The peroxidase activity of a hemin--DNA oligonucleotide complex: free radical damage to specific guanine bases of the DNA.

A specific DNA oligonucleotide--hemin complex (PS2.M--hemin complex) that exhibits DNA-enhanced peroxidative activity was studied by EPR and UV--visible spectroscopy and by chemical probing analysis. EPR data obtained from low-temperature experiments on the PS2.M--hemin complex showed both a low-field g approximately 6 and a high-field g approximately 2 signal. These EPR signals are typical of high-spin ferric heme with axial symmetry as judged by the EPR spectrum of six-coordinate heme iron in acidic Fe(III)-myoglobin. This similarity is consistent with the presence of two axial ligands to the heme iron within the PS2.M--hemin complex, one of which is a water molecule. Optical analyses of the acid-base transition for the hemin complex yielded a pK(a) value for the water ligand of 8.70 +/- 0.03 (mean +/- SD). Low-temperature EPR analysis coupled with parallel spin-trapping investigations following the reaction of the PS2.M--hemin complex and hydrogen peroxide (H(2)O(2)) indicated the formation of a carbon-centered radical, most likely on the PS2.M oligonucleotide. Chemical probing analysis identified specific guanine bases within the PS2.M sequence that underwent oxidative damage upon reaction with H(2)O(2). These and other experimental findings support the hypothesis that the interaction of specific guanines of PS2.M with the bound hemin cofactor might contribute to the superior peroxidative activity of the PS2.M--hemin complex.     

DNA-enhanced peroxidase activity of a DNA aptamer-hemin complex

Background: In vitro selection (SELEX) previously identified short single-stranded DNAs that specifically bound N-methylmesoporphyrin IX (NMM), a stable transition-state analogue for porphyrin-metallation reactions. Interestingly, iron (III)-protoporphyrin (hemin) was a good competitive inhibitor for the DNA-catalyzed metallation reaction, and appeared to bind strongly to the NMM-binding DNA aptamers. We investigated the peroxidase activity of the aptamer-hemin complexes to see if the DNA component of the complex, like the apoenzymes in protein peroxidases, could enhance the low intrinsic peroxidatic activity of hemin.Results: Two porphyrin-bind ing DNA aptamers bound hemin with submicromolar affinity. The aptamer-hemin complexes had significantly higher peroxidase activity than hemin alone, under physiological conditions. The V_obs of the PS2.M-hemin complex was 250 times greater than that of hemin alone, and significantly superior to a previously reported hemin—catalytic-antibody complex. Preliminary spectroscopic evidence suggests the coordination of the hemin iron in the complex changes, such that the complex more closely resembles horseradish peroxidase and other heme proteins rather than hemin.Conclusions: A new class of catalytic activity for nucleic acids is reported. The aptamer-hemin complexes described are novel DNA enzymes and their study will help elucidate the structural and functional requirements of peroxidase enzymes in general and the ways that a nucleic acid ‘apoenzyme’ might work to enhance the intrinsic peroxidatic ability of hemin. These aptamer-hemin complexes could be regarded as prototypes for redox-catalyzing ribozymes in a primordial ‘RNA world’.     

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.     

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.     

Enzyme Mimic Based on a Self-Assembled Chitosan/DNA Hybrid Exhibits Superior Activity and Tolerance

Nature has evolved enzymes with exquisite active sites that catalyze biotransformations with high efficiency. However, the exploitation of natural enzymes is often hampered by poor stability, and natural enzyme production and purification are costly. Supramolecular self-assembly allows the construction of biomimetic active sites, although it is challenging to produce such artificial enzymes with catalytic activity and stability that rival those of natural enzymes. We report herein a strategy to produce a horseradish peroxidase (HRP) mimic based on the assembly of chitosan with a G-quadruplex DNA (G-DNA)/hemin complex. A network-like morphology of the assembled nanomaterial was observed together with a remarkable enhancement of peroxidase activity induced by the chitosan and G-DNA components. The turnover frequency and catalytic efficiency of the enzyme-mimicking material reached or even surpassed those of HRP. Moreover, the catalytic complex exhibited higher tolerance than HRP to harsh environments, such as extremely low pH or high temperatures. In accord with the experimental and simulated results, it is concluded that the spatial distribution of the G-DNA and chitosan components and the exposure of the catalytic center may facilitate the coordination of substrates by the hemin iron, leading to the superior activity of the material. Our work provides a simple and affordable avenue to produce highly active and robust enzyme-mimicking catalytic nanomaterials.     

