Cyclic dinucleotides are second messenger molecules produced by both prokaryotes and eukaryotes in response to external stimuli. In bacteria, these molecules bind to RNA riboswitches and several protein receptors ultimately leading to phenotypic changes such as biofilm formation, ion transport and secretion of virulence factors. Some cyclic dinucleotide analogs bind differentially to biological receptors and can therefore be used to better understand cyclic dinucleotide mechanisms in vitro and in vivo. However, production of some of these analogs involves lengthy, multistep syntheses. Here, we describe a new, simple method for enzymatic synthesis of several 3′, 5′ linked cyclic dinucleotide analogs of c-di-GMP, c-di-AMP and c-AMP-GMP using the cyclic-AMP-GMP synthetase, DncV. The enzymatic reaction efficiently produced most cyclic dinucleotide analogs, such as 2′-amino sugar substitutions and phosphorothioate backbone modifications, for all three types of cyclic dinucleotides without the use of protecting groups or organic solvents. We used these novel analogs to explore differences in phosphate backbone and 2′-hydroxyl recognition between GEMM-I and GEMM-Ib riboswitches.

中文翻译：

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混杂的环状AMP-GMP合成酶酶促合成环状二核苷酸类似物并分析环状二核苷酸反应性核糖开关

环状二核苷酸是原核生物和真核生物响应外部刺激而产生的第二信使分子。在细菌中，这些分子与RNA核糖开关和几种蛋白质受体结合，最终导致表型改变，例如生物膜形成，离子转运和毒力因子的分泌。一些环状二核苷酸类似物与生物受体的结合不同，因此可用于更好地了解体外和体内环状二核苷酸的机制。但是，其中一些类似物的生产涉及冗长的多步合成。在这里，我们描述了一种新的简单方法，可使用环AMP-GMP合成酶酶促合成c-di-GMP，c-di-AMP和c-AMP-GMP的几个3'，5'连接的环状二核苷酸类似物， DncV。对于所有三种类型的环状二核苷酸，酶反应均有效地产生了大多数环状二核苷酸类似物，例如2'-氨基糖取代和硫代磷酸酯骨架修饰，而无需使用保护基或有机溶剂。我们使用这些新颖的类似物来探索GEMM-1和GEMM-1b核糖开关之间的磷酸盐主链和2'-羟基识别的差异。