Data-Rich Strategies for Programming Ligand-Responsive RNA Regulatory Systems

项目摘要 遗传编码技术，使系统的设计，接收，处理， 传输分子信息对于推进基础生物学研究、应用 生物医学研究和生物技术。RNA开关是一类配体反应性遗传开关， 这些控制器正在不同的生物系统中实施，以改变我们的能力， 监测动态细胞状态、与动态细胞状态接口并对其进行编程。虽然应用合成 在过去的十年里，调控RNA的数量显著增加，目前设计调控RNA的方法是， 新的RNA调控元件是低效的、费力的，并且通常不会产生对RNA调控元件的深入了解。 序列-结构-功能的关系，这些分子的活动，在复杂的 生物系统。 拟议项目的目标是制定新的战略，以接近设计， 测量和分析一类重要的RNA开关，其包含核酶作为 基因控制元件该项目的目标将通过三个具体目标实现。第一 具体目标将侧重于开发和验证一个多路复用的自动化演进管道， 能够大规模发现和表征新的RNA开关。第二个具体目标 将致力于开发新的定量方法来检查二级结构和三级结构 这些相互作用是RNA开关活性的关键。第三个具体目标将适用新的 进化管道和分析方法，以产生新的RNA开关和探针的多样性， 工程开关功能的潜在机制。 该项目的成功执行将改变我们快速、可靠地建设的能力 这些遗传工具用于不同的生物系统。此外，通过 新开发的方法将被用来揭示序列活性的新见解 这类重要的功能性RNA分子的基本结构， 现场提问。这些见解将更广泛地推进我们对RNA序列的理解- 结构与功能的关系，并最终大大提高我们设计功能的能力 为生物医学应用定制的RNA分子。通过以下途径和办法制定的管道和办法： 该项目将改变研究界研究功能性RNA的范式 设计，从而对该领域产生更广泛的影响。

PROJECT SUMMARY Genetically-encoded technologies that enable the design of systems that receive, process, and transmit molecular information are essential to advancing basic biological research, applied biomedical research, and biotechnology. RNA switches are a class of ligand-responsive genetic controllers that are being implemented in diverse biological systems to transform our ability to monitor, interface with, and program the dynamic cellular state. While the application of synthetic regulatory RNAs has grown remarkably over the past decade, current approaches to the design of new RNA regulatory elements are inefficient, laborious, and typically do not yield insight into the sequence-structure-function relationships underlying the activities of these molecules in complex biological systems. The goal of the proposed project is to develop new strategies for approaching the design, measurement, and analysis of an important class of RNA switches that incorporate ribozymes as the gene-control element. The goal of the project will be achieved through three specific aims. The first specific aim will focus on developing and validating a multiplexed, automated evolution pipeline to enable the scalable discovery and characterization of new RNA switches. The second specific aim will focus on developing new quantitative methods to examine the secondary structures and tertiary interactions that are key to the activity of RNA switches. The third specific aim will apply the new evolution pipeline and analysis methods to generate new RNA switches and probe the diversity of mechanisms underlying the function of engineered switches. The successful execution of the project will transform our capacity to rapidly and reliably build these genetic tools for diverse biological systems. In addition, the rich datasets generated through the newly developed methods will be leveraged to uncover new insight into the sequence-activity landscapes underlying this important class of functional RNA molecules and answer long-standing questions in the field. These insights will more broadly advance our understanding of RNA sequence- structure-function relationships and ultimately dramatically improve our capacities to design functional RNA molecules tailored to biomedical applications. The pipelines and approaches developed through this project will change the paradigm by which the research community approaches functional RNA design, thereby having a substantially broader impact on the field.