Ideal eukaryotic tetrazine ligations for imaging protein dynamics in live cells

在不影响其功能的情况下修改和可视化活细胞中的生物分子的能力对于理解生命系统至关重要。本研究的目标是开发一个完整的生物正交标记系统，该系统可以编码到蛋白质的任何位置，并在生命系统内对蛋白质进行标记。该项目将开发非常快速和高度特异性的蛋白质标记方法，使真核细胞的无水洗蛋白质标记和单分子成像成为可能。此外，该项目将使新的蛋白质工程方法继续整合到本科实验课程中，并将为参加年度非自然蛋白质设施GCE研讨会的代表性不足的科学家提供奖学金。非自然蛋白质设施使用美国国家科学基金会的资金来开发和集中遗传工具，并接待来自世界各地的本科生、研究生和研究人员，协助将蛋白质工程方法整合到他们的实验室中。这些技术也具有巨大的经济发展潜力，公司正在与俄勒冈州立大学合作，以许可该项目中开发的技术。在不影响其功能的情况下修改和可视化活细胞中的生物分子的能力对于理解生命系统至关重要。该项目的目标是开发一个完整的生物正交标记系统，该系统可以被遗传编码到生命系统中的任何蛋白质中，并且非常快速和高度特异性。该项目将证明使用遗传密码扩增（GCE）工具在活真核细胞中进行免洗蛋白标记和单分子成像是可能的。该提案的第一个目标是通过基因编码含四氮的非规范氨基酸（Tet-ncAAs）到蛋白质中，将定量蛋白质标记率提高1000倍以上。第二个目标是通过优化标签的生物利用度和特异性来开发与含四氮蛋白反应的标签，以实现超低背景。第三个目标是利用位点特异性理想标记系统的优势，开发一类新的蛋白质标签，当它们与DNA结合时“开启”，允许人们监测DNA-蛋白质相互作用。第四个目标是通过将理想的蛋白质标记系统与共聚焦显微镜和单分子跟踪相结合，解决围绕转录因子STAT3（信号换能器和转录激活器3）的定位，动力学和DNA结合能力的三个关键问题。除了开发通用荧光标记外，本研究还将通过工程蛋白标记开辟标记模式，使其在结合DNA时具有荧光性，并可用于报告DNA结合蛋白的功能。这些实验的结果将填补现有知识中的重要空白，这些空白不能用当前的荧光标记方法充分解决。这项研究的成功完成将提供一套GCE工具，这将推动细胞内标记领域的广泛应用，并将在缺乏N-或c端蛋白标签的情况下产生STAT3功能的第一个结果。该项目由分子和细胞生物科学部的分子生物物理集群资助。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。

The ability to modify and visualize biomolecules in live cells without compromising their function is critical to understanding living systems. The goal of this research is to develop a complete bioorthogonal labeling system that can be encoded into any location in a protein and functions to label proteins inside living systems. This project will develop protein labeling methods that are extremely fast and highly specific, enabling wash-free protein labeling and single molecule imaging in live eukaryotic cells. In addition, this project will enable the continued integration of new protein engineering methods into undergraduate lab courses and will provide fellowships to underrepresented scientists to attend the annual Unnatural Protein Facility GCE workshops. The Unnatural Protein Facility uses NSF funding to develop and centralize genetic tools, and hosts undergraduate students, graduate students and researchers from around the world to assist in integrating protein engineering methods into their labs. These technologies also have significant potential for economic development and companies are working with Oregon State University to license the technologies developed in this project. The ability to modify and visualize biomolecules in live cells without compromising their function is critical to understanding living systems. The goal of this project is to develop a complete bioorthogonal labeling system that can be genetically encoded into any protein in living systems and is extremely fast and highly specific. This project will demonstrate that wash-free protein labeling and single molecule imaging is possible in live eukaryotic cells using Genetic Code Expansion (GCE) tools. The first aim of this proposal focuses on increasing quantitative protein labeling rates by over 1000-fold by genetically encoding tetrazine-containing non-canonical amino acids (Tet-ncAAs) into proteins. The second aim seeks to develop labels that react with tetrazine-containing proteins by optimizing label bioavailability and specificity to achieve ultralow background. The third aim leverages the advantages of the site-specific ideal labeling systems to develop a new class of protein labels that “turn-on” when they bind to DNA, allowing one to monitor DNA-Protein interactions. The fourth aim tackles three key questions surrounding the localization, dynamics and DNA binding ability of the transcription factor STAT3 (Signal Transducer and Activator of Transcription 3) by combining the ideal protein labeling system with confocal microscopy and single molecule tracking. In addition to developing general fluorescent labels, this research will open access to labeling modalities by engineering protein labels that are fluorogenic upon binding DNA and can be used to report the function of DNA binding proteins. The results of these experiments will fill important gaps in existing knowledge that cannot be adequately addressed using current fluorescent labeling methods. The successful completion of this research will provide a set of GCE tools that will advance a broad range of applications in the field of in-cell labeling and will yield the first results of STAT3 function in the absence of an N- or C-terminal protein tag. This project is funded by the Molecular Biophysics Cluster in the Division of Molecular and Cellular Biosciences.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.