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

项目总结 重组蛋白生产(Rpp)已成为生产救命食品的有力工具。 治疗药物，如胰岛素、单抗和其他关键生物药物。不过，这个 前景看好的技术严重受限于有效扩展遗传密码以整合 异国情调的单体和骨架，增强治疗功能。目前绝大多数生物聚合物 由平移机械制作的显示聚酰胺主干；因此，可能的次要和 通过RPP合成的蛋白质组学可用的三级确认仅限于这些支架。自.以来 单体序列定义结构，结构定义功能，扩展了可用的单体池 翻译将产生结构更复杂的生物聚合物，从而增加功能 蛋白质仿制疗法的能力。解决这个问题的一个主要限制是没有得到充分的探索 核糖体结合非天然单体的能力，尤其是那些不形成多肽的单体 (酰胺)债券。为了实现这一目标，该提案旨在开发基因编码的化学物质， 在核糖体的催化下合成结构多样的序列确定聚合物 骨架(非肽键)。 具体地说，我将设计并合成一个a-肼基酮基酯单体的库，对它们进行充电 在正交tRNA上，将其引入体外翻译机器中，并评估其能力 核糖体催化它们的聚合。已知联氨和酮酯部分在 形成各种杂环产品的溶液。重要的是，杂环形成的最后一个机械步骤是 酰胺键形成，核糖体的一种特长。因此，我假设双官能团单体 由这两个部分组成的分子能够通过核糖体介导形成杂环连接 催化作用。我们的协作和跨学科研究团队取得了令人鼓舞的初步成果 暗示这一目标是可以实现的，因为我们发现核糖体比以前更容易适应 想着。这个方案中的实验将(1)扩大我们对分子翻译的理解，(2) 阐明管理遗传密码重新编程的限制和原则，以及(3)扩展合成的 通过核糖体开发生物衍生SDPs的工具箱。实现这一目标 提议将增加翻译机构目前可实现的骨干多样性，并可能 生产可能表现出更大治疗效果的SDP。

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.