Karanja Caroline W, Yeboah Kofi S, Ong Wilson W S, Sintim Herman O
Department of Chemistry 560 Oval Drive West Lafayette Indiana 47907-2084 USA.
Institute for Drug Discovery, Purdue University 720 Clinic Drive West Lafayette IN 47907 USA
RSC Chem Biol. 2020 Dec 17;2(1):206-214. doi: 10.1039/d0cb00187b. eCollection 2021 Feb 1.
Cyclic dinucleoties, such as cGAMP, c-di-GMP and c-di-AMP, are fascinating second messengers with diverse roles in both prokaryotes and eukaryotes. Consequently there is a need for simple and inexpensive methods for profiling these compounds in biological media, monitoring their synthesis or degradation by enzymes and for identifying inhibitors of proteins that metabolize or bind to these dinucleotides. Since 2011, when we reported the first simple method to detect c-di-GMP (S. Nakayama, I. Kelsey, J. Wang, K. Roelofs, B. Stefane, Y. Luo, V. T. Lee and H. O. Sintim, , 2011, , 4856) or in 2014 when we revealed another surprisingly simple assay to detect c-di-AMP (J. Zhou, D. A. Sayre, Y. Zheng, H. Szmacinski and H. O. Sintim, , 2014, , 2412), there have been efforts to develop assays to detect cyclic dinucleotides by others. However a unified and simple assay, which can be used for all cyclic dinucleotides is lacking. Here, we investigate STING binding by various fluorescein-labeled c-di-GMP, c-di-AMP and cGAMP, using fluorescent polarization (FP). Fluorescein-labeled c-di-GMP (F-c-di-GMP) was found to be the best binder of STING. This probe could be displaced by unlabeled cGAMP, c-di-AMP or c-di-GMP and hence it is a universal probe, which can be used to monitor all three dinucleotides. HPLC analysis was used to validate the new F-c-di-GMP-based FP assay.
环二核苷酸，如cGAMP、c-di-GMP和c-di-AMP，是一类迷人的第二信使，在原核生物和真核生物中都发挥着多种作用。因此，需要简单且廉价的方法来分析生物介质中的这些化合物，监测它们被酶合成或降解的过程，以及鉴定代谢或结合这些二核苷酸的蛋白质的抑制剂。自2011年我们报道了第一种检测c-di-GMP的简单方法（S. Nakayama、I. Kelsey、J. Wang、K. Roelofs、B. Stefane、Y. Luo、V. T. Lee和H. O. Sintim，《美国化学会志》，2011年，第133卷，第4856页），或者在2014年我们揭示了另一种惊人简单的检测c-di-AMP的方法（J. Zhou、D. A. Sayre、Y. Zheng、H. Szmacinski和H. O. Sintim，《美国化学会志》，2014年，第136卷，第2412页）以来，其他人一直在努力开发检测环二核苷酸的分析方法。然而，目前缺乏一种统一且简单的、可用于所有环二核苷酸的分析方法。在这里，我们使用荧光偏振（FP）技术研究了各种荧光素标记的c-di-GMP、c-di-AMP和cGAMP与干扰素基因刺激蛋白（STING）的结合情况。发现荧光素标记的c-di-GMP（F-c-di-GMP）是STING的最佳结合剂。该探针可以被未标记的cGAMP、c-di-AMP或c-di-GMP取代，因此它是一种通用探针，可用于监测所有这三种二核苷酸。使用高效液相色谱（HPLC）分析来验证基于新的F-c-di-GMP的FP分析方法。
