CAREER: Uncovering Quantitative Design Principles of RNA Regulators For Synthetic Biology

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Nontechnical Description: Cells have an amazing ability to process information, make decisions, and change their state in response to changing environments. This ability is encoded within the cellular DNA genome, which is converted into RNA and protein molecules through the basic processes of gene expression. Among the many functions these RNAs and proteins perform is regulating their own expression. In fact RNAs are now known to regulate almost all aspects of gene expression, and play central roles in controlling some of life's most basic processes. A key question in biotechnology and synthetic biology is then: How can RNA molecules be designed to control the expression of target genes inside cells to facilitate applications ranging from using cells as chemical factories all the way to using them as environmental sensors? As with many biomolecules, RNA function is intimately related to its structure. In this work, the investigator builds off of his development of a new small synthetic RNA mechanism called Small Transcription Activating RNAs, or STARs. STARs are hypothesized to allow the construction of unique gene expression control techniques since they represent a brand new function for sRNAs. This project seeks to use STARs as a test-bed for uncovering the principles for engineering RNA molecules to precisely control gene expression inside cells. This work will help realize the potential of RNA as a powerful substrate for cellular engineering. It will also shed new light on our scientific understanding of the fundamental sequence/structure/function relationship that underlies RNA's central role in natural biological systems. The projected studies and research training activities are thus expected to have a broad impact on society, ranging from the science of cellular gene regulation and the engineering science of RNA gene regulation that can directly connect to a broad array of biotechnological applications. This project will also cultivate the next generation of highly trained graduate students and teachers of synthetic biology who will be introduced to the broad, interdisciplinary nature of biotechnology research. Moreover, this program will actively engage the broader community to help create an informed public that is equipped to make important decisions about the future of synthetic biology.Technical Description: The overall goal of this project is to build an integrated research and education program focused on uncovering quantitative design principles that link small RNA sequence, structure, and function, and to use these principles to design synthetic RNAs that can precisely regulate gene expression. The education plan focuses on integrating research into the training of the next generation of synthetic biology students and teachers, and informing and exciting the broader public about synthetic biology.Trans-acting bacterial small RNAs (sRNA) exert regulation via direct RNA-RNA interactions with target messenger RNAs (mRNAs) that cause structural changes in the target. These changes in turn regulate many aspects of gene expression including transcription, translation and mRNA degradation. The central hypotheses of this project are that: 1) quantitative sRNA structure/function design principles can be discovered and used to rationally optimize and expand the functionality of RNA regulators, and 2) circuit-level design rules can be discovered and used to engineer new synthetic RNA genetic modules that control the timing and pattern of gene expression. The central goal of this CAREER project is to use Small Transcription Activating RNAs (STARs) as a test-bed for uncovering the quantitative design principles of RNA regulators for synthetic biology. This will be pursued using a multi-faceted approach that includes using cutting-edge RNA structure measurement technologies to elucidate the molecular level design principles of STARs. Once molecular-level principles are learned, the project will focus on using these techniques to learn the design rules for integrating STARs into decision making regulatory networks. In addition, new experiments and computational tools that can quantitatively model the kinetics of gene expression mediated by STARs will be used to understand how different aspects of STAR function propagate through RNA networks. Successful completion of these studies will forward the broader goal of creating a quantitative discipline of biological design that can be used to program cellular systems to solve important problems in sustainable energy, and biomanufacturing. In addition, the work may provide insight to mechanisms of native small RNA regulation of naturally occurring biological systems.Educational activities include developing a long term plan for the continuation of the Cold Spring Harbor Synthetic Biology summer course as a training center for a broad range of students and teachers across the globe; developing quantitative curricular materials for training future generations of synthetic biologists; and performing hands-on activities aimed towards families with school-age children to excite and inform them about synthetic biology.