Development of an improved core technology for efficient genetic code expansion in biomedical research

标题：生物医学中高效遗传密码扩展的改进核心技术的开发 研究 主要调查者：瑞安·梅尔 摘要 遗传密码扩展(GCE)允许将非规范氨基酸(NCAA)引入蛋白质中。 这为操纵和扩展生化活动提供了一个强大的工具箱，打开了新的可能性 为了控制细胞中的蛋白质功能，并创造出针对许多人类病理的新疗法， 包括癌症、自身免疫综合症和代谢性疾病。最初是在细菌系统中开发的， 由于最近扩展的能力，GCE的生物医学应用现在正在迅速增加 在哺乳动物细胞培养和模型多细胞生物体中。尽管有这些进步和 该技术的巨大潜力，是全面实施GCE的关键挑战--特别是在体内 应用-在重新编码的细菌和哺乳动物中低效地合成含有NCAA的蛋白质 细胞。特别是，近年来的各种研究都集中在找出 技术：创建高效的正交氨酰-tRNA合成酶(o-RS)并转移 RNA(o-tRNAs)。事实上，研究表明，现有的o-RS/tRNA对是两到五个数量级的 比自然产生的配对效率低得多。我们在这项提案中的目标是消除这种情况 瓶颈：(1)开发一种普遍适用的优化工程功能的方法 O-RS/tRNA对，以及(2)为当前的主力O-RS/tRNA创建改进的“高效率”底盘 成对的。有了这些进展，在体内进行GCE研究以探索细胞功能的方式可以 产生含有一种或多种NCAA的蛋白质，其浓度和调控类似于 天然细胞，在没有大量截断产物或过量GCE的情况下始终如一地这样做 造成混乱的组件。到目前为止还没有人试图系统地攻击这一点 有问题。与该领域以前的所有工作相比，我们的方法有两个关键特点。首先，我们将 建立一个新的、详细的数据库，将纯化的o-RS/tRNA的体外动力学性质与 相同的o-RS/tRNA对在体内和在无细胞细菌和 哺乳动物提取物。第二，我们将开发和应用迭代过程来改进现有的o- RS/tRNA对，在计算方法和实验测定变构的指导下 途径，以优化覆盖蛋白质-tRNA复合体部分的文库设计 在探索得很好的初级NCAA结合位点之外。然后将这些库合并到DIRECTED 选择催化改良配对的进化方法。我们在以下方面提出了这一创新方法 对侧重于技术开发的特定NIGMS资助机会的回应，以实现 生物医学研究，它充分体现了PAR-17-045为创建广泛的 适用的新研究工具，是具有真正转型潜力的高风险企业。

Title: Development of an improved core technology for efficient genetic code expansion in biomedical research Principal Investigator: Ryan Mehl Summary Genetic code expansion (GCE) allows the introduction of noncanonical amino acids (ncAAs) into proteins. This provides a powerful toolbox to manipulate and expand biochemical activities, opens new possibilities to control protein function in cells, and creates new therapeutics against many human pathologies, including cancer, autoimmune syndromes, and metabolic diseases. First developed in bacterial systems, biomedical applications of GCE are now rapidly increasing as a consequence of recently expanded abilities in both mammalian cell culture and model multicellular organisms. Despite these advances and the enormous potential of the technology, a key challenge to fully implementing GCE – especially for in vivo applications – is the inefficient synthesis of ncAA-containing proteins in recoded bacterial and mammalian cells. In particular, a variety of studies in recent years have converged on identifying the key bottleneck in the technology: creating efficient pairs of orthogonal aminoacyl-tRNA synthetases (o-RS) and transfer RNAs (o-tRNAs). Indeed, studies indicate that existing o-RS/tRNA pairs are two to five orders of magnitude less efficient than naturally occurring pairs. Our goal in this proposal is to eliminate this bottleneck by (1) developing a generally applicable approach for optimizing the function of engineered o-RS/tRNA pairs, and (2) creating improved “high-efficiency” chassis for current workhorse o-RS/tRNA pairs. With these advances, in vivo GCE studies to probe cellular functions can be carried out in ways that generate proteins containing one or more ncAAs at similar concentrations and regulatory control as in natural cells, and do it consistently without massive amounts of truncation products or excess GCE components that cause confounding disturbances. No one has yet attempted to systematically attack this problem. Our approach has two key features as compared to all prior work in the field. First, we will generate a novel, detailed database correlating in vitro kinetic properties of purified o-RS/tRNA with the performance of the same o-RS/tRNA pairs in protein synthesis in vivo and in cell-free bacterial and mammalian extracts. Second, we will develop and apply an iterative process to improve existing o- RS/tRNA pairs, guided by both computational approaches and experimental determination of allosteric pathways, in order to optimize the design of libraries covering portions of the protein-tRNA complexes outside the well-explored primary ncAA binding site. These libraries are then incorporated into directed evolution approaches to select for catalytically improved pairs. We propose this innovative approach in response to a specific NIGMS funding opportunity focused on technology development to enable biomedical research, and it fully embodies the qualities specified by PAR-17-045 for the creation of broadly applicable new research tools that are higher-risk ventures with truly transformational potential.