Fluorescent modification of proteins of interest (POI) in living cells is desired to study their behaviour and functions in their natural environment. In a perfect setting it should be easy to perform, inexpensive, efficient and site-selective. Although multiple chemical and biological methods have been developed, only a few of them are applicable for cellular studies thanks to their appropriate physical, chemical and biological characteristics. One such successful system is a tetracysteine tag/motif and its selective biarsenical binders (e.g. FlAsH and ReAsH). Since its discovery in 1998 by Tsien and co-workers, this method has been enhanced and revolutionized in terms of its efficiency, formed complex stability and breadth of application. Here, we overview the whole field of knowledge, while placing most emphasis on recent reports. We showcase the improvements of classical biarsenical probes with various optical properties as well as multifunctional molecules that add new characteristics to proteins. We also present the evolution of affinity tags and motifs of biarsenical probes demonstrating much more possibilities in cellular applications. We summarize protocols and reported observations so both beginners and advanced users of biarsenical probes can troubleshoot their experiments. We address the concerns regarding the safety of biarsenical probe application. We showcase examples in virology, studies on receptors or amyloid aggregation, where application of biarsenical probes allowed observations that previously were not possible. We provide a summary of current applications ranging from bioanalytical sciences to allosteric control of selected proteins. Finally, we present an outlook to encourage more researchers to use these magnificent probes.

中文翻译：

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用于生命科学中蛋白质多功能位点特异性修饰的双砷荧光探针：概述和未来展望。

需要在活细胞中对感兴趣的蛋白质 (POI) 进行荧光修饰，以研究它们在自然环境中的行为和功能。在完美的环境中，它应该易于执行、价格低廉、高效且具有站点选择性。尽管已经开发了多种化学和生物学方法，但由于其适当的物理、化学和生物学特性，其中只有少数适​​用于细胞研究。一个这样成功的系统是四半胱氨酸标签/基序及其选择性双砷结合剂（例如Flash 和 ReAsH）。自 1998 年由 Tsien 及其同事发现以来，该方法在效率方面得到了增强和革命，形成了复杂的稳定性和应用范围。在这里，我们概述了整个知识领域，同时将重点放在最近的报告上。我们展示了具有各种光学特性的经典双砷探针的改进以及为蛋白质添加新特性的多功能分子。我们还展示了双砷探针的亲和标签和基序的演变，展示了在细胞应用中的更多可能性。我们总结了协议和报告的观察结果，因此双砷探针的初学者和高级用户都可以对他们的实验进行故障排除。我们解决了有关双砷探针应用安全性的问题。我们展示了病毒学、受体或淀粉样蛋白聚集研究中的例子，其中双砷探针的应用允许以前不可能的观察。我们总结了从生物分析科学到选定蛋白质的变构控制的当前应用。最后，我们提出了一个展望，以鼓励更多的研究人员使用这些宏伟的探测器。