Nucleotides modified at the terminal phosphate position have been proven to be interesting entities to study the activity of a variety of different protein classes. In this chapter, we present various types of modifications that were attached as reporter molecules to the phosphate chain of nucleotides and briefly describe the chemical reactions that are frequently used to synthesize them. Furthermore, we discuss a variety of applications of these molecules. Kinase activity, for instance, was studied by transfer of a phosphate modified with a reporter group to the target proteins. This allows not only studying the activity of kinases, but also identifying their target proteins. Moreover, kinases can also be directly labeled with a reporter at a conserved lysine using acyl-phosphate probes. Another important application for phosphate-modified nucleotides is the study of RNA and DNA polymerases. In this context, single-molecule sequencing is made possible using detection in zero-mode waveguides, nanopores or by a Förster resonance energy transfer (FRET)-based mechanism between the polymerase and a fluorophore-labeled nucleotide. Additionally, fluorogenic nucleotides that utilize an intramolecular interaction between a fluorophore and the nucleobase or an intramolecular FRET effect have been successfully developed to study a variety of different enzymes. Finally, also some novel techniques applying electron paramagnetic resonance (EPR)-based detection of nucleotide cleavage or the detection of the cleavage of fluorophosphates are discussed. Taken together, nucleotides modified at the terminal phosphate position have been applied to study the activity of a large diversity of proteins and are valuable tools to enhance the knowledge of biological systems.

中文翻译：

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磷酸修饰的核苷酸，用于监测酶的活性

已经证明在末端磷酸盐位置修饰的核苷酸是研究多种不同蛋白质类别活性的有趣实体。在本章中，我们介绍了各种类型的修饰，这些修饰作为报告分子连接到核苷酸的磷酸酯链上，并简要描述了经常用于合成它们的化学反应。此外，我们讨论了这些分子的各种应用。例如，通过将经报告基团修饰的磷酸酯转移至靶蛋白来研究激酶活性。这不仅允许研究激酶的活性，而且可以鉴定其靶蛋白。此外，还可以使用酰基磷酸探针在保守的赖氨酸上用报道分子直接标记激酶。磷酸修饰核苷酸的另一个重要应用是RNA和DNA聚合酶的研究。在这种情况下，通过在零模波导，纳米孔中进行检测或通过聚合酶和荧光团标记的核苷酸之间基于傅斯特共振能量转移（FRET）的机制进行检测，就可以进行单分子测序。另外，已经成功地开发了利用荧光团与核碱基之间的分子内相互作用或分子内FRET效应的荧光核苷酸，以研究多种不同的酶。最后，还讨论了一些应用基于电子顺磁共振（EPR）的核苷酸裂解检测或氟磷酸盐裂解检测的新颖技术。在一起