Secondary structures formed by expanded CUG RNA are involved in the pathobiology of myotonic dystrophy type 1. Understanding the molecular basis of toxic RNA structures can provide insights into the mechanism of disease pathogenesis and accelerate the drug discovery process. Here, we report the crystal structure of CUG repeat RNA containing three U–U mismatches between C–G and G–C base pairs. The CUG RNA crystallizes as an A-form duplex, with the first and third U–U mismatches adopting a water-mediated asymmetric mirror isoform geometry. We found for the first time that a symmetric, water-bridged U-H₂O-U mismatch is well tolerated within the CUG RNA duplex, which was previously suspected but not observed. The new water-bridged U–U mismatch resulted in high base-pair opening and single-sided cross-strand stacking interactions, which in turn dominate the CUG RNA structure. Furthermore, we performed molecular dynamics simulations that complemented the structural findings and proposed that the first and third U–U mismatches are interchangeable conformations, while the central water-bridged U–U mismatch represents an intermediate state that modulates the RNA duplex conformation. Collectively, the new structural features provided in this work are important for understanding the recognition of U–U mismatches in CUG repeats by external ligands such as proteins or small molecules.

由扩增的 CUG RNA 形成的二级结构参与 1 型强直性肌营养不良的病理生物学。了解有毒 RNA 结构的分子基础可以深入了解疾病发病机制并加速药物发现过程。在这里，我们报道了 C-G 和 G-C 碱基对之间含有三个 U-U 错配的 CUG 重复 RNA 的晶体结构。CUG RNA 结晶为 A 型双链体，第一个和第三个 U-U 错配采用水介导的不对称镜像异构体几何形状。我们首次发现，对称的水桥 UH ₂ O-U 错配在 CUG RNA 双链体中具有良好的耐受性，这一点之前曾被怀疑，但并未观察到。新的水桥U-U错配导致高碱基对开放和单侧交叉链堆积相互作用，这反过来又主导了CUG RNA结构。此外，我们进行了分子动力学模拟，补充了结构发现，并提出第一个和第三个U-U错配是可互换的构象，而中央水桥U-U错配代表了调节RNA双链体构象的中间状态。总的来说，这项工作提供的新结构特征对于理解蛋白质或小分子等外部配体对 CUG 重复中 U-U 错配的识别非常重要。