Positive effects of ATP on G-quadruplex-hemin DNAzyme-mediated reactions.

Some G-quadruplex-hemin complexes can be used as peroxidase-mimicking DNAzymes, catalyzing H(2)O(2)-mediated reactions such as the oxidation of 2,2'-azinobis (3-ethylbenzothiozoline)-6-sulfonic acid (ABTS) by H(2)O(2). However, some challenges, for example, the relatively low catalytic activity and the disproportionation of the reaction product ABTS*(+), may seriously restrict further development and applications of these complexes. Here, we demonstrated the positive effect of adenosine triphosphate (ATP) on G-quadruplex-hemin DNAzyme-mediated catalytic reactions. The presence of ATP not only improved the catalytic activity of G-quadruplex-hemin DNAzymes, but also inhibited the disproportionation of ABTS*(+). These observations may improve the performance of existing G-quadruplex-hemin DNAzyme-based chemical sensors, for example, the Ag(+)-detection method that uses G-quadruplex-hemin DNAzymes, and widen the application range of G-quadruplex-hemin DNAzymes. We also demonstrated that the phosphate groups, nucleobase, and sugar of ATP determine the reaction-promoting ability of ATP. These observations may be helpful in the design of highly efficient enhancers for G-quadruplex-hemin DNAzymes.

Ag+ and cysteine quantitation based on G-quadruplex-hemin DNAzymes disruption by Ag+.

Some G-quadruplex-hemin complexes are DNAzyme peroxidases that efficiently catalyze H(2)O(2)-mediated reactions, such as the oxidation of ABTS (2,2'-azinobis(3-ethylbenzothiozoline)-6-sulfonic acid) by H(2)O(2). Since Ag(+) chelates guanine bases at the binding sites are involved in G-quadruplex formation, the presence of Ag(+) may disrupt these structures and inhibit the peroxidase activity of G-quadruplex-hemin DNAzymes. On the basis of this principle, a highly sensitive and selective Ag(+)-detection method was developed. The method allows simple detection of aqueous Ag(+) with a detection limit of 64 nM and a linear range of 50-3000 nM. Cysteine (Cys) is a strong Ag(+)-binder and competes with quadruplex-forming G-rich oligonucleotides for Ag(+)-binding, promoting the reformation of G-quadruplexes and increasing their peroxidase activity. Therefore, the Ag(+)-sensing system was also developed as a Cys-sensing system. This "turn-on" process allowed the detection of Cys at concentrations as low as 50 nM using a simple colorimetric technique. The Cys-sensing system could also be used for the detection of reduced glutathione (GSH). Neither the Ag(+)-sensing nor the Cys-sensing systems required labeled oligonucleotides. In addition, both gave large changes in absorbance signal that could be observed by the naked eye. Thus, a simple visual method for Ag(+)- or Cys-detection was developed.

Quantitative detection of Ag(+) and cysteine using G-quadruplex-hemin DNAzymes.

A highly sensitive and selective Ag(+) detection method was developed based on the Ag(+)-mediated formation of G-quadruplex-hemin DNAzymes. In this method, two unlabelled oligonucleotides with different lengths are used. In the absence of Ag(+), the two oligonucleotides hybridize to each other to form an intermolecular duplex. The addition of Ag(+) can disrupt the intermolecular duplex and promote a part of the sequence of the longer oligonucleotide to fold into an intramolecular duplex, in which cytosine-cytosine (C-C) mismatches are stabilized by C-Ag(+)-C base pairs. As a result, the G-rich sequence of the same oligonucleotide can fold into a G-quadruplex, which is able to bind hemin to form a catalytically active G-quadruplex-hemin DNAzyme. This can be reflected by an absorbance increase when monitored in the H(2)O(2)-ABTS (2,2'-azinobis(3-ethylbenzothiozoline)-6-sulfonic acid) reaction system by using UV-vis absorption spectroscopy. This 'turn-on' process allows the detection of aqueous Ag(+) at concentrations as low as 20 nM using a simple colorimetric technique. Considering that Cysteine (Cys) is a strong binder of Ag(+), the presence of Cys may disrupt the C-Ag(+)-C base pairs in the intramolecular duplex, resulting in the reformation of the intermolecular duplex and the decrease of the catalytic activity of the sensing system. Therefore, the Ag(+)-sensing system can be further developed as a Cys-sensing system. This method allows the detection of Cys with a detection limit of 25 nM. With the development of the studies on DNA-metal base pairs, this Ag(+)-sensing method can be easily extended to the analysis of other metal ions.

