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

在化学、生物工程、环境和运输系统学部生物传感项目的支持下，以及化学学部化学测量和成像项目的部分共同资助下，尤明旭博士和他在马萨诸塞大学阿默斯特分校的研究团队正在简化一种用于活细胞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。该项目的成果将为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来测量各种分析物的细胞水平和动态。该项目还将传感器工程研究与科学教育和推广相结合。在本项目中，我们将新兴的荧光RNA适体、系统进化方法、天然RNA机制与核酸纳米技术相结合，开发了一种新的通用传感器系统。荧光RNA适配体，如所谓的“菠菜”和“西兰花”，作为基因编码的荧光报告者。系统进化用于筛选能够选择性地与活细胞内目标小分子结合的适体。最后，RNA器件和电路存在于自然界中或通过纳米技术实现，为目标分析物的比率和多路成像和定量提供了信号放大和支架。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)设计出灵敏、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)建立一个高通量筛选平台，直接在细胞环境中快速进化功能性RNA传感器。总的来说，基于rna的荧光探针将通过该项目从根本上推进对活细胞中广泛的小分子分析物的定量成像。尤博士是尤里卡！霍利奥克女孩公司的项目，并将继续在这个项目中为女中学生提供暑期研究机会。You研究团队还将为当地中学生和教师提供实验室参观。此外，该项目还为美国培养的下一代科学家提供了培训机会。参与本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。基于提出的传感器设计原则，尤博士将组建并指导麻省大学阿默斯特分校本科生团队参加国际基因工程机器（iGEM）竞赛。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识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.