Saran Runjhun, Kleinke Kimberly, Zhou Wenhu, Yu Tianmeng, Liu Juewen
Department of Chemistry, Waterloo Institute for Nanotechnology, University of Waterloo , Waterloo, Ontario N2L 3G1, Canada.
Biochemistry. 2017 Apr 11;56(14):1955-1962. doi: 10.1021/acs.biochem.6b01131. Epub 2017 Mar 31.
Most RNA-cleaving DNAzymes require a metal ion to interact with the scissile phosphate for activity. Therefore, few unmodified DNAzymes work with thiophilic metals because of their low affinity for phosphate. Recently, an Ag-specific Ag10c DNAzyme was reported via in vitro selection. Herein, Ag10c is characterized to rationalize the role of the strongly thiophilic Ag. Systematic mutation studies indicate that Ag10c is a highly conserved DNAzyme and its Ag binding is unrelated to C-Ag-C interaction. Its activity is enhanced by increasing Na concentrations in buffer. At the same metal concentration, activity decreases in the following order: Li > Na > K. Ag10c binds one Na ion and two Ag ions for catalysis. The pH-rate profile has a slope of ∼1, indicating a single deprotonation step. Phosphorothioate substitution at the scissile phosphate suggests that Na interacts with the pro-R oxygen of the phosphate, and dimethyl sulfate footprinting indicates that the DNAzyme loop is a silver aptamer binding two Ag ions. Therefore, Ag exerts its function allosterically, while the scissile phosphate interacts with Na, Li, Na, or Mg. This work suggests the possibility of isolating thiophilic metal aptamers based on DNAzyme selection, and it also demonstrates a new Ag aptamer.
大多数RNA切割型DNA酶需要金属离子与可切割磷酸基团相互作用以发挥活性。因此，由于对磷酸基团亲和力低，很少有未修饰的DNA酶能与亲硫金属起作用。最近，通过体外筛选报道了一种特异性识别银的Ag10c DNA酶。在此，对Ag10c进行表征以阐明强亲硫性银的作用。系统突变研究表明，Ag10c是一种高度保守的DNA酶，其与银的结合与C-Ag-C相互作用无关。在缓冲液中增加钠离子浓度可增强其活性。在相同金属浓度下，活性按以下顺序降低：Li＞Na＞K。Ag10c结合一个钠离子和两个银离子以进行催化。pH-速率曲线的斜率约为1，表明存在单个去质子化步骤。在可切割磷酸基团处进行硫代磷酸酯取代表明，钠离子与磷酸基团的前-R氧相互作用，硫酸二甲酯足迹法表明DNA酶环是一个结合两个银离子的银适配体。因此，银通过变构发挥其功能，而可切割磷酸基团与钠、锂、钠或镁相互作用。这项工作表明基于DNA酶筛选分离亲硫金属适配体的可能性，同时也展示了一种新的银适配体。
