The Peroxidase Activity of a Hemin−DNA Oligonucleotide Complex:  Free Radical Damage to Specific Guanine Bases of the DNA

Abstract

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.

Cleavage-based signal amplification of RNA

“…Two kinds of catalytic DNA sequences, RNA-cleaving DNAzyme (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23) 26,27 and G-quadruplex horseradish peroxidase-mimicking DNAzyme (PW17) [28][29][30] were applied in our research. The 10-23 RNAcleaving DNAzyme has been reported to cleave any purinepyrimidine (RY) junction under simulated physiological conditions 26 ; hence, it was applied in our strategy for the cleaving of target RNA molecule to initiate the following exponential amplification.…”

Visual Detection ofrpoBMutations in Rifampin-Resistant Mycobacterium tuberculosis Strains by Use of an Asymmetrically Split Peroxidase DNAzyme

“…In the detection system, the initial tool for SNP analysis is the molecular-beacon probe (19); however, the requirement for the instrument and the individually fluorescence-labeled probe is definitely costly. The peroxidase DNAzyme was selected due to its robustness, its sensitivity, and its relatively low cost (3,12,14,25,26,27,32). Here, we apply the 3:1 split DNAzyme to detect target DNA.…”

“…Sen and coworkers (25)(26)(27)) discovered a DNAzyme that integrates hemin into the DNA G-quadruplex and has peroxidase activity that allows it to oxidize 2,2=-azinobis-(3-ethylbenzthiazoline-6-sulfonate) dianion (ABTS 2Ϫ ) using H 2 O 2 to produce the colored radical anion ABTS ·Ϫ . The reaction then undergoes a detectable color change.…”

A Novel Colorimetric PCR-Based Biosensor for Detection and Quantification of Hepatitis B Virus

“…This method takes the key advantage of the TaqMan technology (i.e., the elegant use of the 5 0 -exonuclease activity of Taq polymerase) [8], and applies a low-cost catalytic DNA molecular beacon as probe [9]. In the process of PCR amplification, Taq DNA polymerases cleave the probe and release a DNAzyme sequence [10][11][12], which has been embedded in the probe. After PCR amplification, the DNAzyme can form G-quadruplex and bind with hemin, possessing a peroxidase-like activity which catalyzes the oxidation of different substrates by H 2 O 2 to generate either colorimetric or fluorometric signals [13][14][15].…”