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

项目总结 基因编码技术，使系统能够接收、处理和 传输分子信息对推进基础生物学研究至关重要，应用 生物医学研究和生物技术。RNA开关是一类配基反应基因 在不同的生物系统中实施的控制器，以改变我们的能力 监控动态蜂窝状态、与之交互并对其进行编程。而合成纤维的应用 监管RNA在过去十年中显著增长，目前设计的方法 新的RNA调节元件效率低、费力，通常不能深入了解 复合体中这些分子活性的序列-结构-功能关系 生物系统。 拟议项目的目标是制定接近设计的新战略， 测量和分析一类重要的RNA开关，这些开关将核酶作为 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 具体目标将集中在开发和验证多路复用的自动化演进管道，以 支持对新的RNA交换机进行可扩展的发现和表征。第二个具体目标 将专注于开发新的定量方法来检查二级结构和三级结构 对RNA开关的活性至关重要的相互作用。第三个具体目标将适用于新的 进化流水线和分析方法以产生新的RNA开关并探索 工程交换机功能的潜在机制。 该项目的成功实施将使我们的能力转变为快速可靠地建设 这些基因工具适用于不同的生物系统。此外，通过 新开发的方法将被用来发现对序列活动的新见解 这类重要的功能性RNA分子的基础景观和答案由来已久 在现场的问题。这些见解将更广泛地推动我们对RNA序列的理解- 结构-功能关系，并最终显著提高我们设计功能的能力 为生物医学应用量身定做的RNA分子。通过开发的管道和方法 该项目将改变研究界研究功能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.