Structure-function study of peroxidase-like G-quadruplex-hemin complexes.

The structure-function relationship of G-quadruplex-hemin complexes with peroxidase activity was studied by comparing peroxidase activity and circular dichroism (CD) spectra of 22 oligonucleotides with the sequence of d(G(2)T(n))(3)G(2), d(G(3)T(n))(3)G(3) (n = 1-4) and dG(3)T(i)G(3)T(j)G(3)T(k)G(3). According to the experimental results, some conclusions can be drawn, such as the addition of hemin may promote the conversion of some G-quadruplexes from antiparallel structures to parallel structures; the formation of G-quadruplexes is a crucial factor in determining the peroxidase activity of G-quadruplex-hemin complexes; and the complexes formed by hemin and parallel G-quadruplexes have much higher peroxidase activity than those formed by hemin and antiparallel G-quadruplexes.

'Turn-on' detection of Hg2+ ion using a peroxidase-like split G-quadruplex-hemin DNAzyme.

A highly sensitive and selective Hg(2+) detection method was developed based on the Hg(2+)-mediated formation of split G-quadruplex-hemin DNAzymes. In this method, two label-free oligonucleotides are used. In the presence of Hg(2+), the two oligonucleotides hybridize to each other to form a duplex, in which T-T mismatches are stabilized by T-Hg(2+)-T base pair. As a result, the G-rich sequences of the two oligonucleotides can associate to form a split G-quadruplex, which is able to bind hemin to form the catalytically active G-quadruplex-hemin DNAzymes. This can be reflected by an absorbance increase when monitored in the H(2)O(2)-ABTS (2,2'-azinobis(3-ethylbenzothiazoline)-6-sulfonic acid) reaction system by using UV-vis absorption spectroscopy. This 'turn-on' process allows the detection of aqueous Hg(2+) at concentrations as low as 19 nM using a simple colorimetric technique. With the development of the studies on metal-base pairs, this Hg(2+)-sensing method can be easily extended to the analysis of other metal ions.

Fluorescent sensor for monitoring structural changes of G-quadruplexes and detection of potassium ion.

G-rich sequences with the potential for quadruplex formation are common in genomic DNA. Considering that the biological functions of G-quadruplexes may well depend on their structures, the development of a sensitive structural probe for distinguishing different types of quadruplexes has received great attention. Crystal violet (CV) is a triphenylmethane dye, which can stack onto the two external G-quartets of a G-quadruplex. The ability of CV to discriminate G-quadruplexes from duplex and single-stranded DNAs has been reported by us. Herein, the ability of CV to discriminate parallel from antiparallel structures of a G-quadruplex was studied. The binding of CV to an antiparallel G-quadruplex can make its fluorescence intensity increase to a high level because of the protection of bound CV from the solvent by quadruplex end loops. The presence of side loops in parallel G-quadruplexes cannot provide bound CV such protection, causing the fluorescence intensity of CV/G-quadruplex mixture to be obviously weaker when the G-quadruplex adopts a parallel structure than that when the G-quadruplex adopts an antiparallel structure. Therefore, CV can be developed as a sensitive fluorescent biosensor for the discrimination of antiparallel G-quadruplexes from parallel G-quadruplexes and for monitoring the structural interconversion of G-quadruplexes. In addition, considering that some G-rich DNA sequences can adopt different G-quadruplex structures under Na(+) or K(+) ion conditions, a novel, cheap and simple K(+) ion detection method was developed. This method displays a high K(+) ion selectivity against Na(+) ion, the change of 200 mM in Na(+) ion concentration only causes a similar fluorescent signal change to 0.3 mM K(+) ion.

Discrimination of G-quadruplexes from duplex and single-stranded DNAs with fluorescence and energy-transfer fluorescence spectra of crystal violet.

G-rich nucleic acid sequences with the potential to form G-quadruplex structures are common in biologically important regions. Most of these sequences are present with their complementary strands, so the development of a sensitive biosensor to distinguish G-quadruplex and duplex structures and to determine the competitive ability of quadruplex to duplex structures has received a great deal of attention. In this work, the interactions between two triphenylmethane dyes (malachite green (MG) and crystal violet (CV)) and G-quadruplex, duplex, or single-stranded DNAs were studied by fluorescence spectroscopy and energy-transfer fluorescence spectroscopy. Good discrimination between quadruplexes and duplex or single-stranded DNAs can be achieved by using the fluorescence spectrum of CV or the energy-transfer fluorescence spectra of CV and MG. In addition, by using energy-transfer fluorescence titrations of CV with G-quadruplexes, the binding-stoichiometry ratios of CV to G-quadruplexes can be determined. By using the fluorescence titrations of G-quadruplex-CV complexes with C-rich complementary strands, the fraction of G-rich oligonucleotide that engages in G-quadruplex structures in the presence of the complementary sequence can be measured. This study may provide a simple method for discrimination between quadruplexes and duplex or single-stranded DNAs and for measuring G-quadruplex percentages in the presence of the complementary C-rich sequences.

