Specific labeling of proteins within living cells with single residue precision through genetically encoded click chemistry

无催化剂化学的发展，即一种无毒的化学反应，甚至在活的有机体中也能容易地、特异性地进行，已经彻底改变了化学生物学。通过将烷基非天然氨基酸（UAAs）编码到细胞的遗传库中，我们利用这种化学方法在细菌和哺乳动物细胞中标记具有单残基特异性的蛋白质。在过去的几年中，我们将该方法扩展到更多的点击型反应，即Diels-Alder型反应，改进了一些技术缺陷，并将新型UAAs应用于完整哺乳动物细胞表面的时间依赖性生长因子受体标记。在本提案的扩展中，我们旨在通过合成新的UAAs以及与细胞区室和蛋白质表位非特异性相互作用倾向降低的荧光标记试剂来进一步改进细胞蛋白质标记技术。我们将继续改进将uaa引入蛋白质所需的tRNA合成酶，我们将提供表达tRNA和tRNA合成酶的稳定细胞系。这些综合改进将有助于为包括超分辨率显微镜在内的广泛应用提供特定位点点击介导的荧光标记。

The development of catalyst-free click chemistry, i.e. a chemical reaction that is non-toxic and proceeds readily and specifically even within living organisms, has revolutionized chemical biology. By encoding strained alkyne unnatural amino acids (UAAs) into the genetic repertoire of the cell, we exploited this chemistry to label proteins with single residue specificity in bacteria and mammalian cells. In the past years we extended the method to more click type reactions, namely of the Diels-Alder type, improved some of the technical shortcomings, and applied the novel UAAs to time-dependent growth factor receptor labeling on the surface of intact mammalian cells. In this proposal extension, we aim to further improve the cellular protein labeling technique by synthesizing new UAAs as well as fluorescent labeling reagents with a reduced tendency to interact with cell compartments and protein epitopes unspecifically. We will continue to improve the tRNA synthetases necessary for introducing UAAs to proteins and we will provide stable cell lines expressing tRNA and tRNA synthetases. The combined improvements will help to provide site-specific click-mediated fluorescent labeling to a broad range of applications, including superresolution microscopy.