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.

抽象的 近年来蛋白质设计的出现使我们有能力开发“纳米级编程” 语言”，其中分子作为操作数，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将允许 我们使用纳米级计算代理（NCA）编写和执行“代码”。 NCA 提供正交和 控制细胞表型的补充手段。 12年来，我们集团不断发展 为此目的，通过工程改造可以由光和小分子控制的蛋白质。我们 设计的功能原型已经在细胞运动领域提供了宝贵的见解。焦点 母体 R35 的目标是开发 NCA 以靶向调节细胞活性。本次活动的主要目标 补充建议是： (1) 扩大蛋白质调节的输入范围。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。为了方便创建更高阶 变构调节的蛋白质系统并在细胞中进行复杂的计算，我们一直致力于 构建响应不同分子线索（例如温度和 pH 值）的新型蛋白质调控工具。使用 这样的监管工具，我们要构建一个多输入的NCA原型。目前，我们在这个方向上 使用化学遗传学和光遗传学成功设计了两种候选蛋白质的双输入蛋白质系统 工具。使用设计的双输入系统，我们控制了特定的细胞表型。使用各种 我们计划构建一个多输入 NCA 原型的监管工具。应对这些挑战将提供 活细胞编程技术的重大飞跃。我们研究的重要方面之一是测试 NCA 使用体外技术。在这里，我们建议使用微量热泳法在体外测试 NCA。 我们请求为微型热泳设备提供补充资金以进行体外相互作用 分析。添加微型热泳设备对于我们设计新输入传感器的能力至关重要 并验证设计的 NCA。