Genetic Encoding of Tetrazine Amino Acids to Develop Ideal Bioorthogonal Ligations

生物过程通过大分子之间极其复杂的相互作用发挥作用，这些相互作用发生在巨大的时间尺度和分子特异性范围内。生物分子的可控修饰使科学家能够探索生命系统，设计新的生物功能并产生新的生物材料。目前，生物和非生物系统中修饰（标记）蛋白质的反应的速度和选择性限制了研究蛋白质动力学、加工和相互作用以及设计新蛋白质功能的能力。开发一种理想的标记蛋白质的反应，这种反应在生物条件下是有选择性的，并且足够快，可以与自然生物过程竞争，将克服目前的许多限制。氨基酸是蛋白质的组成部分，在蛋白质合成过程中，它们以精确定义的顺序插入。因此，基因编码修饰的氨基酸，包含所需的标记反应成分，并结合到蛋白质中，允许蛋白质的位点特异性修饰，并使许多新的研究途径和潜在的生物技术应用成为可能。在这个项目中，将携带四氮基团的修饰氨基酸（满足理想标记反应的要求）纳入蛋白质中。这种四氮蛋白在生物和非生物系统中的反应速度和特异性允许与各种针对不同选择功能的标记物偶联。这个扩展遗传密码的平台将被纳入本科课程，作为化学生物学教学的一种方法，并说明工程对生物聚合物的化学修饰对于理解和操纵它们的功能是多么的可能和关键。每年将举办一次研讨会，供高中科学教师和研究人员（本科生、研究生和研究生）学习遗传密码扩展方法。理想的生物正交连接将被开发，关键的功能基团将通过遗传密码扩展特异性地结合到蛋白质中。含四氮胺的氨基酸将被设计和合成为与带标签的张力反式环烯官能团高度反应，但与其他细胞成分选择性不反应。四氮氨基酸也将遗传地纳入真核生物和原核生物并表征。在亚化学计量浓度的标记下，将测量标记和二聚化蛋白质在体内的四氮蛋白稳定性和反应速率。该反应具有改变蛋白质研究和蛋白质工程的潜力，因为它将在生物体内以秒到分钟的生物时间尺度和四氮蛋白和标记的生物浓度进行定量。为了证明这种生物正交标记反应在体内的效用，双组分信号系统的关键功能将在精确控制下打开和关闭，以了解其调节转录的机制。该项目由生物科学理事会分子和细胞生物科学部的分子生物物理集群和数学与物理科学理事会化学部的生命过程化学项目共同资助。

Biological processes function through extremely complex interactions of macromolecules that take place over an enormous range of timescales and molecular specificity. Controlled modification of biomolecules enables scientists to explore living systems, engineer new biological functions and generate new biomaterials. Currently the speed and selectivity of the reactions that modify (label) proteins in living and non-living systems limit the ability to study protein dynamics, processing and interactions as well as to engineer novel protein function. Development of an ideal reaction for labeling proteins that is selective in biological conditions and is fast enough to compete with natural biological processes will overcome many of the current limitations. Amino acids are building blocks of proteins and they are inserted in a precisely defined sequence during protein synthesis. Thus, genetically encoding a modified amino acid that contains the needed labeling reaction components and is incorporated into a protein allows for site-specific modification of proteins and enables many new research avenues and potential biotechnological applications. In this project, a modified amino acid carrying a tetrazine group (which meets the demands of an ideal labeling reaction) will be incorporated into proteins. The speed and specificity of reaction of this tetrazine-protein in both living and non-living systems permits coupling with a variety of markers targeted for different selected functions. This platform for expansion of the genetic code will be incorporated into undergraduate courses as an approach to teaching chemical biology and to illustrate how engineering the chemical modification of biopolymers is both possible and critical for understanding and manipulating their function. An annual workshop will be held for high school science teachers and researchers (undergraduate, graduate and post-graduate) to learn genetic code expansion methodology. The ideal bioorthogonal ligation will be developed and key functional groups will be site-specifically incorporated into proteins via genetic code expansion. Tetrazine containing amino acids will be designed and synthesized to be highly reactive with strained trans-cyclooctene functionalities bearing labels, but selective unreactive with other cellular components. Tetrazine-amino acids will also be genetically incorporated into eukaryotes and prokaryotes and characterized. Tetrazine-protein stability and reaction rates will be measured for labeling and dimerizing proteins in vivo at sub-stoichiometric concentrations of label. This reaction has the potential to transform protein studies and protein engineering because it will be quantitative in vivo on biological time scales of seconds to minutes and at biological concentrations of tetrazine-protein and label. To demonstrate the utility of this bioorthogonal labeling reaction in vivo, key functions integral to two-component signaling systems will be switched on and off with precise control to understand their mechanism of regulating transcription.This project is jointly funded by the Molecular Biophysics Cluster in the Division of Molecular and Cellular Biosciences in the Directorate for Biological Sciences and the Chemistry of Life Processes Program in the Division of Chemistry in the Directorate of Mathematical and Physical Sciences.