through hybridization with a target DNA strand, and thus forms a three-way junction architecture and allows a dimeric G-quadruplex to form, which, upon binding with hemin, has a synergistic enhancement of catalytic activities. This provides a high-sensitivity colorimetric readout by the catalyzing H₂O₂-mediated oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline -6-sulfonic acid) diammonium salt (ABTS). Up to 10 nM of target DNA can be detected through colorimetric observation with the naked eye using our strategy. Hence, our approach provides a non-amplifying, non-labeling, simple-operating, cost-effective colorimetric biosensing method for target nucleic acids, such as norovirus-conserved sequence detection, and highlights the further implication of higher-order multimerized G-quadruplex structures in the design of high-sensitivity biosensors.