Genetically-encoded technologies that enable the construction 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 measurement, analysis, and design 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 new data-rich, massively-parallel measurement strategies that leverage next generation sequencing (NGS)-based assays to obtain gene-regulatory and cleavage activity information on millions of RNA switch sequences in a single experiment. The second specific aim will focus on developing new computational methods to perform analyses of large NGS datasets on RNA switch activities to gain and apply new insight into the sequence-structure-function relationships of functional RNA molecules. The third specific aim will apply these new measurement and analysis methods to specific libraries and cellular systems to advance our understanding of the sequence-activity landscape of RNA 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 for rational design of functional RNA molecules. The assay and analysis methods 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.

Public Health Relevance

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, interfac 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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM086663-07
Application #
9438910
Study Section
Genomics, Computational Biology and Technology Study Section (GCAT)
Program Officer
Reddy, Michael K
Project Start
2009-01-02
Project End
2020-02-29
Budget Start
2018-03-01
Budget End
2019-02-28
Support Year
7
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Stanford University
Department
Biomedical Engineering
Type
Schools of Medicine
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94304
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Wang, Yen-Hsiang; McKeague, Maureen; Hsu, Tammy M et al. (2016) Design and Construction of Generalizable RNA-Protein Hybrid Controllers by Level-Matched Genetic Signal Amplification. Cell Syst 3:549-562.e7
McKeague, Maureen; Wong, Remus S; Smolke, Christina D (2016) Opportunities in the design and application of RNA for gene expression control. Nucleic Acids Res 44:2987-99
Townshend, Brent; Kennedy, Andrew B; Xiang, Joy S et al. (2015) High-throughput cellular RNA device engineering. Nat Methods 12:989-94
Church, George M; Elowitz, Michael B; Smolke, Christina D et al. (2014) Realizing the potential of synthetic biology. Nat Rev Mol Cell Biol 15:289-94
Chang, Andrew L; McKeague, Maureen; Smolke, Christina D (2014) Facile characterization of aptamer kinetic and equilibrium binding properties using surface plasmon resonance. Methods Enzymol 549:451-66
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
Galloway, Kate E; Franco, Elisa; Smolke, Christina D (2013) Dynamically reshaping signaling networks to program cell fate via genetic controllers. Science 341:1235005
Galanie, Stephanie; Siddiqui, Michael S; Smolke, Christina D (2013) Molecular tools for chemical biotechnology. Curr Opin Biotechnol 24:1000-9
Kennedy, Andrew B; Liang, Joe C; Smolke, Christina D (2013) A versatile cis-blocking and trans-activation strategy for ribozyme characterization. Nucleic Acids Res 41:e41

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