A new method for the study of G-quadruplex ligands.

A new method for the study of G-quadruplex ligands was developed, in which the interaction of G-quadruplexes with ligands can be judged by the naked eye, eliminating the need for any expensive machines.

Oxidative DNA cleavage by Schiff base tetraazamacrocyclic oxamido nickel(II) complexes.

Nickel is considered a weak carcinogen. Some researches have shown that bound proteins or synthetic ligands may increase the toxic effect of nickel ions. A systematic study of ligand effects on the interaction between nickel complexes and DNA is necessary. Here, we compared the interactions between DNA and six closely related Schiff base tetraazamacrocyclic oxamido nickel(II) complexes NiL(1-3a,1-3b). The structure of one of the six complexes, NiL(3b) has been characterized by single crystal X-ray analysis. All of the complexes can cleave plasmid DNA under physiological conditions in the presence of H(2)O(2). NiL(3b) shows the highest DNA cleavage activity. It can convert supercoiled DNA to nicked DNA then linear DNA in a sequential manner as the complex concentration or reaction time is increased. The cleavage reaction is a typical pseudo-first-order consecutive reaction with the rate constants of 3.27+/-0.14h(-1) (k(1)) and 0.0966+/-0.0042h(-1) (k(2)), respectively, when a complex concentration of 0.6mM is used. The cleavage mechanism between the complex and plasmid DNA is likely to involve hydroxyl radicals as reactive oxygen species. Circular dichronism (CD), fluorescence spectroscopy and gel electrophoresis indicate that the complexes bind to DNA by partial intercalative and groove binding modes, but these binding interactions are not the dominant factor in determining the DNA cleavage abilities of the complexes.

[Fluorescence investigation on interaction between artemisinin (qinghaosu) and hemin and its analytical application].

Hemin-catalytic decomposition of artemisinin (qinghaosu, QHS) was studied using pyronine B (PB) as an indicator. The interaction between hemin and QHS was an enzyme-substrate model, and the action sites were the endoperoxide moiety of QHS and the central metal ion of enzyme respectively. The kinetic catalytic constant depends upon enzyme and substrate concentrations, and the Michaelis-Menten parameters Km, Vmax and Kcat was 8.4 x 10(-5) mol x L(-1), 7.4 x 10(-6) mol x L(-1) s(-1) and 50.23 s(-1) respectively. The catalytic activity of hemin was inhibited in the presence of deactivated agents and at high temperature. Under optimal conditions, the change in fluorescence intensity (Fo-F) of pyronine B was proportional to the QHS concentration from 0.0 to 1.27 x 10(-6) mol x L(-1), and the detection limit (3sigma) was as low as 2.3 x 10(-8) mol x L(-1). The proposed method was applied to detect the concentration of QHS in the media of plasma and urine.

A modified molecular beacon combining the properties of TaqMan probe.

A modified molecular beacon that possesses a stem-hairpin structure as seen in conventional molecular beacons and can be cleaved during PCR in designed, and it can specifically recognize the presence of the target and was obviously more sensitive than conventional molecular beacons.

Spectrofluorimetric determination of hydrogen peroxide with 2-hydroxy-1-naphthaldehyde salicyloylhydrazone (HNSH) as the substrate for horseradish peroxidase (HRP).

The oxidation reaction of HNSH with H2O2 under the catalysis of HRP was studied in detail. The possible reaction mechanism was discussed. Under optimum experimental conditions, the oxidized product of HNSH had excitation and emission maxima at 296 and 414 nm, respectively. A study to prove the existence of -O-O-H in polyethylene glycols was carried out. The proposed method was successfully applied to the determination of -O-O-H in polyethylene glycols.

Amplified detection of DNA ligase and polynucleotide kinase/phosphatase on the basis of enrichment of catalytic G-quadruplex DNAzyme by rolling circle amplification.