Highly effective colorimetric and visual detection of nucleic acids using an asymmetrically split peroxidase DNAzyme

G-quadruplex containing peroxidase DNAzyme is a complex of hemin and a single-stranded guanine-rich DNA (hemin-binding DNA aptamer), which is used as an attractive catalytic label for biosensing recently. Therein, the hemin-binding DNA aptamer contains four GGG repeats and can fold into a G-quadruplex structure. In this paper, we have developed a new split mode to divide the hemin-binding DNA aptamer into two parts: one possesses three GGG repeats, and another part possesses one GGG repeat, namely, the 3:1 split mode. The combination of G-quadruplex and hemin binding could be used as a sensitive probe for the identification of single nucleotide polymorphisms by giving a color signal, visible to the naked eye at room temperature. The G-quadruplex containing peroxidase DNAzyme utilizes the 3:1 split mode and can be directly used for the identification of SNPs with a detection limit in the nM range when the matching length of the probe is short enough. When the matching length of the probe is relatively long, another method adding competition sequences to the probe could also operate effectively for the identification of SNPs. The results also suggested that we could detect the signal when the mutation sample was only 5% in the total target DNA with a competition strategy.     

A label-free DNA reduced graphene oxide-based fluorescent sensor for highly sensitive and selective detection of hemin

G-quadruplex structure aptamer (PS2.M) can capture acridine orange (AO) from reduced graphene oxide (rGO). When the AO-PS2.M/rGO mixture is incubated with hemin, the specific binding of hemin with PS2.M results in a release of AO from PS2.M and return of AO back to rGO. Based on the quenching of fluorescence, the target hemin was detected sensitively and selectively, giving a detection limit of 50 nM.     

An Electrochemical Sensor for the Detection of Cu2+ Based on Gold Nanoflowers‐modifed Electrode and DNAzyme Functionalized Au@MIL‐101 (Fe)

In this study, we developed an electrochemical sensor for sensitive detection of Cu²⁺ based on gold nanoflowers (AuNFs)‐modified electrode and DNAzyme functionalized Au@MIL‐101(Fe) (MIL: Materials of Institute Lavoisier). The AuNFs‐modified indium tin oxide modified conductive glass electrode(AuNFs/ITO) prepared via electrodeposition showed improved electronic transport properties and provided more active sites to adsorb large amounts of oligonucleotide substrate (DNA1) via thiol‐gold bonds. The stable Au@MIL‐101(Fe) could guarantee the sensitivity because of its intrinsic peroxidase mimic property, while the Cu²⁺‐dependent DNA‐cleaving DNAzyme linked to Au@MIL‐101(Fe) achieved the selectivity toward Cu²⁺. After the DNAzyme substrate strand (DNA2) was cleaved into two parts due to the presence of Cu²⁺, the oligonucleotide fragment linked to MIL‐101(Fe) was able to hybridize with DNA1 adsorbed onto the surface of AuNFs/ITO. Due to the peroxidase‐like catalytic activity of MIL‐101(Fe) and the affinity recognition property of DNAzyme toward Cu²⁺, the electrochemical biosensor showed a sensitive detection range from 0.001 to 100 μM, a detection limit of 0.457 nM and a high selectivity, demonstrating its potential for Cu²⁺ detection in real environmental samples.     

Investigation of 3,3′,5,5′-tetramethylbenzidine as colorimetric substrate for a peroxidatic DNAzyme

In this work, the suitability of 3,3′,5,5′-tetramethylbenzidine sulfate (TMB) as the substrate of a DNAzyme catalytic system composed of a guanine-quadruplex DNA molecule and hemin was investigated. In the presence of H₂O₂, the hemin-DNA complex catalyzes the oxidation of TMB to produce two colored products, much like a peroxidase. The color-generating activity of this system could be influenced by several factors such as buffer type, pH value, DNA sequence, reaction time, and concentrations of both the hemin and H₂O₂. To illustrate the utility of this catalytic system, we designed a colorimetric assay, in which a synthetic oligonucleotide with a sequence complementary to the G-quadruplex DNA was used as the target. A detection limit of 1.86 nM was obtained. Our data have shown that TMB was an excellent colorimetric indicator that reported the peoxidase activities of the widely studied hemin-G-quadruplex DNAzyme system.     

