SITE SPECIFIC HYDROLYSIS OF DNA AND RNA

该项目的目标是发展金属过氧物的化学。 DNA和RNA“水解性”裂解的复合体。这一点的基础 该项目是我们最近观察到的双核的过氧化氢加合物 配合物Fe2(HPTB)OH(NO3)4(HPTB=N，N，N‘，N’-四基(2- (苯并咪唑甲基)-1，3-二氨基-2-羟基丙烷)解离超螺旋 PBR322在室温下混合。在以下情况下得到相同的结果 过氧化氢被氧气和一种还原剂取代。这种卵裂化学是 在几个方面是独一无二的：它是双链的；它发生在特定的 它提供3‘-OH和5’-OPO3末端，涉及水解物， 而不是氧化机制。我们建议进行试验，以 了解卵裂的机制和卵裂的潜在基础 这种相对简单的试剂的特异性。乳沟是 假设是金属激活的过氧化氢的亲核攻击 在敏感的磷酸二酯部分上。过氧化氢或氧气的作用将是 以确定磷酸盐产品是否被标记 当使用H_218O2或18O_2时。这些标签实验将被进行 带有小分子的OUT以及使用多链接剂修饰的pBR322 结合在裂解部位附近，以提供足够小的片段用于 高分辨率质谱分析。去核配体将是 系统地变化，以揭示赋予特定的 乳沟。PBR322将以多种方式进行修改，以确定是否 存在与序列相关的切割专一性的模式 或拓扑学。这些变化包括在 裂解位点，将裂解位点移动到质粒的另一部分， 将裂解部位附近的裂解部位或残基从 质粒组。PBR322将作为连锁的函数被分解成物种 研究超卷程度如何影响卵裂的数字 专一性。相关和不相关的DNA分子将被 裂解试剂及其裂解特性的测定。核糖核酸 还将检查分子以确定切割试剂是否 对于探测丰富的RNAs拓扑可能很有用。最后，复杂的 其他路易斯酸性(但非氧化还原活性)金属离子将被研究以 提高“水解性”裂解的效率 二铁络合物产生的氧化副反应。

The goal of this project is to develop the chemistry of metal-peroxo complexes for the "hydrolytic" cleavage of DNA and RNA. The basis for this project is our recent observation that the H2O2 adduct of the dinuclear complex Fe2(HPTB)OH(NO3)4 (HPTB = N,N,N',N'-tetrakis(2- benzimidazolylmethyl)- 1,3-diamino-2-hydroxypropane) cleaves supercoiled pBR322 upon mixing at room temperature. The same result is obtained when H2O2 is replaced with O2 and a reductant. This cleavage chemistry is unique in several respects: it is double stranded; it occurs at a specific site; and it affords 3'-OH and 5'-OPO3 ends implicating a hydrolytic, rather than an oxidative mechanism. We propose to carry out experiments to understand the mechanism of cleavage and the underlying basis for the specificity of this relatively simple reagent. The cleavage is hypothesized to be the nucleophilic attack of a metal-activated peroxide on a susceptible phosphate diester moiety. The role of H2O2 or O2 will be ascertained in order to determine whether the phosphate product is labeled when H218O2 or 18O2 is used. These labeling experiments will be carried out with small molecules as well as pBR322 modified with a polylinker incorporated near the cleavage site to afford small enough fragments for high resolution mass spectral analysis. The dinucleating ligand will be varied systematically to uncover features that confer specificity of cleavage. pBR322 will be modified in a number of ways to determine if there is a pattern to the cleavage specificity that is related to sequence or topology. These variations include altering the sequence at the cleavage site, moving the cleavage site to another section of the plasmid, and deleting the cleavage site or residues near the cleavage site from the plasmid. pBR322 will be resolved into species as a function of linkage number to study how the extent of supercoiling affects the cleavage specificity. Related and unrelated DNA molecules will be treated with the cleavage reagent and their cleavage specificities determined. RNA molecules will also be examined to determine whether the cleavage reagent may be useful for probing the rich topology of RNAs. Lastly, complexes of other Lewis acidic (but non-redox active) metal ions will be studied to improve the efficiency of the "hydrolytic" cleavage by eliminating the oxidative side reactions engendered by the diiron complexes.