Ideal eukaryotic tetrazine ligations for imaging protein dynamics in live cells

在活细胞中修饰和可视化生物分子而不损害其功能的能力对于理解生命系统至关重要。这项研究的目标是开发一个完整的生物正交标记系统，可以编码到蛋白质的任何位置，并在生命系统内标记蛋白质。 该项目将开发非常快速和高度特异性的蛋白质标记方法，使活真核细胞中的免清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室课程中，并将为代表性不足的科学家提供奖学金，以参加年度非天然蛋白质设施GCE研讨会。非天然蛋白质设施使用NSF资金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，以协助将蛋白质工程方法整合到他们的实验室中。这些技术对经济发展也有很大的潜力，公司正在与俄勒冈州州立大学合作，许可该项目中开发的技术。在活细胞中修饰和可视化生物分子而不损害其功能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）工具在活的真核细胞中进行免清洗蛋白质标记和单分子成像是可能的。 该提案的第一个目标集中于通过将含四嗪的非规范氨基酸（Tet-ncAAs）遗传编码到蛋白质中来将定量蛋白质标记率提高超过1000倍。第二个目标是通过优化标签生物利用度和特异性来开发与含四嗪蛋白反应的标签，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记，当它们与DNA结合时“打开”，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，解决转录因子STAT 3（信号转导和转录激活因子3）的定位，动力学和DNA结合能力的三个关键问题。 除了开发一般的荧光标记外，这项研究还将通过工程蛋白标记开放标记方式，这些标记在结合DNA时是荧光的，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，这些空白无法使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，将推进细胞内标记领域的广泛应用，并将在没有N-或C-末端蛋白质标签的情况下产生STAT 3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学小组资助。该奖项反映了NSF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.