A DNA Oligonucleotide−Hemin Complex Cleavest-Butyl Hydroperoxide through a Homolytic Mechanism

Abstract

Both electron paramagnetic resonance (EPR) and electronic absorption spectroscopy have been employed to investigate the reaction of a guanine-rich DNA nucleotide-hemin complex (PS2.M-hemin complex) and organic peroxide (t-Bu-OOH). Incubation of the PS2.M-hemin complex with t-Bu-OOH resulted in the time-dependent decrease in the heme Soret with concomitant changes to the visible bands of the electronic absorbance spectrum for the PS2.M-hemin complex. Parallel EPR studies using the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) combined with spectral simulation demonstrated the presence of tert-butyloxyl, carbon-centered methyl, and methyl peroxyl radicals as well as a simple nitroxide (triplet) signal. Experiments, performed by maintaining a constant ratio of t-Bu-OOH/PS2.M-hemin complex ( approximately 35 mol/mol) while varying DMPO concentration, indicated that the relative contributions of each radical adduct to the composite EPR spectrum were significantly influenced by the DMPO concentration. For example, at DMPO/PS2.M-hemin of 10-50 mol/mol, a complex mixture of radicals was consistently detected, whereas at high trapping efficiency (i.e., DMPO/PS2.M-hemin of approximately 250 mol/mol) the tert-butyloxyl-DMPO adduct was predominant. In contrast, at relatively low DMPO/PS2.M-hemin complex ratios of < or =5 mol/mol, a simple nitroxide three-line EPR signal was detected largely in the absence of all other radicals. Together, these data indicate that tert-butyloxyl radical is the primary radical likely formed from the homolytic cleavage of the O-O peroxy bond of t-Bu-OOH, while methyl and methyl peroxyl radicals result from beta-scission of the primary tert-butyloxyl radical product.

DNA‐Based Peroxidation Catalyst—What Is the Exact Role of Topology on Catalysis and Is There a Special Binding Site for Catalysis?

“…[30] The majority of Gquadruplex peroxidases reported to date were generally inactivated in the first few minutes of the peroxidation reaction. [15,17,18] The identification of G-quadruplexes that are stable to oxidizing conditions will be important for the construction of a more efficient G-quadruplex-based bioassays that rely on the peroxidation reaction as well as to provide a model to study how nucleic acids catalyze reactions in an oxidative environment. Interestingly, when ABTS was used as the organic reductant, HT hemin1 was significantly more resistant to oxidative inactivation in buffers containing excess potassium cations than the rest of the DNAzymes used in this study (see Figure 4 A).…”

“…[12][13][14][15][16] Thus far, the majority of G-quadruplex peroxidases reported to date are readily inactivated under the oxidizing reaction condition. [15,17,18] It is thought that hydroxyl radicals or the high-valent iron(IV)-oxo species generated during the peroxidation reaction are responsible for oxidizing the guanine tracts in G-quadruplexes to oxidized nucleobase analogues such as 8-oxo-guanines. 8-oxo-guanines do not support the G-quadruplex structure and hence peroxidations rates of G-quadruplex DNAzymes diminish as the guanines get oxidized during the course of the reaction.…”

“…8-oxo-guanines do not support the G-quadruplex structure and hence peroxidations rates of G-quadruplex DNAzymes diminish as the guanines get oxidized during the course of the reaction. [17,18] Non guanine-rich oligonucleotide peroxidases, which might be less prone to oxidative inactivation, would be ideal biolabels for sensing applications.…”

“…We identify a new DNAzyme peroxidase (HT hemin1 ) (HT: human telomere repeat fragment), which is more resistant to oxidative inactivation compared to other G-quadruplexes. [15][16][17][18] These new findings challenge our current understanding of how nucleic acids catalyze cofactor oxidation reactions and also provide a starting point in the quest to Abstract: In the last decade, there has been growing interests in studies aimed at delineating the strategies used by various nucleic acid enzymes to facilitate catalysis. Insights gained from such studies would enable the design of better DNA/RNA catalysts for various applications such as biosensing.…”

Porphyrins in complex with DNA: Modes of interaction and oxidation reactions

“…The peroxidase reactivity remains when hemin is covalently bound to a nucleic acid vector [185]. DNA aptamers capable of recognizing heme derivatives [180] and exhibiting peroxidase activity have been first obtained through in vitro selection techniques [189][190][191].…”