Design and Synthesis of Robust and Tunable Nucleic Acid-Based Oscillators for Bionanotechnology

该项目的目标是利用核酸和蛋白质产生稳健和可调的分子钟。振荡器是生物学和计算学的基本组成部分：分子钟同步各种细胞过程，就像计算机时钟协调数百万硅器件的操作一样。本研究将结合动态系统工具和核酸纳米技术的最新进展，系统地研究体外生化时钟的设计和合成。一系列反应原语将被生成，用于核酸和蛋白质组分的动态生产、降解和转化。这些反应原语将用于构建候选时钟，其稳健性将在计算机上使用结构方法（图形和几何条件）和数值分岔分析进行评估。特别是，研究将集中在制造频率和幅度分别可调的时钟上。最佳候选振荡子将通过使用可用的软件工具指定原语的核酸序列来合成。实验筛选将采用依赖微滴的高通量随机实验装置进行。这项研究将扩展关于鲁棒可调谐振荡器结构的科学知识。特别是，该项目将弥合旨在发现生化网络中的周期性行为的理论研究与系统实验实施之间的差距。由于核酸网络的可编程性，可以建立这种桥梁。我们的研究结果将在两个主要领域产生影响：1)结合动力系统理论和高通量实验技术，将验证新的自下而上设计原则。该平台将用于生成其他自主生化电路，如多平稳系统和复杂非线性网络；2)可编程定时器件的可用性将使逻辑电路、分子机器、可重构纳米结构和模式形成的协调成为可能。生成的时钟将在包括纳米制造、生物材料和药物开发在内的各种环境中发挥作用，因为核酸可以选择性地与各种有机和无机材料相结合。此外，理论和计算结果将为研究自然振荡器的特征提供新的标准，并将为使用可编程核酸电路在细胞中产生周期性行为开辟新的途径。这项研究的结果将通过出版材料和国际会议发言加以传播。将为参与的研究生和本科生提供跨学科培训。PI的小组将在当地服务不足的学校开展针对K8-12学生的推广活动，包括制作关于周期性现象和技术和生物学计时的迷你课程和视频。

The objective of this project is to generate robust and tunable molecular clocks using nucleic acids and proteins. Oscillators are fundamental components both in biology and in computation: molecular clocks synchronize a variety of cellular processes, as much as computer clocks orchestrate the operations of millions of silicon devices. This research will combine dynamical systems tools and recent advances in nucleic acids nanotechnology to systematically study the design and synthesis of biochemical clocks in vitro. An array of reaction primitives will be generated for dynamic production, degradation and transformation of nucleic acids and protein components. These reaction primitives will be used to build candidate clocks, whose robustness will be evaluated in silico using structural methods (graphical and geometric conditions) and numerical bifurcation analysis. In particular, research will focus on creating clocks with separately tunable frequency and amplitude. The best candidate oscillators will be synthesized by specifying nucleic acid sequences of the primitives with available software tools. Experimental screening will be performed with a high-throughput, randomized experimental setup relying on microdroplets.This research will expand scientific knowledge regarding the structure of robust and tunable oscillators. In particular, this project will bridge the gap between theoretical studies aimed at discovering periodic behaviors in biochemical networks and their systematic experimental implementation. This bridge can be built because of the programmability of nucleic acid networks. Our results will have an impact in two main areas: 1) New bottom-up design principles will be validated integrating dynamical systems theory and high-throughput experimental techniques. This platform will be used to generate other autonomous biochemical circuits, such as multistationary systems and complex nonlinear networks; 2) The availability of programmable timing devices will enable the coordination of logic circuits, molecular machines, reconfigurable nanostructures, and pattern formation. The generated clocks will be useful in a variety of contexts including nanofabrication, biomaterials, and drug development, because nucleic acids can be selectively interfaced with a variety of organic and inorganic materials. In addition, theoretical and computational results will provide novel criteria to investigate the features of natural oscillators, and will open new avenues to the use of programmable nucleic acid circuits for generating periodic behaviors in cells. The results of this research will be disseminated through published materials and international conference presentations. Interdisciplinary training will be provided to participating graduate and undergraduate students. The PI's group will pursue outreach activities targeting K8-12 students at local underserved schools, including the creation of mini-courses and videos about periodic phenomena and timekeeping in technology and biology.