New cofactors and inhibitors for a DNA-cleaving DNAzyme: superoxide anion and hydrogen peroxide mediated an oxidative cleavage process.

Herein, we investigated the effects of new cofactors and inhibitors on an oxidative cleavage of DNA catalysis, known as a pistol-like DNAzyme (PLDz), to discuss its catalytic mechanism. PLDz performed its catalytic activity in the presence of ascorbic acid (AA), in which Cu promoted, whereas Fe significantly inhibited the catalytic function. Since Fe/AA-generated hydroxyl radicals are efficient on DNA damage, implying that oxidative cleavage of PLDz had no relation with hydroxyl radical. Subsequently, we used Fe/HO and Cu/HO to identify the role of hydroxyl radicals in PLDz catalysis. Data showed that PLDz lost its activity with Fe/HO, but exhibited significant cleavage with Cu/HO. Because Fe/HO and Cu/HO are popular reagents to generate hydroxyl radicals and the latter also produces superoxide anions, we excluded the possibility that hydroxyl radical participated in oxidative cleavage and confirmed that superoxide anion was involved in PLDz catalysis. Moreover, pyrogallol, riboflavin and hypoxanthine/xanthine oxidase with superoxide anion and hydrogen peroxide generation also induced self-cleavage of PLDz, where catalase inhibited but superoxide dismutase promoted the catalysis, suggesting that hydrogen peroxide played an essential role in PLDz catalysis. Therefore, we proposed a catalytic mechanism of PLDz in which superoxide anion and hydrogen peroxide mediated an oxidative cleavage process.