Exploring the Limits of Ribosome Mediated Polymerizations for Expanding the Genetic Code

项目摘要 重组蛋白生产（RPP）已成为生产救命的有力工具 治疗药物，如胰岛素、单克隆抗体和其他重要的生物药物。但这 有前途的技术受到有效扩展遗传密码的能力的严重限制， 用于增强治疗功能的外来单体和主链。目前绝大多数生物聚合物 由翻译机器产生的显示聚酰胺主链;因此，可能的第二和 可用于通过RPP合成的蛋白质模拟物的三级确认限于这些支架。以来 单体序列定义结构，结构定义功能，扩展可用单体池， 翻译将产生具有更大结构复杂性的生物聚合物， 蛋白质模拟疗法的能力。解决这个问题的一个主要限制是未充分探索的 核糖体结合非天然单体的能力，特别是那些不形成肽的单体 （酰胺）键。为了实现这一目标，该提案旨在开发基因编码的化学物质， 由核糖体催化合成具有结构多样性的序列限定的聚合物（SDPs） 主链（非肽键）。 具体来说，我将设计并合成一个α-肼基酮酯单体库， 在正交tRNA上，将它们引入体外翻译机器，并评估 核糖体来催化它们的聚合。已知肼和酮基酯部分在以下条件下反应： 形成各种杂环产物。重要的是，杂环形成的最后一个机械步骤是 酰胺键的形成是核糖体的一种特性。因此，我假设双官能单体 由这两个部分组成的多核苷酸能够通过核糖体介导形成杂环连接 催化作用我们的合作和跨学科研究团队取得了令人鼓舞的初步成果， 我认为这一目标是可以实现的，因为我们发现核糖体比以前更具适应性 想的本实验将（1）拓宽我们对分子翻译的理解，（2） 阐明控制遗传密码重编程的限制和原则，以及（3）扩大合成 通过核糖体开发生物衍生SDPs的工具箱。实现这一目标 该提案将增加翻译机器目前可实现的主干多样性，并且可以 产生可能表现出更大疗效的SDPs。

PROJECT SUMMARY Recombinant protein production (RPP) has become a powerful tool for producing life-saving therapeutics such as insulin, monoclonal antibodies, and other critical biopharmaceuticals. However, this promising technology is severely limited by the ability to efficiently expand the genetic code to incorporate exotic monomers and backbones for enhanced therapeutic function. The vast majority of biopolymers currently produced by the translation machinery display polyamide backbones; therefore, the possible secondary and tertiary confirmations available to proteomimetics synthesized via RPP are limited to these scaffolds. Since monomer sequence defines structure and structure defines function, expanding the available monomer pool for translation will produce biopolymers with greater structural complexity and thus increase the functional capabilities for proteomimetic therapeutics. A major limitation to addressing this issue is the underexplored capability of the ribosome to incorporate unnatural monomers, especially those that do not form peptide (amide) bonds. Toward this goal, this proposal aims to develop genetically encoded chemistries that can be catalyzed by the ribosome to synthesize sequence defined polymers (SDPs) with structurally diverse backbones (non-peptide bonds). Specifically, I will design and synthesize a library of a-hydrazino-keto ester monomers, charge them onto orthogonal tRNA, introduce them to the translation machinery in vitro, and evaluate the ability of the ribosome to catalyze their polymerizations. The hydrazine and keto ester moieties are known to react in solution to form various heterocyclic products. Importantly, the last mechanistic step in heterocyclic formation is amide bond formation, a specialty of the ribosome. Therefore, I hypothesize that bifunctional monomers comprised of both these moieties are capable of forming heterocyclic linkages via ribosome mediated catalysis. Encouraging preliminary results by our collaborative and interdisciplinary research team have suggested this goal is achievable as we have found the ribosome to be more accommodating than previously thought. The experiments in this proposal will (1) broaden our understanding of molecular translation, (2) elucidate the limitations and principles that govern genetic code reprogramming, and (3) expand the synthetic toolbox for the development of biologically derived SDPs via the ribosome. Accomplishing the aims in this proposal will increase the backbone diversity currently attainable by the translation machinery and could produce SDPs that might exhibit greater therapeutic efficacy.