MECHANISM & CHARGE CONTROL OF METAL CATALYZED RNA TRANSESTERIFICATION

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• 批准号: 6665800
• 负责人: James K. Bashkin
• 金额: $ 15.75万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-08-01 至 2003-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6665800
• 关键词: biological products; biomedical resource; nucleic acids; spectrometry

We have begun to develop a detailed description of the metal-catalyzed cleavage (transesterification) of single-stranded RNA through the use of the embedded RNA (embRNA) assay.1-3 EmbRNA contains a single RNA residue in a DNA oligomer. The relative effectiveness of catalysts reported by different groups is often assessed by comparison of these kobs values. We have found that, in a number of cases, the kobs values for cleavage of RNA dimers do not correlate with kobs for cleavage of related RNA oligomers. This makes the dimeric assays potentially misleading in the choice of reagents to cleave high MW, biological RNA samples. We are investigating which factors account for this behavior. Possible contributors include the multitude of metal binding sites on high MW RNA (at phosphodiester, O-4'-ether, 2'-hydroxyl and nucleobase sites), the potential for more than one kinetically-important metal ion, the greater variety of conformations available to oligomeric RNA vs . di meric substrates, and polyelectrolyte effects. Furthermore, as part of our kinetic investigations of RNA cleavage, we have determined the effect of catalyst concentration on kobs for several catalysts, including Ce(III) and La(III) ions and Ce(III) macrocycles. We found that, for aqueous Ce(III), the order of the reaction with respect to Ce(III) varied dramatically. Thus, small changes in catalyst concentration can have huge effects on the observed rate constant. In contrast, the transesterification of embRNA by Ce(III) macrocycles was found to be first order with respect to catalyst.4 This difference is likely due to the suppression of polynuclear species by the macrocyclic ligand. We have determined pH-rate profiles and reaction orders for both "free" Ln3+ ions and the macrocycles, and we are now able to compare these reagents in a meaningful way to Cu(II)- and Zn(II)-based RNA transesterification catalysts. The pH-rate profile helps determine which protonation state of the ca talyst is kinetically important. We have also begun to use multiply-chimeric embRNA substrates (i.e. DNA/RNA/methyl-phosphonate combinations) to determine the role of specific phosphate residues in catalyst binding. Mass spectrometry is required for the identification of cleavage products

我们已经开始对 单链RNA的金属催化裂解（酯交换） 通过使用嵌入的RNA（EMBRNA）测定法。1-3Embrna包含 DNA低聚物中的单个RNA残基。 相对有效性 经常通过比较评估不同组报告的催化剂 这些KOB值。 我们发现，在许多情况下 RNA二聚体裂解的KOBS值与KOB不相关 相关RNA低聚物的切割。 这使二聚体测定 可能会误导选择裂解高MW的试剂， 生物RNA样品。 我们正在研究哪些因素说明 为此行为。 可能的贡献者包括众多 高MW RNA上的金属结合位点（在磷酸二酯，O-4'-醚， 2'-羟基和核碱基位点），这是一个以上的潜力 动力学至关重要的金属离子，更多的构象 可用于寡聚RNA与。 Di Meric底物，以及 聚电解质效应。 此外，作为我们动力学的一部分 对RNA裂解的研究，我们确定了 催化剂浓度是多种催化剂的KOB，包括 CE（III）和LA（III）离子和CE（III）大环。 我们发现，因为 CE（III），相对于CE（III）的反应顺序 变化很大。 因此，催化剂浓度的小变化 可以对观察到的速率常数产生巨大影响。 相反， 发现CE（III）大环的交叉疗法是 关于催化剂的第一阶。4此差异可能是由于 通过大环配体抑制多核物种。 我们已经确定了两者的pH率曲线和反应顺序 “免费” LN3+离子和大环，我们现在能够比较 这些试剂以铜（II）和Zn（II）的RNA的有意义方式 式催化剂。 pH率配置文件有助于确定 Ca talyst的哪种质子化状态在动力学上很重要。 我们 也已经开始使用多晶型包裹底物（即 DNA/RNA/甲基膦酸酯组合）以确定 催化剂结合中的特定磷酸盐残基。 质谱是 识别裂解产品所必需的