Nanoscale programming of cellular and physiological phenotypes: Equipment

细胞和生理表型的纳米级编程：设备

• 批准号: 10382641
• 负责人: NIKOLAY DOKHOLYAN
• 金额: $ 18.05万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-01-01 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10382641
• 关键词: Address; Binding; Biochemical; Cells; Code; Complex; Cues; Equipment; Evaluation; Funding; Goals; Image; In Vitro; Language; Light; Methods; Molecular; Molecular Conformation; Parents; Phenotype; Physiological; Process; Protein Engineering; Proteins; Rapid screening; Regulation; System; Techniques; Technology; Temperature; Testing; Validation; Writing; cell growth regulation; cell motility; design; genetic regulatory protein; in vitro testing; insight; nano; nanoscale; novel; optogenetics; programs; prototype; sensor; small molecule; targeted agent; tool

ABSTRACT The advent of protein design in recent years has brought us within reach of developing a “nanoscale programing language,” in which molecules serve as operands with their conformational states functioning as logical gates. Combining these operands into larger molecules and molecular complexes through protein engineering will allow us to write and execute “code” using nanoscale computing agents (NCAs). NCAs offer an orthogonal and complementary means for controlling cellular phenotypes. In the past 12 years, our group has developed technology toward this end, by engineering proteins that can be controlled by light and small molecules. We designed functional prototypes that have already offered valuable insights in the cellular motility field. The focus of the parent R35 is to develop NCAs for the targeted regulation of cellular activity. The main goals of this supplementary proposal are: (1) Extend the repertoire of inputs for regulation of proteins. We plan to utilize/design proteins that respond to pH and temperature via conformational change in order to modulate the activities of target proteins. (2) Build a multi-input NCA prototype. In order to facilitate the creation of higher order allosterically regulated protein systems and to conduct complex computations in cells, we have been working on building novel protein regulatory tools responding to diverse molecular cues, such as temperature and pH. Using such regulatory tools, we want to build a multi-input NCA prototype. In this direction, currently, we have successfully designed two-input protein systems in two candidate proteins using chemogenetic and optogenetic tools. Using the designed two-input systems we have controlled specific cellular phenotypes. Using various regulatory tools we plan to build a multi-input NCA prototype. Addressing these challenges will provide a significant leap in technology for programming living cells. One of the important aspects in our study is to test the NCAs using in vitro techniques. Here, we propose to test the NCAs in vitro using MicroScale Thermophoresis. We request supplemental funding for MicroScale Thermophoresis equipment to perform in vitro interaction analysis. The addition of MicroScale Thermophoresis equipment is vital to our ability to design new input sensors and validate the designed NCAs.

摘要 近年来，蛋白质设计的出现使我们能够开发“纳米级编程”， 在这种语言中，分子充当操作数，其构象状态充当逻辑门。 通过蛋白质工程将这些操作数组合成更大的分子和分子复合物， 我们使用纳米级计算代理（NCAs）编写和执行“代码”。NCA提供了一个正交和 用于控制细胞表型的互补手段。在过去的12年里，我们的团队已经发展到 通过设计可以被光和小分子控制的蛋白质来实现这一目标。我们 设计的功能原型已经在细胞运动领域提供了有价值的见解。重点 亲本R35的目的是开发用于靶向调节细胞活性的NCAs。其主要目标是 补充的建议是：（1）扩大输入的剧目调节蛋白质。我们计划 利用/设计通过构象变化响应pH和温度的蛋白质，以调节 目标蛋白的活性。(2)构建多输入NCA原型。为了促进更高秩序的建立 变构调节的蛋白质系统，并在细胞中进行复杂的计算，我们一直在努力 建立新的蛋白质调控工具，响应不同的分子线索，如温度和pH值。 这样的监管工具，我们要建立一个多输入NCA原型。在这个方向上，目前， 利用化学遗传学和光遗传学方法， 工具.使用设计的双输入系统，我们已经控制了特定的细胞表型。使用各种 监管工具，我们计划建立一个多输入NCA原型。应对这些挑战将提供一个 活细胞编程技术的重大飞跃。我们研究的一个重要方面是测试 使用体外技术的NCA。在这里，我们建议使用MicroScale Thermophoresis在体外测试NCAs。 我们要求为MicroScale热泳设备提供补充资金，以进行体外相互作用 分析.添加微量热泳设备对于我们设计新输入传感器的能力至关重要 并验证所设计的NCA。