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Directed evolution of functional synthetic and biological macromolecules.

机译：指导功能性合成和生物大分子的进化。

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摘要

The selection of functional macromolecules from diversified pools of polymeric sequences is a central feature of natural and laboratory-based molecular evolution. However, the monomers that are available for evolution are largely limited to those that are tolerated by polymerise enzymes or by the ribosome. We have investigated the DNA-templated polymerization of peptide nucleic acid building blocks, with the purpose of applying this reaction towards the directed evolution of functional synthetic polymers. Exploiting the high fidelity, efficiency, and rapid kinetics of DNA-templated reductive amination of PNA peptide aldehydes, we created libraries of DNA-encoded synthetic PNA polymers with diversities exceeding 109 members. Selection of such a library for binding to the protein papain revealed a sequence that was shown to have measurable affinity and specificity toward the target. Additionally, the mutation and reselection of this initial sequence led to second-generation synthetic polymers with improved protein-binding affinity. These findings represent the first in vitro evolution of a synthetic polymer.; The second part of this thesis describes the application of directed evolution principles to the study of natural proteins. Combinatorial and selection-based approaches are increasingly enabling the rapid and efficient characterization of protein complexes, which play a central role in almost all biological functions. Beyond characterization, the ability to re-engineer the specificity of protein-protein interactions can help us better understand complex networks of interacting proteins in living organisms. We have optimized an in vivo protein-protein selection system in E. coli based on fragment complementation of the enzyme dihydrofolate reductase. As opposed to existing in vitro systems such as phage display and in vivo systems such as the yeast two-hybrid, this system allowed for the randomization and selection of a complex, marginally stable mammalian protein, the nuclear receptor PPARgamma. We have used this system to characterize the interface between the PPARgamma ligand binding domain and several of its coactivator binding partners, with the ultimate goal of reengineering specificity in these biologically important complexes.
机译：从聚合物序列的多样化集合中选择功能性大分子是自然和基于实验室的分子进化的主要特征。但是，可用于进化的单体在很大程度上限于聚合酶或核糖体可耐受的单体。我们已经研究了以DNA为模板的肽核酸构件的聚合反应，目的是将该反应应用于功能性合成聚合物的定向进化。利用DNA模板化的PNA肽醛还原胺化的高保真度，效率和快速动力学，我们创建了DNA编码的合成PNA聚合物库，其多样性超过109个成员。选择与木瓜蛋白酶结合的文库揭示了对靶标具有可测量的亲和力和特异性的序列。另外，该初始序列的突变和重新选择导致具有改进的蛋白结合亲和力的第二代合成聚合物。这些发现代表了合成聚合物的首次体外进化。本文的第二部分描述了定向进化原理在天然蛋白质研究中的应用。基于组合和选择的方法越来越能够快速，有效地表征蛋白质复合物，而蛋白质复合物在几乎所有生物学功能中都发挥着核心作用。除了表征之外，重新改造蛋白质与蛋白质相互作用的特异性的能力还可以帮助我们更好地了解生物体中相互作用的蛋白质的复杂网络。我们基于二氢叶酸还原酶的片段互补，在大肠杆菌中优化了体内蛋白质-蛋白质选择系统。与现有的体外系统（例如噬菌体展示）和体内系统（例如酵母双杂交）相反，该系统允许对复杂的，边缘稳定的哺乳动物蛋白核受体PPARgamma进行随机化和选择。我们已经使用该系统表征了PPARgamma配体结合结构域和其一些共活化剂结合伴侣之间的界面，其最终目的是重新设计这些生物学上重要的复合物的特异性。

著录项

  • 作者

    Rosenbaum, Daniel Mark.;

  • 作者单位

    Harvard University.;

  • 授予单位 Harvard University.;
  • 学科 Chemistry Organic.; Chemistry Biochemistry.
  • 学位 Ph.D.
  • 年度 2005
  • 页码 130 p.
  • 总页数 130
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 有机化学;生物化学;
  • 关键词

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