Nanoscale programming of cellular and physiological phenotypes: Equipment

摘要 近年来蛋白质设计的出现使我们能够开发出一种纳米级的程序设计 在语言中，分子充当操作数，其构象状态起逻辑门的作用。 通过蛋白质工程将这些操作数结合成更大的分子和分子复合体 美国使用纳米级计算代理(NCA)编写和执行“代码”。NCAS提供了一个正交和 控制细胞表型的补充手段。在过去的12年里，我们集团发展了 通过改造可以由光和小分子控制的蛋白质来实现这一目标的技术。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。焦点 亲本R35的目的是开发NCA，用于靶向调节细胞活动。这次会议的主要目标是 补充建议有：(1)扩大蛋白质调控的输入范围。我们计划 利用/设计通过构象变化对pH和温度做出反应的蛋白质，以调节 目的蛋白的活性。(2)构建多输入NCA原型。为了促进更高秩序的建立 为了在细胞中进行复杂的计算，我们一直致力于 构建新的蛋白质调节工具，以响应不同的分子信号，如温度和pH。vbl.使用 这样的监管工具，我们要构建一个多输入的NCA原型。在这个方向上，目前我们有 利用化学遗传学和光遗传学在两种候选蛋白质中成功地设计了双输入蛋白质系统 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 监管工具我们计划构建一个多输入NCA原型。应对这些挑战将提供 活细胞编程技术的重大飞跃。我们研究的一个重要方面是测试 NCA使用体外技术。在这里，我们建议使用微型热电泳法在体外测试NCA。 我们要求为进行体外相互作用的微型热电导入设备提供补充资金。 分析。增加微型热电导入设备对我们设计新的输入传感器的能力至关重要 并对所设计的NCA进行验证。

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.