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

我们已经开始详细描述 单链RNA的金属催化裂解（酯交换） 通过使用包埋RNA（embRNA）分析。1 -3 EmbRNA包含 DNA寡聚体中的单个RNA残基。 的相对有效性 由不同团体报道的催化剂通常通过比较来评估 这些Kobs值。 我们发现，在许多情况下， RNA二聚体切割的kobs值与 切割相关的RNA寡聚体。 这使得二聚体测定法 在选择裂解高MW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起作用 为这种行为。 可能的贡献者包括众多的 高分子量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），潜在的多个 动力学上重要的金属离子，更大的各种构象 寡聚RNA与 二梅里克底物，和 影响。 此外，作为我们动力学的一部分， RNA切割的研究，我们已经确定了 对于几种催化剂，kobs上的催化剂浓度，包括 Ce（III）和La（III）离子和Ce（III）大环。 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 会对观测到的速率常数产生巨大的影响。 而反观 发现通过Ce（III）大环的embRNA的酯交换是 关于催化剂的第一顺序。4这种差异可能是由于 涉及通过大环配体抑制多核物质。 我们已经确定了pH速率曲线和反应级数， “自由”Ln 3+离子和大环，我们现在能够比较 这些试剂以有意义的方式对基于Cu（II）和Zn（II）的RNA进行分析， 酯交换催化剂 pH速率曲线有助于确定 催化剂的质子化状态在动力学上是重要的。 我们 也已经开始使用多重嵌合的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。 质谱法是 鉴定裂解产物所需的