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.
Basic biological research, applied biomedical research, and biotechnology are limited by our ability to get information into and out from living systems, and to act on information inside living systems. This research will result in transformative advances in capabilities to design genetically- encoded technologies that receive, process, and transmit molecular information. Such technologies will significantly impact our ability to monitor, interface with, and program the dynamic cellular state, thereby resulting in an enhanced understanding of biological processes leading to disease and new approaches for constructing targeted molecular therapeutics.
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| Kennedy, Andrew B; Vowles, James V; d'Espaux, Leo et al. (2014) Protein-responsive ribozyme switches in eukaryotic cells. Nucleic Acids Res 42:12306-21 |
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