Quadruplet Decoding for Multiplexed Non-Canonical Amino Acid Incorporation

项目摘要/摘要 在过去的20年里，蛋白质疗法在药物发现中发挥了越来越大的作用， 导致今天市场上大约有380种FDA批准的药物。所有这些生物制剂只使用了很小一部分 化学空间可达标准的20个氨基酸。整合非典范氨基酸的能力 将酸(NCAA)引入蛋白质疗法是一种很有前途的策略，它将极大地改善这种化学物质 通过环化或聚乙二醇化提高生物制剂的生物利用度，并促进 化学偶联产生免疫偶联物。NCAA的掺入可以在体外和 但体外系统的成本使其不适合大规模生产治疗药物，以及 由于缺乏免费的三联体密码子，细胞的生产受到了阻碍。我建议利用强大的新导演 进化技术开发一种能够有效地将大量NCAA整合到蛋白质中的方法 体内，从而为低成本、化学多样化的蛋白质疗法铺平了道路。 目前使用的技术仅限于在同一蛋白质中掺入不超过两个NCAA 链条。在这里，我的目标是提高这一技术能力。如果成功，这种方法将为 在生产完全由NCAA组成的基因编码材料方面，一种技术能力 将立即应用于治疗学以及更广泛的材料科学。我专注于 帧移位抑制，这是一种用于NCAA整合的可扩展技术，提供256个密码子，而不仅仅是 二。我建议开发一种用于帧移位抑制的记者，使我能够量化抑制效率 比以前可能的更强劲。接下来，我建议进化出两个独立的分子靶点 限制移码抑制效率：抑制tRNA和核糖体rRNA，有利于提高效率。 与传统工程和小型图书馆的现有工作已经在实现这一目标方面取得了一定的成功 提高了效率，这表明我的方法利用了强大的持续进化 技术，将是成功的。总而言之，该项目研究了一种可扩展的方法，以实现 NCAA的整合能力，旨在开发能够缓解技术困难的技术 这目前限制了这种方法的实用性。

Project Summary/Abstract Protein therapeutics have played an ever-increasing role in drug discovery during the past 20 years, leading to some 380 FDA-approved drugs on the market today. All these biologics use only the tiny fraction of chemical space accessible to the standard 20 amino acids. The ability to incorporate Non-Canonical Amino Acids (NCAAs) into protein therapeutics is a promising strategy that would greatly improve the chemical sophistication of biologics, increase their bioavailability through cyclization or PEGylation, and facilitate chemical conjugation to create immunoconjugates. NCAA incorporation can be accomplished both in vitro and in vivo, but the cost of in vitro systems renders them unsuitable for production of therapeutics at scale, and cellular production is crippled by the lack of free triplet codons. I propose to harness powerful new directed evolution techniques to develop a method capable of efficiently incorporating numerous NCAAs into proteins in vivo, thereby paving the way toward low-cost, chemically-diverse protein therapeutics. Techniques in use today are limited to incorporation of no more than two NCAAs into the same protein chain. Here, I aim to improve upon this technical capability. If successful, this approach will pave the way toward production of genetically-encoded materials entirely composed of NCAAs, a technical capability that would have immediate applications for therapeutics as well as material science more broadly. I focus on frameshift suppression, an extensible technique for NCAA incorporation that offers 256 codons, rather than just two. I propose to develop a reporter for frameshift suppression, allowing me to quantify suppression efficiency more robustly than was previously possible. Next, I propose to evolve two independent molecular targets that limit frameshift suppression efficiency: suppressor tRNAs and ribosomal rRNA, in favor of improved efficiency. Existing work with traditional engineering and small libraries has seen modest success already toward the goal of improved efficiency, suggesting that my approach, which leverages a powerful continuous evolution technique, will be successful. Together, this project investigates an extensible approach to the important capability of NCAA incorporation, and aims to develop technology capable of alleviating technical difficulties that presently limit the utility of this approach.