An oxidative DNA-cleaving DNAzyme (PL) employs a double-cofactor model “X/Cu²⁺” for catalysis. Herein, we verified that reduced nicotinamide adenine dinucleotide (NADH), flavin mononucleotide, cysteine, dithiothreitol, catechol, resorcinol, hydroquinone, phloroglucinol, o-phenylenediamine, 3,3′,5,5'-tetramethylbenzidine, and hydroxylamine acted as cofactor X. According to their structural similarities or fluorescence property, we further confirmed that reduced nicotinamide adenine dinucleotide phosphate (NADPH), 2-mercaptoethanol, dopamine, chlorogenic acid, resveratrol, and 5-carboxyfluorescein also functioned as cofactor X. Superoxide anions might be the commonality behind these cofactors. We subsequently determined the conservative change of individual nucleotides in the catalytic core under four different cofactor X. The nucleotides A4 and C5 are highly conserved, whereas the conservative levels of other nucleotides are dependent on the types of cofactor X. Moreover, we observed that the minor change in the PL's secondary structure affects electrophoretic mobility. Finally, we characterized a highly efficient variant T3G and converted its double-cofactor NADH/Cu²⁺ to sole-cofactor NADH.

氧化性DNA裂解DNA酶（PL）采用双辅因子模型“ X / Cu ²⁺ ”进行催化。在此，我们验证了原型烟酰胺腺嘌呤二核苷酸（NADH），黄素单核苷酸，半胱氨酸，二硫苏糖醇，儿茶酚，间苯二酚，间苯三酚，Ô-苯二胺，3,3'，5,5'-四甲基联苯胺和羟胺充当辅因子X。根据它们的结构相似性或荧光性质，我们进一步证实了烟酰胺腺嘌呤二核苷酸磷酸（NADPH），2-巯基乙醇，多巴胺，绿原酸，白藜芦醇和5-羧基荧光素也可作为辅因子X。超氧阴离子可能是这些辅因子的共性。我们随后确定了在四个不同的辅因子X下催化核心中各个核苷酸的保守变化。核苷酸A4和C5是高度保守的，而其他核苷酸的保守水平取决于辅因子X的类型。此外，我们观察到PL二级结构的微小变化会影响电泳迁移率。最后，²⁺为唯一辅因子NADH。