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

开发改进的核心技术，用于生物医学研究中有效的遗传密码扩展

• 批准号: 10093096
• 负责人: RYAN A MEHL
• 金额: $ 35.91万
• 依托单位: OREGON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-02-01 至 2022-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10093096
• 关键词: 3-nitrotyrosine; Active Sites; Adopted; Affinity; Algorithm Design; Amber; Amino Acids; Amino Acyl-tRNA Synthetases; Autoimmune; Bacteria; Binding Sites; Biochemical; Biological Assay; Biological Models; Biomedical Research; Cell Culture Techniques; Cell model; Cell physiology; Cells; Codon Nucleotides; Complex; Coupling; Cross Reactions; Databases; Development; Directed Molecular Evolution; Distal; Engineering; Epitopes; Escherichia coli; Eukaryota; Funding Opportunities; GTP Binding; Genetic Code; Genetic Engineering; Goals; Green Fluorescent Proteins; Human Pathology; In Vitro; Kinetics; Laboratories; Libraries; Malignant Neoplasms; Mammalian Cell; Maps; Measurement; Measures; Metabolic Diseases; Methanosarcina barkeri; Methodology; Methods; Modeling; Mutagenesis; Mutate; National Institute of General Medical Sciences; Organism; Pathway interactions; Peptides; Performance; Phylogenetic Analysis; Physiological; Positioning Attribute; Principal Investigator; Procedures; Process; Property; Protein Biosynthesis; Protein Engineering; Proteins; Protocols documentation; RNA; Reporter; Research; Research Personnel; Sampling; Scheme; Source; Specific qualifier value; Structure; Syndrome; System; Technology; Transfer RNA; Tyrosine-tRNA Ligase; Work; base; cellular engineering; chemical group; design; direct application; high risk; human disease; improved; in vivo; innovation; molecular sequence database; novel; novel therapeutics; overexpression; premature; protein expression; protein function; research and development; response; screening; stem; technology development; therapeutic development; therapy development; tool

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.

标题：生物医学领域高效遗传密码扩展的改进核心技术的开发 研究 主要研究者：Ryan Mehl 总结 遗传密码扩展（GCE）允许将非经典氨基酸（ncAAs）引入蛋白质中。 这提供了一个强大的工具箱来操纵和扩展生物化学活动， 来控制细胞中的蛋白质功能，并创造了针对许多人类疾病的新疗法， 包括癌症、自身免疫综合征和代谢疾病。最初是在细菌系统中发展起来的， 由于最近能力的扩展，GCE的生物医学应用正在迅速增加 在哺乳动物细胞培养物和多细胞生物模型中。尽管取得了这些进步， 该技术的巨大潜力，是全面实施GCE的关键挑战-特别是对于体内 应用-是重新编码的细菌和哺乳动物中含ncAA的蛋白质的低效合成 细胞特别是，近年来的各种研究都集中在确定关键的瓶颈， 该技术：创建高效的正交氨酰-tRNA合成酶对（o-RS）， RNA（o-tRNA）。事实上，研究表明，现有的o-RS/tRNA对是两到五个数量级， 比自然发生的对效率低。我们在这个提案中的目标是消除这一点 瓶颈（1）开发一种普遍适用的方法，用于优化工程的功能， o-RS/tRNA对，以及（2）为当前的主力o-RS/tRNA创建改进的“高效”底盘 对.有了这些进展，体内GCE研究，以探测细胞功能可以进行的方式， 产生含有一种或多种ncAA的蛋白质，其浓度和调节控制与 天然细胞，并始终没有大量的截断产物或过量的GCE 造成干扰的部件。还没有人试图系统地攻击这一点 问题.我们的方法有两个关键特征相比，在该领域的所有先前的工作。一是 生成一个新的、详细的数据库，将纯化的o-RS/tRNA的体外动力学特性与 相同的o-RS/tRNA对在体内和无细胞细菌中的蛋白质合成中的性能， 哺乳动物提取物。第二，我们将开发和应用一个迭代过程，以改善现有的o- RS/tRNA对，由计算方法和变构的实验测定指导 途径，以优化覆盖蛋白质-tRNA复合物部分的文库的设计 在充分探索的主要ncAA结合位点之外。然后将这些库并入定向 进化方法来选择催化改进的对。我们提出了这种创新的方法， 对专注于技术开发的特定NIGMS资助机会的响应，以实现 生物医学研究，它充分体现了PAR-17-045规定的广泛创造的品质 适用的新研究工具，是真正具有变革潜力的高风险企业。