Adding new covalent bonds to proteins in live cells

项目概要/摘要 蛋白质通过与不同的双分子家族相互作用来发挥多种生物学功能。这些 相互作用本质上主要是非共价的。尽管这种非共价相互作用提供了多样性和 对于生物学动力学而言，其可逆性和弱点也对研究和应用造成了很大的限制。 工程蛋白质-双分子相互作用。在这个应用中，自然屏障将通过设计被打破 并将生物反应性非天然氨基酸（Uaas）基因整合到蛋白质中，这将使蛋白质 以共价模式与核糖核酸和碳水化合物结合。创新的根本在于开发 生物相容性化学，使 Uaa 选择性地与目标核糖核酸或碳水化合物发生反应 仅在结合时通过接近启用的反应性，导致蛋白质之间稳定且不可逆的连接 和目标。为了实现这些目标，适合共价靶向核糖核酸的新化学物质和 将开发细胞和生理条件下的碳水化合物。所需的化学功能将 被合成到Uaa的侧链中，并且Uaa将被定点地整合到蛋白质中 通过遗传密码扩展技术的活细胞。这种新的共价键合策略将应用于现场 细胞了解RNA-蛋白质识别特异性，识别糖基化的底物糖蛋白 修饰酶并拮抗细胞间通讯。通过利用新的共价键 由于天然蛋白质无法获得，该项目将为研究和合理利用开辟一个新的维度 工程蛋白质-RNA 和蛋白质-碳水化合物相互作用。因为这样的互动是不可或缺的 生物功能及其异常与各种人类疾病、新 该项目产生的技术将从根本上影响基础生物学研究和治疗 应用程序。

Project Summary/Abstract Proteins perform a variety of biological functions by interacting with different families of bimolecules. These interactions are largely noncovalent in nature. Although such noncovalent interactions provide diversity and dynamics for biology, their reversibility and weakness also impose strong limitations on researching and engineering protein-bimolecule interactions. In this application the natural barrier will be broken by designing and genetically incorporating bioreactive unnatural amino acids (Uaas) into proteins, which will enable proteins to bind with ribonucleic acids and carbohydrates in the covalent mode. The underlying innovation is to develop biocompatible chemistry, so that the Uaa will selectively react with the target ribonucleic acid or carbohydrate via proximity-enabled reactivity only upon binding, resulting in stable and irreversible linkage between protein and the target. To achieve these goals, new chemistries suitable for covalently targeting ribonucleic acids and carbohydrates in cellular and physiological conditions will be developed. The desired chemical functionality will be synthesized into the side chain of a Uaa, and the Uaa will be site-specifically incorporated into proteins in live cells via the genetic code expansion technology. This new covalent bonding strategy will be applied in live cells to understand RNA-protein recognition specificity, to identify substrate glycoproteins for glycosylation modifying enzymes, and to antagonize cell-cell communication. By harnessing the new covalent linkages inaccessible to natural proteins, this project will initiate a new dimension for researching and rationally engineering protein-RNA and protein-carbohydrate interactions. As such interactions are indispensable for biological functions and their aberrations are extensively implicated in various human diseases, new technologies resulting from this project will fundamentally impact both basic biological research and therapeutic applications.