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.

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.

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In this paper, we describe a turn-on fluorescent probe for Zn(2+) and Cd(2+) ion detection and discrimination. This probe, N'-bis-(N,N-di-(2-pyridylmethyl)-ethane-1,2-diamine)-perylene-3,4,9,10-tetracarboxylic-diimide (PDI-DIDPA), exhibits very low fluorescence at pH above 6.0 due to the photo-induced electron transfer (PET) process from 2-pyridylmethyl-amine (DPA) to perylene tetracarboxylic diimide (PDI). Zn(2+) and Cd(2+) can chelate PDI-DIDPA to form a stable complex at pH 6.0-7.0 and 9.0 respectively, and inhibit the PET process, which result in the fluorescence recovery of PDI-DIDPA. This fluorescence turn-on behavior allows the detection of Zn(2+) in the range of 0.1-4.0 μM and Cd(2+) in the range of 0.1-5.0 μM. The limit of detection for Zn(2+) and Cd(2+) is as low as 32 nM and 48 nM. The high selectivity, high sensitivity and easy operation make this probe suitable for the rapid detection of Zn(2+) and Cd(2+) respectively. The different response of PDI-DIDPA to Zn(2+) and Cd(2+) at different pH makes it possible to discriminate Zn(2+) and Cd(2+) by simply adjusting the pH of the working solution.