A STING-based fluorescent polarization assay for monitoring activities of cyclic dinucleotide metabolizing enzymes,RSC Chemical Biology

当前位置： X-MOL 学术 › RSC Chem. Biol. › 论文详情

Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)

A STING-based fluorescent polarization assay for monitoring activities of cyclic dinucleotide metabolizing enzymes
RSC Chemical Biology ( IF 4.2 ) Pub Date : 2020-12-17 , DOI: 10.1039/d0cb00187b
Caroline W Karanja ₁ , Kofi S Yeboah ₁ , Wilson W S Ong ₁ , Herman O Sintim _{1,

2,

3}

Affiliation

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, J. Am. Chem. Soc., 2011, 133, 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, Anal. Chem., 2014, 86, 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.

中文翻译：

基于 STING 的荧光偏振测定，用于监测环状二核苷酸代谢酶的活性

环状二核，例如 cGAMP、c-di-GMP 和 c-di-AMP，是令人着迷的第二信使，在原核生物和真核生物中发挥着不同的作用。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、监测它们的合成或酶的降解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 年起，我们报道了第一个检测 c-di-GMP 的简单方法（S. Nakayama、I. Kelsey、J. Wang、K. Roelofs、B. Stefane、Y. Luo、VT Lee 和 HO Sintim, J. Am. Chem. Soc. , 2011, 133 , 4856）或在 2014 年，我们发现了另一种检测 c-di-AMP 的方法（J. Zhou、DA Sayre、Y. Cheng、H. Szmacinski 和 HO Sintim， Anal）。 . Chem. , 2014, 86 , 2412），其他人一直在努力开发检测环状二核苷酸的方法。然而，缺乏可用于所有环状二核苷酸的统一且简单的测定法。在这里，我们使用荧光偏振 (FP) 研究了各种荧光素标记的 c-di-GMP、c-di-AMP 和 cGAMP 与 STING 的结合。发现荧光素标记的 c-di-GMP (Fc-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，可用于监测所有三种二核苷酸。 HPLC 分析用于验证新的基于 Fc-di-GMP 的 FP 测定。

更新日期：2020-12-17

点击分享查看原文

点击收藏

公开下载

阅读更多本刊最新论文