CAREER: A Generalized Quantitative Imaging Approach for Small Molecules using Genetically Engineered RNA Sensors

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With support from the Biosensing Program in the Chemical, Bioengineering, Environmental, and Transport Systems Division, and partial co-funding from the Chemical Measurement and Imaging Program in the Division of Chemistry, Dr. Mingxu You and his research team at the University of Massachusetts Amherst are streamlining a generalized approach for imaging small molecules in living cells. Revolutionary fluorescent biosensing probes have been engineered for the live-cell imaging of proteins, nucleic acids, and small molecules. The outcome of this project will provide ground-breaking methodologies in the field of live-cell imaging of small molecules. The proposed project will allow researchers from the life sciences, engineering, and physical sciences to measure cellular levels and dynamics of a wide range of analytes with readily accessible sensors. This project will also integrate sensor engineering research with scientific education and outreach. In this project, a new generalized sensor system is developed by integrating emerging fluorogenic RNA aptamers, a systematic evolution approach, and natural RNA machineries with nucleic acid nanotechnology. Fluorogenic RNA aptamers, such as so-called "Spinach" and "Broccoli", serve as the genetically encoded fluorescent reporters. Systematic evolution is used to screen for aptamers that can selectively bind with target small molecules inside live cells. Finally, RNA devices and circuits existing in nature or enabled by nanotechnology provides signal amplification as well as scaffolds for the ratiometric and multiplexed imaging and quantification of target analytes. The major research objectives are: (1) to develop quantitative RNA sensors with multiplexing ability for the cellular imaging of small-molecule levels; (2) to engineer sensitive and robust RNA circuits and devices for detecting low-abundance analytes in both prokaryotic and eukaryotic cells; and (3) to generate a high-throughput screening platform to rapidly evolve functional RNA sensors directly in a cellular milieu. Overall, RNA-based fluorescent probes will be radically advanced through this project for quantitative imaging of a wide range of small-molecule analytes in living cells. Dr. You is an active faculty participant in the Eureka! Program of Girls Inc. of Holyoke, and will continue to provide summer research opportunities for female middle school students in this program. The You research team will also provide lab tours for local middle school students and teachers. In addition, this project provides training opportunities for the next-generation of U.S. trained scientists. Undergraduate and graduate students involved in this project will gain experience in bioengineering, analytical chemistry, and chemical biology. Based on the proposed sensor design principles, Dr. You will assemble and mentor a team of UMass Amherst undergrads for the International Genetically Engineered Machine (iGEM) competition.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.