NUCLEASE ACTIVITY OF 1, 10-PHENANTHROLINE-COPPER ION

1，10-邻菲咯啉-铜的化学核酸酶活性导致 A)DNA结合蛋白转化为位点特异性核酸酶； B)发现与开放结合的转录抑制物- 在RNA合成开始时形成的复合体；以及c)一种试剂，用于 分析RNA的二级结构/配体结合。 A)果蝇的同源结构域基序和亮氨酸拉链 爱泼斯坦-巴尔斑马的主题将转换为特定地点 1，10-二氮杂菲与半胱氨酸偶联的裂解试剂 通过定点突变。反转因子的结合位点 在大肠杆菌和斑马中的刺激(FIS)蛋白和色氨酸抑制物 爱泼斯坦-巴尔病毒中的蛋白质将通过它们的 使用连接介导的聚合酶链式反应策略进行切割。它的作用机制 将使用脱氧核糖-重氢核苷酸来研究断裂 以确定化学物质氧化攻击的初始位置 核酸酶。由于Trp抑制因子嵌合体切割其野生型位点 以100%的效率，基于Trp的稀有刀具家族 通过诱变DNA制备抑制子结构支架 色氨酸抑制因子E49C-OP的结合域。 B)与启动子和RNA聚合酶形成的开放复合体将是 用作两种不同类型转录抑制物的靶标： I)2：12，9-二甲基-1，10-菲咯啉-亚铜络合物((NC)2Cu+)； 和ii)与单链DNA互补的寡核苷酸 开放的综合体。 I)类四面体配体(NC)2Cu+与TO的相互作用 单链DNA在酶活性部位或在溶液中游离 没有先例。因此，(NC)2Cu+与两种原核生物的结合 而真核开放复合体将使用立体化学进行探索， 定点突变、错配DNA和DNA的体外筛选 亲和力标签。 Ii)与(NC)2Cu+相反，后者是一种全局转录 抑制物、核寡核苷酸和非生物类似物(例如2-OME 寡核苷酸)能够进行基因特异性抑制，并可以 为抗病毒、抗菌药物的设计提供了一条新途径 DNA级别的特工。的长度、目标和主干结构 最具抑制力的寡核苷酸将被确定。派生的 有了反应性基团，它们将作为亲和配体进行修饰 转录复合体的相邻成分。 C)2：1，10-菲咯啉-亚铜络合物对RNA的切割 ((OP2)Cu+))将与在 体外研究它们与异构体(NC)2Cu+的结合亲和力。装订 这些RNA对(NC.)2Cu+及其衍生物的特异性将是 下定决心。(OP2)Cu+Will对核糖氚核糖核酸的切割作用 提出氧化裂解反应的化学机制。

The chemical nuclease activity of 1,10-phenanthroline-copper has led to a) the Conversion of DNA binding proteins into site specific nucleases; b) the discovery of transcription inhibitors which bind to the open- complex formed at the initiation of RNA synthesis; and c) a reagent to analyze the secondary structure/ligand binding of RNA. a) The homeodomain motif of Drosophila engrailed and the leucine zipper motif of Epstein-Barr zebra will be converted into site specific scission reagents by linking 1,10-phenanthroline to cysteines introduced by site directed mutagenesis. Binding sites for the factor for inversion stimulation (fis) protein and Trp repressor, in E. coli, and the zebra protein, in Epstein-Barr virus, will be identified by their sites of scission using a ligation mediated PCR strategy. The mechanism of scission will be investigated using deoxyribose-deuterated nucleotides to identify the initial site of oxidative attack by the chemical nuclease. Since the Trp repressor chimera cleaves its wild-type sites with 100% efficiency, a family of rare cutters based on the Trp repressor structural scaffold will be prepared by mutagenizing the DNA binding domain of Trp repressor E49C-OP. b) The open complexes formed with promoters and RNA polymerase will be used as the target for two distinct types of transcription inhibitors: i) the 2:1 2,9-dimethyl-1,10-phenanthroline-cuprous complex ((NC)2Cu+); and ii) oligonucleotides complementary to the single-stranded DNA of the open complex. i) The interaction of a tetrahedral ligand like (NC)2Cu+ to single stranded DNA either in an enzyme active site or free in solution has no precedent. Therefore, the binding of(NC)2Cu+ to both procaryotic and eucaryotic open complexes will be explored using stereochemistry, site directed mutagenesis, in vitro selection of mismatched DNAs and affinity labelling. ii) In contrast to (NC)2Cu+ which is a global transcription inhibitor, ribooligonucleotides and abiological analogs (e.g. 2-OMe oligonucleotides) are capable of gene specific inhibition and could provide a new approach for the design of antiviral and antibacterial agents at the DNA level. The length, target, and backbone structure of the most inhibitory oligonucleotides will be determined. Derivatized with reactive groups, they will serve as affinity ligands to modify neighboring components of transcription complexes. c) The scission of RNA by the 2:1 1,10-phenanthroline-cuprous complex ((OP2)Cu+)) will be investigated with RNAs which have been selected in vitro for their binding affinity for the isosteric (NC)2Cu+. The binding specificity of these RNAs for (NC.)2Cu+ and its derivatives will be determined. Cleavage of ribose-deuterated RNAs with (OP2)Cu+ will suggest chemical mechanisms for the oxidative scission reaction.