Repurposing the Ribosome for Exotic Polymers

项目摘要： 用于模板引导蛋白质合成的机器-核糖体及其相关的 翻译因子-已被利用超过20年，以网站-具体纳入> 150 非天然α-氨基酸和> 10 α-羟基酸转化为蛋白质，在体外、细胞和动物中。 更多的外来单体，包括N-烷基α-氨基酸、D-氨基酸和一些β-氨基 已经使用无细胞的体外系统掺入酸。这些努力加深了我们的 了解蛋白质和RNA的功能，并提供了现实世界的商品，如 抗体-药物缀合物、修饰的治疗剂和生物材料，以造福社会。但即使 经过二十多年的研究，直到最近， 应用-没有核糖体能够将β-氨基酸引入蛋白质的报道 在体内，也没有任何复杂的生物信息学工具的核糖体重新设计。这个目标 应用是发展和应用基因工程、生物信息学和化学生物学 工具，以重新利用细菌翻译装置的模板，在体内生物合成的 含有主链修饰的单体的蛋白质和多肽。主要关注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 奖项，但开发的战略，见解和工具将提供强大的基础设施， 不同种类的可进化物质的序列模板生物合成-真正的异国情调 生物聚合物-其功能仅受我们集体想象力的限制。通过创造新 在体内将β-氨基酸有效地结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持久问题的工具，但也为以下方面提供了真实的好处： 生物医学研究引擎的不同部分。

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.