Adding new covalent bonds to proteins in live cells

项目概要/摘要 蛋白质通过与不同家族的双分子相互作用来执行各种生物学功能。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 生物学的动力学，其可逆性和弱点也对研究和 工程化蛋白质-双分子相互作用。在这种应用中，通过设计， 并将生物活性非天然氨基酸（Uaas）基因整合到蛋白质中，这将使蛋白质 以共价方式与核糖核酸和碳水化合物结合。潜在的创新是开发 生物相容性化学，使得Uaa将选择性地与靶核糖核酸或碳水化合物反应 通过仅在结合时的邻近使能反应性，导致蛋白质之间的稳定和不可逆的连接， 和目标为了实现这些目标，适合于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。所需的化学官能度将 被合成到Uaa的侧链中，并且Uaa将被位点特异性地掺入蛋白质中， 通过基因密码扩展技术获得活细胞。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，识别糖基化底物糖蛋白 修饰酶，并拮抗细胞-细胞通讯。通过利用新的共价键 天然蛋白质无法获得，该项目将开创一个新的研究领域， 工程化蛋白质-RNA和蛋白质-碳水化合物相互作用。因为这种互动是必不可少的， 生物学功能及其畸变广泛涉及各种人类疾病，新的 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.

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.