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

在化学，生物工程，环境和运输系统部门的生物传感计划的支持下，以及化学部门化学测量和成像计划的部分共同资助下，马萨诸塞州阿默斯特大学的Mingxu You博士和他的研究团队正在简化活细胞中小分子成像的通用方法。 革命性的荧光生物传感探针已经被设计用于蛋白质、核酸和小分子的活细胞成像。 该项目的成果将在小分子活细胞成像领域提供突破性的方法。 拟议的项目将允许来自生命科学，工程和物理科学的研究人员使用易于访问的传感器测量各种分析物的细胞水平和动态。 该项目还将把传感器工程研究与科学教育和推广相结合。 在这个项目中，一个新的广义传感器系统是通过整合新兴的荧光RNA适体，系统进化的方法，和天然RNA机器与核酸纳米技术。 荧光RNA适体，如所谓的“菠菜”和“花椰菜”，充当遗传编码的荧光报告基因。 系统进化用于筛选可以选择性地与活细胞内的靶小分子结合的适体。 最后，自然界中存在的或通过纳米技术实现的RNA装置和电路提供了信号放大以及用于靶分析物的比率和多重成像和定量的支架。 主要研究目标是：（1）开发具有多路复用能力的定量RNA传感器，用于小分子水平的细胞成像;（2）设计灵敏和稳健的RNA电路和设备，用于检测原核和真核细胞中的低丰度分析物;（3）生成高通量筛选平台，以直接在细胞环境中快速进化功能性RNA传感器。 总体而言，基于RNA的荧光探针将通过该项目从根本上推进，用于活细胞中各种小分子分析物的定量成像。尤博士是尤里卡的积极参与者！Girls Inc.的霍利奥克，并将继续提供夏季研究机会，为女中学生在这一计划。 You研究团队还将为当地中学生和教师提供实验室图尔斯参观。 此外，该项目还为美国下一代训练有素的科学家提供培训机会。 参与该项目的本科生和研究生将获得生物工程，分析化学和化学生物学方面的经验。 根据所提出的传感器设计原则，尤博士将召集和指导一个麻省大学阿默斯特分校的本科生团队参加国际遗传工程机器（iGEM）竞赛。该奖项反映了NSF的法定使命，并通过使用基金会的智力价值和更广泛的影响审查标准进行评估，被认为值得支持。

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.