Riboswitches are mRNA domains that make gene-regulatory decisions upon binding their cognate ligands. Bacterial riboswitches that specifically recognize 5-aminoimidazole-4-carboxamide riboside 5′-monophosphate (ZMP) and 5′-triphosphate (ZTP) regulate genes involved in folate and purine metabolism. Now, we have developed synthetic ligands targeting ZTP riboswitches by replacing the sugar-phosphate moiety of ZMP with various functional groups, including simple heterocycles. Despite losing hydrogen bonds from ZMP, these analogs bind ZTP riboswitches with similar affinities as the natural ligand, and activate transcription more strongly than ZMP in vitro. The most active ligand stimulates gene expression ∼3 times more than ZMP in a live Escherichia coli reporter. Co-crystal structures of the Fusobacterium ulcerans ZTP riboswitch bound to synthetic ligands suggest stacking of their pyridine moieties on a conserved RNA nucleobase primarily determines their higher activity. Altogether, these findings guide future design of improved riboswitch activators and yield insights into how RNA-targeted ligand discovery may proceed.

核糖开关是 mRNA 结构域，可在与其同源配体结合时做出基因调控决定。特异性识别 5-氨基咪唑-4-甲酰胺核苷 5'-单磷酸 (ZMP) 和 5'-三磷酸 (ZTP) 的细菌核糖开关调节参与叶酸和嘌呤代谢的基因。现在，我们通过用各种官能团（包括简单的杂环）替换 ZMP 的糖磷酸部分，开发了靶向 ZTP 核糖开关的合成配体。尽管从 ZMP 失去了氢键，但这些类似物以与天然配体相似的亲和力结合 ZTP 核糖开关，并在体外比 ZMP 更强烈地激活转录。最活跃的配体在活大肠杆菌中刺激基因表达的程度是 ZMP 的 3 倍记者。溃疡梭杆菌ZTP 核糖开关与合成配体结合的共晶结构表明，它们的吡啶部分堆积在保守的 RNA 核碱基上主要决定了它们的更高活性。总之，这些发现指导了改进的核糖开关激活剂的未来设计，并深入了解了 RNA 靶向配体的发现可能如何进行。