As two commonly used tool enzymes, DNA ligase and polynucleotide kinase/phosphatase (PNKP) play important roles in DNA metabolism. More and more studies show that regulation of their activity represents promising means for cancer therapy. To detect the activity of DNA ligase with high sensitivity and specificity, a G-quadruplex DNAzyme-based DNA ligase sensor was developed. In this sensor, the use of G-quadruplex DNAzyme eliminated the needs for any labeled oligonucleotide probes, thus making label-free detection possible. The introduction of rolling circle amplification (RCA) reaction could lead to the formation of multimeric G-quadruplexes containing thousands of G-quadruplex units, which can provide highly active hemin-binding sites, thus significantly improving the sensitivity of the sensor. The proposed sensor allowed specific detection of T4 DNA ligase with a detection limit of 0.0019 U/mL. By adding a PNKP-triggered 5'-phosphroylation step of the template DNA, the above sensing strategy could be easily extended to the design of PNKP sensor. The established sensor allowed specific detection of T4 PNKP with a detection limit of 0.0018 U/mL. In addition, these two sensors could also be used for the studies on inhibitors of these two enzymes.

Fluorogenic substrate screening for G-quadruplex DNAzyme-based sensors.

Due to the inherent higher sensitivity of fluorescence detection than colorimetric detection, it is necessary to screen out a suitable fluorogenic substrate for G-quadruplex DNAzymes to improve the sensitivities of G-quadruplex DNAzyme-based sensors. Herein, seven candidates were tested to determine the possibilities of them as fluorogenic substrates. Among these candidates, tyramine hydrochloride gave the maximum signal-to-background ratio for the sensing systems with and without G-quadruplexes, and thus was recommended as the fluorogenic substrate for the sensors that are developed on the basis of target-triggered G-quadruplex formation or destruction. 10-acetyl-3,7-dihydroxyphenoxazine gave the maximum fluorescence signal change between the sensing systems without and with H2O2, thus was recommended as the fluorogenic substrate for the sensors targeting the detection of H2O2 or H2O2-related analytes. In a model system of G-quadruplex DNAzyme-based Cu(2+) sensor, fluorescence detection using tyramine hydrochloride as fluorogenic substrate could decrease the detection limit from 4 nM to 0.7 nM compared with the colorimetric detection.

Ultrasensitive, high temperature and ionic strength variation-tolerant Cu²âº fluorescent sensor based on reconstructed Cu²âº-dependent DNAzyme/substrate complex.

A previously reported Cu²âº-dependent DNAzyme/substrate complex was reconstructed in this work, which makes possible the use of an intramolecular stem-loop structure and is, therefore, a good choice for the design of Cu²âº sensors. To demonstrate this, a fluorescent sensor was designed on the basis of the reconstructed complex. In this sensor, the fluorophore/quencher pair was caged tightly in an intramolecular double-helix structure; thus, the background signal was greatly suppressed. Cu²âº-dependent cleavage of the complex could cause the release of the fluorophore, leading to restoration of the fluorescence signal. High quenching efficiency provides the sensor with three important characteristics: high sensitivity, high temperature variation tolerance and high ionic strength tolerance. The proposed sensor allows specific detection of aqueous Cu²âº down to a limit of 0.6 nM, and the performance is independent of temperature and ionic strength in the range of 4-40 °C and 0.8-3.0 M NaCl, respectively. This work identifies a good choice for sensor design on the basis of DNAzymes containing triple-helix structures.

Sequence-specific detection of nucleic acids utilizing isothermal enrichment of G-quadruplex DNAzymes.

G-quadruplex DNAzymes are peroxidase-like complexes formed by nucleic acid G-quadruplexes and hemin. Various chemical sensors and biosensors have been developed, based on such DNAzymes. Here we report a novel, specific nucleic acid detection method utilizing the isothermal amplification strategy of G-quadruplex DNAzymes. In this method, an unlabeled oligonucleotide probe was used. The probing sequence of the oligonucleotide was in the form of a stem-loop structure. A G-rich sequence, containing three GGG repeats, was linked to the 5'-end of the stem-loop structure. In the presence of target, the probing sequence hybridized to the target, and a G(n) (n≥2) repeat was extended from its 3'-end. This G(n) repeat, together with the three GGG repeats at the 5'-end, folded into a G-quadruplex, and displayed enhanced peroxidase acitivity upon hemin binding. Utilizing the dynamic binding interaction between the probe and its target, the enrichment of G-quadruplex DNAzymes was achieved. Using this method, simple, rapid and cost-effective nucleic acid detection could be achieved. This method displayed high target-length tolerance and good detection specificity; one-base mismatch could be judged easily, even by visual inspection. This method may be used as an auxiliary tool for amplified detection of specific DNA targets in some situations, in which isothermal detection is desirable.

