MECHANISM & CHARGE CONTROL OF METAL CATALYZED RNA TRANSESTERIFICATION

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• 批准号: 6336750
• 负责人: James K. Bashkin
• 金额: $ 0.25万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-08-01 至 2001-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6336750
• 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

我们已经开始制定详细的描述 金属催化的单链RNA裂解(酯交换) 通过使用嵌入的RNA(EmbRNA)分析1-3 EmbRNA包含 DNA寡聚体中的单个核糖核酸残基。的相对有效性。 不同组织报告的催化剂通常通过比较进行评估 这些科布斯值。我们发现，在许多情况下， RNA二聚体裂解的Kobs值与Kobs值 相关RNA寡聚体的切割。这使得二聚体分析 在选择裂解高MW的试剂时可能具有误导性， 生物RNA样本。我们正在调查哪些因素导致了 为这一行为负责。可能的贡献者包括众多 高相对分子质量RNA上的金属结合部位(在磷酸二酯，O-4‘-醚， 2‘-羟基和碱基位置)，一个以上的可能性 动力学上重要的金属离子，构象的更大变化 可用于寡聚RNA VS。Di Meric衬底，以及 聚电解质效应。此外，作为我们动力计划的一部分 对RNA裂解的研究，我们已经确定了 几种催化剂在KOBS上的催化剂浓度，包括 Ce(III)、La(III)离子和Ce(III)大环。我们发现，对于 Ce(III)水溶液与Ce(III)的反应级数 变化很大。因此，催化剂浓度的微小变化 会对观测到的速率常数有很大的影响。相比之下， 用Ce(III)大环催化embRNA的酯交换反应 相对于催化剂的第一顺序。这种差异很可能是由于 大环配体对多核物种的抑制作用。 我们已经测定了两者的pH速率分布和反应级数 “自由”的Ln3+离子和大循环，我们现在能够比较 这些试剂对以铜(II)和锌(II)为基础的RNA具有重要意义 酯交换催化剂。酸碱度曲线有助于确定 催化剂的哪种质子化状态在动力学上是重要的。我们 也开始使用多重嵌合embRNA底物(即 DNA/RNA/甲基膦酸盐组合)，以确定 催化剂结合中的特定磷酸盐残留物。质谱学是 鉴定裂解产物所需的