NUCLEASE ACTIVITY OF 1, 10-PHENANTHROLINE-COPPER ION

1，10-菲咯啉-铜的化学核酸酶活性导致 a）将DNA结合蛋白转化为位点特异性核酸酶; B）发现了与开放的 在RNA合成起始时形成的复合物;和c）试剂， 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 Epstein-Barr斑马的基序将被转化为位点特异性的 通过将1，10-菲咯啉连接到半胱氨酸上的断裂试剂 通过定点诱变。转化因子的结合位点 刺激蛋白（fis）和色氨酸阻遏蛋白，在E.大肠杆菌和斑马 蛋白质，在EB病毒，将确定其网站的 使用连接介导的PCR策略进行切割。的机理 将使用脱氧核糖氘代核苷酸研究断裂 以确定化学物质氧化攻击的初始部位 核酸酶。由于色氨酸阻遏物嵌合体切割其野生型位点 100%的效率，一个基于Trp的稀有切割机家族 阻遏物结构支架将通过诱变DNA来制备 Trp阻遏物E49 C-OP的结合结构域。 B）与启动子和RNA聚合酶形成的开放复合物将是 用作两种不同类型的转录抑制剂的靶标： i）2：1的2，9-二甲基-1，10-菲咯啉-亚铜络合物（（NC）2Cu+）; 和ii）与所述单链DNA互补的寡核苷酸， 开放式复合体 i）四面体配体如（NC）2Cu+与 在酶活性位点或溶液中游离的单链DNA 没有先例。因此，（NC）2Cu+与两种原核细胞的结合 和真核开放复合物将使用立体化学进行探索， 定点诱变，体外选择错配DNA， 亲和标记 ii）与（NC）2Cu+相反，其是全局转录 抑制剂、核糖寡核苷酸和非生物类似物（例如2-OMe 寡核苷酸）能够基因特异性抑制，并且可以 为抗病毒和抗菌药物的设计提供了新的途径 在DNA水平上。的长度、靶标和主链结构。 将确定最具抑制性的寡核苷酸。衍生化 与反应性基团，它们将作为亲和配体来修饰 转录复合物的相邻组分。 c）通过2：1的1，10-菲咯啉-亚铜络合物切割RNA （（OP 2）Cu+））将与RNA进行研究，这些RNA已在 体外测定其对电子等排（NC）2Cu+的结合亲和力。的结合 这些RNA对（NC.）2Cu+及其衍生物将 测定用（OP 2）Cu+切割核糖氘代RNA将 提出了氧化断裂反应的化学机制。

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.