A simple, post-additional antioxidant capacity assay using adenosine triphosphate-stabilized 2,2'-azinobis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) radical cation in a G-quadruplex DNAzyme catalyzed ABTS-H2O2 system.

The scavenging of 2,2'-azinobis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) radical cation (ABTS(+)) by antioxidants has been widely used in antioxidant capacity assay. Because of ABTS(+) disproportionation, however, this radical cannot be prepared on a large scale and stored long-term, making it unsuitable for high-throughput detection and screening of antioxidants. We developed a modified "post-additional" antioxidant capacity assay. This method possessed two remarkable features: First, instead of natural peroxidases, an artificial enzyme, G-quadruplex DNAzyme, was used for the preparation of ABTS(+), thus greatly reducing the cost of the assay, and eliminating the strict demand for the storage of enzymes. Second, an ABTS(+) stabilizer, adenosine triphosphate (ATP), was used. In the presence of ATP, the disproportionation of ABTS(+) was effectively inhibited, and the lifetime of this radical cation was prolonged about 6-fold (12 days versus 2 days), making the large-scale preparation of ABTS(+) possible. Utilizing this method, the antioxidant capacities of individual antioxidants and real samples can be quantified and compared easily. In addition, this method can be developed as a high-throughput screening method for antioxidants. The screening results could even be judged by the naked eye, eliminating the need for expensive instruments.

Single-stranded DNAzyme-based Pb2+ fluorescent sensor that can work well over a wide temperature range.

DNAzymes have become an excellent choice for sensing applications. Based on DNAzymes, three generations of Pb(2+) fluorescent sensors have been reported. In these sensors, two oligonucleotide strands (substrate strand and enzyme strand) were used, which not only increased the complexity of the detection system, but also brought some difficulties for the use of the sensors at elevated temperatures. To overcome this problem, a single-stranded DNAzyme-based Pb(2+) fluorescent sensor was designed by combining the substrate sequence and the enzyme sequence into one oligonucleotide strand. The intramolecular duplex structure of this single-stranded DNAzyme kept the fluorophore and the quencher, labeled at its two ends, in close proximity; thus the background fluorescence was significantly suppressed. Using this fluorescent sensor, Pb(2+) quantitation can be achieved with high sensitivity and high selectivity. In addition, the extraordinary stability of the intramolecular duplex structure could assure a low background fluorescence at high temperature, even if the number of complementary base pairs between the substrate sequence and the enzyme sequence was reduced, allowing the sensor to work well over a wide temperature range. Similar performances of the fluorescent sensor at 4, 25 and 37°C suggested that this sensor has a good ability to resist temperature fluctuations.

Sensitive dual DNAzymes-based sensors designed by grafting self-blocked G-quadruplex DNAzymes to the substrates of metal ion-triggered DNA/RNA-cleaving DNAzymes.

A universal label-free metal ion sensor design strategy was developed on the basis of a metal ion-specific DNA/RNA-cleaving DNAzyme and a G-quadruplex DNAzyme. In this strategy, the substrate strand of the DNA/RNA-cleaving DNAzyme was designed as an intramolecular stem-loop structure, and a G-rich sequence was caged in the double-stranded stem and could not form catalytically active G-quadruplex DNAzyme. The metal ion-triggered cleavage of the substrate strand could result in the release of the G-rich sequence and subsequent formation of a catalytic G-quadruplex DNAzyme. The self-blocking mechanism of the G-quadruplex DNAzyme provided the sensing system with a low background signal. The signal amplifications of both the DNA/RNA-cleaving DNAzyme and the G-quadruplex DNAzyme provided the sensing system with a high level of sensitivity. This sensor design strategy can be used for metal ions with reported specific DNA/RNA-cleaving DNAzymes and extended for metal ions with unique properties. As examples, dual DNAzymes-based Cu(2+), Pb(2+) and Hg(2+) sensors were designed. These "turn-on" colorimetric sensors can simply detect Cu(2+), Pb(2+) and Hg(2+) with high levels of sensitivity and selectivity, with detection limits of 4 nM, 14 nM and 4 nM, respectively.

Amplification of G-quadruplex DNAzymes using PCR-like temperature cycles for specific nucleic acid and single nucleotide polymorphism detection.

A nucleic acid sensor, based on the amplified formation of G-quadruplex DNAzymes by polymerase chain reaction (PCR)-like temperature cycles, was developed. This "turn-on" process allowed effective detection of specific nucleic acid targets and identification of single nucleotide polymorphisms (SNPs).