Genetic Encoding of Tetrazine Amino Acids to Develop Ideal Bioorthogonal Ligations

生物过程通过大分子的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，在生命和非生命系统中修饰（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。开发一种在生物条件下具有选择性并且足够快以与自然生物过程竞争的用于标记蛋白质的理想反应将克服当前的许多限制。氨基酸是蛋白质的组成部分，它们在蛋白质合成过程中以精确定义的序列插入。因此，遗传编码含有所需标记反应组分并掺入蛋白质中的修饰的氨基酸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，一种带有四嗪基团的修饰氨基酸（符合理想标记反应的要求）将被掺入蛋白质中。 这种四嗪蛋白在活体和非活体系统中的反应速度和特异性允许与针对不同选择功能的各种标记物偶联。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，并说明如何工程化生物聚合物的化学修饰对于理解和操纵它们的功能既可能又至关重要。 将为高中科学教师和研究人员（本科生、研究生和研究生）举办年度讲习班，学习遗传密码扩展方法。将开发理想的生物正交连接，并通过遗传密码扩展将关键官能团位点特异性地并入蛋白质中。含四嗪的氨基酸将被设计和合成为与带有标记的应变反式环辛烯官能团高度反应，但与其他细胞组分选择性不反应。四嗪-氨基酸也将被遗传地掺入真核生物和原核生物中并表征。将在亚化学计量浓度的标记物下测量四嗪-蛋白质稳定性和反应速率，用于体内标记和二聚化蛋白质。该反应具有改变蛋白质研究和蛋白质工程的潜力，因为它将在几秒到几分钟的生物时间尺度上以及在四嗪蛋白和标记的生物浓度下进行体内定量。为了证明这种生物正交标记反应在体内的效用，关键功能集成到两个-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学小组和生物科学部的生命过程化学计划共同资助。数学和物理科学理事会的化学。

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.