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首页> 外文学位 >Novel approaches to regulating RNA function: Creation and evaluation of a composite surface-binding RNA targeting system.
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Novel approaches to regulating RNA function: Creation and evaluation of a composite surface-binding RNA targeting system.

机译：调节RNA功能的新方法：创建和评估复合表面结合RNA靶向系统。

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摘要

RNA plays a central role in numerous cellular processes. The control of these events through direct interactions with the RNA of interest presents an attractive strategy for both therapeutic and basic science applications. However, efficient formation of highly specific complexes with RNA has proven exceedingly challenging. A reliable method for generating ligands of high affinity and high specificity for a chosen RNA would represent a crucial advance in the regulation of RNA function. One promising solution to this problem utilizes a composite protein-small molecule surface to capture the RNA. In this approach, a small molecule effector moiety is displayed from the surface of the protein, and protein and small molecule act in consort to bind the RNA target. The effective surface area of the small molecule is enhanced by the presence of the protein, and exquisite control may be achieved by the use of the small molecule ligand. Although composite surface binding systems that target proteins are found in Nature, no similar systems are known for RNA.; To explore the feasibility of using composite surface binding to target RNA, we have engineered a model RNA targeting system in which all components of the system are foreign to the cell, and in which the small molecule is cell permeable. A combination of protein design, chemical synthesis and in vitro selection were used to generate the system, and biochemical and computational techniques were used to extensively characterize the result. These analyses reveal the formation of a high affinity (K d ∼ 5 nM) ternary protein-small molecule-RNA complex. This system demonstrates high specificity over both related proteins and related small molecules, and binds with a high degree of cooperativity. Structural probing of the ternary complex has identified critical interactions between the RNA and the protein-small molecule surface and suggests a mechanism by which recognition may be achieved. Insights gained from this system should enable further efforts to target RNA through composite surface binding, and the system itself provides a convenient tool with which to manipulate cellular processes.
机译：RNA在众多细胞过程中起着核心作用。通过与目的RNA的直接相互作用控制这些事件为治疗和基础科学应用提供了一种有吸引力的策略。然而，事实证明，与RNA高效形成高度特异性的复合物极具挑战性。产生对所选RNA具有高亲和力和高特异性的配体的可靠方法将代表RNA功能调节的关键进展。解决该问题的一种有前途的解决方案是利用复合蛋白-小分子表面捕获RNA。在这种方法中，小分子效应子部分从蛋白质的表面显示出来，蛋白质和小分子共同作用以结合RNA靶标。蛋白质的存在会增强小分子的有效表面积，并且可以通过使用小分子配体来实现精确的控制。尽管在自然界中发现了靶向蛋白质的复合表面结合系统，但尚无类似的RNA系统。为了探索使用复合表面结合靶RNA的可行性，我们设计了一个模型RNA靶向系统，其中系统的所有组件对细胞都是外来的，并且小分子可渗透细胞。结合蛋白质设计，化学合成和体外选择来生成系统，并使用生化和计算技术广泛表征结果。这些分析揭示了高亲和力（K d〜5 nM）三元蛋白质-小分子-RNA复合物的形成。该系统对相关蛋白和相关小分子均显示出高特异性，并以高度协同性结合。三元复合物的结构探测已确定了RNA与蛋白质-小分子表面之间的关键相互作用，并提出了可实现识别的机制。从该系统获得的见识应使人们能够进一步努力通过复合表面结合来靶向RNA，并且该系统本身提供了一种方便的工具来操纵细胞过程。

著录项

  • 作者

    Plummer, Kelly Ann.;

  • 作者单位

    Harvard University.;

  • 授予单位 Harvard University.;
  • 学科 Chemistry Biochemistry.
  • 学位 Ph.D.
  • 年度 2005
  • 页码 123 p.
  • 总页数 123
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 生物化学;
  • 关键词

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