Aptamer-based label-free method for hemin recognition and DNA assay by capillary electrophoresis with chemiluminescence detection

An aptamer-based label-free approach to hemin recognition and DNA assay using capillary electrophoresis with chemiluminescence detection is introduced here. Two guanine-rich DNA aptamers were used as the recognition element and target DNA, respectively. In the presence of potassium ions, the two aptamers folded into the G-quartet structures, binding hemin with high specificity and affinity. Based on the G-quartet–hemin interactions, the ligand molecule was specifically recognized with a K _d ≈ 73 nM, and the target DNA could be detected at 0.1 μM. In phosphate buffer of pH 11.0, hemin catalyzed the H₂O₂-mediated oxidation of luminol to generate strong chemiluminescence signal; thus the target molecule itself served as an indicator for the molecule–aptamer interaction, which made the labeling and/or modification of aptamers or target molecules unnecessary. This label-free method for molecular recognition and DNA detection is therefore simple, easy, and effective. Figure A label-free approach to aptamer-based hemin recognition and DNA detection is introduced, which gives great potential for using a small molecule itself as the indicator for molecular recognition and DNA detection thereby avoiding any labeling or modification step     

Effects of Molecular Crowding on G-Quadruplex-hemin Mediated Peroxidase Activity

The concentration of macromolecules in cells can reach up to 50-400 mg/mL.They occupy 40%(volume fraction)of the whole cellar space,known as molecular crowding.The diluted solution condition in vitro is different from the crowded physiological condition in vivo.Therefore,the simulation of the physiological condition is necessary for obtaining the reliable results.It has been reported that G-quadruplex can bind to hemin to enhance its catalytic function for generating oxygen radicals,which can oxidize the lipids,proteins and DNA,thus leading to the damage of cells and tissues.In this paper,we chose PEG400 as molecular crowding reagent to simulate the molecular crowding environment in vivo.The catalytic characteristics of G-quadruplex-hemin complex in H202-ABTS system have been investigated[ABTS=2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate)].The results showed that the binding affinity of G-quadruplex and hemin was decreased with the increasing of PEG400 concentration.They even lose their binding affinity in the presence of 40%PEG400.As a result,the peroxidase activity of G-quadruplex-hemin also reduced.Therefore,in physiological condition,hemin might not bind to G-quadruplex and it might not be the main reason to cause the damages of cells and tissues.     

Insight into How Telomeric G‐Quadruplexes Enhance the Peroxidase Activity of Cellular Hemin

The toxic oxidative damage of G‐quadruplexes (G4), linked to neurodegenerative diseases, may arise from their ability to bind and oxidatively activate cellular hemin. However, there have been no precise studies on how telomeric G4 enhances the low intrinsic peroxidase activity of hemin. Herein, a label‐free and nanopore‐based strategy was developed to explore the enhancement mechanism of peroxidase activity of hemin induced by telomeric G4 (d(TTAGGG)_n). The nanopore‐based strategy demonstrated that there were simultaneously two different binding modes of telomere G4 to hemin. At the single‐molecule level, it was found that the hybrid structural telomeric G4 directly binds to hemin (the affinity constant (K_a)≈10⁶ m ^?1) to form a tight complex, and some of them underwent a topological change to a parallel structure with an enhancement of K_a to approximately 10⁷ m ^?1. Through detailed analysis of the topology and peroxidase activity and molecular modeling investigations, the parallel telomere G4/hemin DNAzyme structure was proven to be preferable for high peroxidase activity. Upon strong π–π stacking, the parallel structural telomere G4 supplied a key axial ligand to the hemin iron, which accelerated the intermediate compound formation with H₂O₂ in the catalytic cycle. Our studies developed a label‐free and single‐molecule strategy to fundamentally understand the catalytic activity and mechanism of telomeric DNAzyme, which provides some support for utilizing the toxic, oxidative‐damage property in cellular oxidative disease and anticancer therapeutics.     

Programmable and Reversible Regulation of Catalytic Hemin@MOFs Activities with DNA Structures

Metal-organic frameworks(MOFs)-based nanozyme plays an important role in biosensing,therapy and catalysis.In this study,the effects of single-stranded DNA(ssDNA)with programmable sequences and its complementary DNA(Tdna)on the intrinsic peroxidase-like activity of hemin loaded MOFs(UiO-66-NH2),denoted as he-min@UiO-66-NH2,were investigated.The hemin@UiO-66-NH2 exhibited improved catalytic activity compared with free hemin.However,the catalytic activity is inhibited in the presence of ssDNA,as ssDNA can be adsorbed by MOFs and therefore protected the active sites from contact with substrates.Upon the addition of the TDNA,double-stranded DNA(dsDNA)was formed and detached from the MOFs,resulting in the recovery of catalytic activity.Sequentially adding ssDNA or its complementary DNA strands can achieve the reversible regulation of the catalytic activity of MOFs nanozymes.Moreover,the DNA hybridization-based regulation was further applied to a cascaded catalytic system composed of the nanozyme,hemin@UiO-66-NH2,and glucose oxidase.These nanozyme based programmable and reversibly regulated catalytic systems may have potential applications in future smart biosensing and catalysis systems.     

