Repurposing the Ribosome for Exotic Polymers

项目总结： 用于模板引导的蛋白质合成的机器-核糖体及其相关的 翻译因素-已开发了20多年，专门整合了&gt；150 在体外、细胞和动物体内，将非天然α-氨基酸和&GT；10α-羟基酸转化为蛋白质。 更多外来单体，包括N-烷基α-氨基酸、D-氨基酸和一些β-氨基酸 酸已经使用无细胞的体外系统被结合。这些努力加深了我们的 了解蛋白质和RNA的功能，并提供真实世界的商品，如 抗体-药物结合物、改良疗法和造福社会的生物材料。然而，即使是 经过二十多年的研究，直到最近，这方面的初步结果 应用--没有关于核糖体能够将β-氨基酸引入蛋白质的报道 在活体内，也没有任何先进的生物信息学工具来重新设计核糖体。这样做的目的是 应用是开发和应用基因工程、生物信息学和化学生物学 为模板化的体内生物合成重新调整细菌翻译装置的用途的工具 含有骨架修饰单体的蛋白质和多肽。主要关注的是β- 氨基酸，因为初步结果表明，这一目标可以通过4-5年的RO1实现 获奖，但开发的战略、见解和工具将为 不同类别可进化物质的序列模板化生物合成--真正的奇特 生物聚合物--其功能仅限于我们的集体想象力。通过创建新的 在体内有效地将β-氨基酸整合到蛋白质中的机器，我们不仅将提供 解决有关自然翻译的长期问题的工具，但也提供了真正的好处 不同细分领域的生物医学研究引擎。

Project Summary: The machine used for template-guided protein synthesis–the ribosome and its associated translation factors–has been exploited for over 20 years to site-specifically incorporate > 150 unnatural α-amino acids and > 10 α-hydroxy acids into proteins, in vitro, in cells, and in animals. More exotic monomers, including N-alkyl α-amino acids, D-amino acids, and some β-amino acids have been incorporated using cell-free in vitro systems. These efforts have deepened our understanding of protein and RNA function and provided real-world commodities such as antibody-drug conjugates, modified therapeutics, and biomaterials to benefit society. Yet, even after two-plus decades of research, until very recently–the preliminary results for this application–there were no reports of a ribosome able to introduce a β-amino acid into a protein in vivo, nor any sophisticated bioinformatics tools for ribosome redesign. The goal of this application is to develop and apply genetic engineering, bioinformatics, and chemical biology tools to repurpose the bacterial translational apparatus for the templated, in vivo biosynthesis of proteins and polypeptides containing backbone-modified monomers. The primary focus is on β- amino acids, as preliminary results indicate that this goal is attainable with a 4-5 year RO1 award, but the strategies, insights, and tools developed will provide a robust infrastructure for the sequence-templated biosynthesis of diverse classes of evolvable matter–truly exotic biopolymers–whose functions are limited only by our collective imagination. By creating new machines that efficiently incorporate β-amino acids into proteins in vivo, we will not only provide tools to address enduring questions about natural translation, but also provide real benefit to diverse segments of bio-medical research engine.