MECHANISM & CHARGE CONTROL OF METAL CATALYZED RNA TRANSESTERIFICATION

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• 批准号: 6486680
• 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: 2001
• 资助国家: 美国
• 起止时间: 2001-08-01 至 2002-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6486680
• 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 单链 RNA 的金属催化裂解（酯交换） 通过使用嵌入 RNA (embRNA) 检测。1-3 EmbRNA 包含 DNA寡聚体中的单个RNA残基。 的相对有效性 不同团体报告的催化剂通常通过比较进行评估 这些 kobs 值。 我们发现，在很多情况下， RNA 二聚体切割的 kobs 值与 kobs 不相关 相关 RNA 寡聚体的切割。 这使得二聚体检测 在选择裂解高分子量的试剂时可能会产生误导， 生物RNA样本。 我们正在调查哪些因素会影响 对于这种行为。 可能的贡献者包括众多 高 MW RNA 上的金属结合位点（磷酸二酯、O-4'-醚、 2'-羟基和核碱基位点），可能有多个 动力学上重要的金属离子，构象的多样性更大 可用于寡聚 RNA 与 . 二聚体底物，和 聚电解质效应。 此外，作为我们动力的一部分 通过对 RNA 切割的研究，我们确定了 几种催化剂在 kobs 上的催化剂浓度，包括 Ce(III) 和 La(III) 离子以及 Ce(III) 大环化合物。 我们发现，对于 Ce(III) 水溶液，相对于 Ce(III) 的反应顺序 变化很大。 因此，催化剂浓度的微小变化 可以对观察到的速率常数产生巨大影响。 相比之下， Ce(III) 大环化合物对 embRNA 进行酯交换反应 相对于催化剂的一阶。4 这种差异可能是由于 通过大环配体抑制多核物种。 我们已经确定了两者的 pH 速率曲线和反应顺序 “游离”Ln3+离子和大环化合物，我们现在可以比较 这些试剂对基于 Cu(II) 和 Zn(II) 的 RNA 具有有意义的作用 酯交换催化剂。 pH 速率曲线有助于确定 催化剂的哪种质子化状态在动力学上很重要。 我们 也已开始使用多重嵌合 embRNA 底物（即 DNA/RNA/甲基膦酸组合）以确定的作用 催化剂结合中的特定磷酸盐残基。 质谱法是 鉴定裂解产物所需的