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.     

A novel mimetic peroxidase catalyst by using magnetite-containing silica nanoparticles as carriers

A method for coating magnetite and mimetic enzyme (hemin) with amorphous silica to form a novel mimetic peroxidase (magnetite–hemin/SiO₂) has been developed by combining reverse microemulsion and the modified Stöber method. The magnetic silica nanoparticle supported hemin has a long-term stability toward temperature and good reusability. They can be easily separated from the reaction solution by using an external magnetic field and reused directly for next round of reaction. The peroxidase activity of the magnetite–hemin/SiO₂ was studied based on its catalytic effect on the reaction of p-hydroxyphenylacetic acid and H₂O₂. The results indicated that the catalytic activity of the new mimetic enzyme catalyst is higher than that of the free hemin. The possibility of its application was proven by the determination of H₂O₂, with the detection limits of 7.3 nmol L^?1 H₂O₂.     

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.     

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.     

DNA sensing on glassy carbon electrodes by using hemin as the electrochemical hybridization label

The electrochemical behavior of hemin, an iron complex of porphyrin, on binding to DNA at a glassy carbon electrode (GCE) and in solution, is described. Hemin, which interacts with covalently immobilized calf thymus DNA, was detected by use of a bare GCE, a double-stranded DNA-modified GCE (dsDNA-modified GCE), and a single-stranded DNA-modified GCE (ssDNA-modified GCE), in combination with differential pulse voltammetry (DPV). The structural conformation of DNA was determined from changes in the voltammetric signals acquired on reduction of hemin. As a result of its large steric structure and anionic substitution on its porphyrin plane, hemin intercalates between the base pairs of dsDNA. A scan-rate study for hemin and the dsDNA-hemin complex were also performed to determine the electrochemical behavior of the complex. The partition coefficient was obtained from the peak currents measured when different concentrations of hemin were in the presence of dsDNA. By observing the oxidation signals of guanine, damage to DNA after reaction with hemin at the GCE surface was also detected. The electrochemical detection of hybridization between the covalently immobilized probe and its target sequence was detected by use of hemin. These results demonstrate the use of DNA biosensors in conjunction with hemin for electrochemical detection of hybridization and damage to DNA.     

苹果酸酶结合域定点突变对烟酰胺腺嘌呤二核苷酸类似物催化性能的影响

通过对苹果酸酶(ME)辅酶结合域L310、Q401、L404饱和位点突变库与辅酶烟酰胺腺嘌呤二核苷酸(NAD⁺)类似物库的高通量筛选,研究了苹果酸酶结合域位点对NAD⁺及其类似物(B1~B7)催化活性的影响。 结果表明,突变后酶ME-Q401H/L404T对类似物B4的k_cat/K_m是野生型酶的50倍;突变后酶ME-L310M/Q401N对类似物B4的k_cat/K_m是野生型酶的16倍,对类似物B3的k_cat/K_m是野生型酶的5倍,因此通过对结合域定点突变,NAD⁺类似物的催化活性得到提高。     

Catalytical Activities of Reconstructed Hemoglobin with Different Central Ions in Prosthetic Group

Hemoglobin(Hb) was de-prosthetized, which was then reconstructed with the prosthetic groups with different central metal ions including Fe ( Ⅱ ), Co ( Ⅰ ) and Mn( Ⅰ ). The spectral properties along with the catalase and peroxidase activities of the reconstructed hemoglobin were compared with those of Hb and prosthetic groups with different ions. When the central ion is iron, the reconstituted Hb(rHb) has the highest catalase and peroxidase activities. Maybe it is the reason that iron is chosen as the central ion in the prosthetic groups of natural hemoproteins. Different from peroxidase activity, the catalase activity of hemin cannot be enhanced by the microenvironment of apoHb. This result shows that the structure of apoHb is more similar to that of aooHRP than that of apocatalase.