Ren Yangyi, Lin Xiaowei, Liao Wenjian, Peng Xuemei, Deng Jie, Zhang Zhe, Zhan Jian, Zhou Yaoqi, Westhof Eric, Lilley David M J, Wang Jia, Huang Lin
Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Guangdong-Hong Kong Joint Laboratory for RNA Medicine, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou 510120, China.
Department of Urology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou 510120, China.
Nucleic Acids Res. 2025 Jan 24;53(3). doi: 10.1093/nar/gkae1218.
X-ray crystallography is a fundamental technique that provides atomic-level insights into RNA structures. However, obtaining crystals of RNA structures diffracting to high resolution is challenging. We introduce a simple strategy to enhance the resolution limit of RNA crystals by the selective substitution of Watson-Crick pairs by GU pairs within RNA sequences. Our approach has successfully yielded high-resolution structures for eight unique RNA crystals. Notably, six instances showed marked resolution enhancement upon GC/AU to GU base pair substitution, with two cases achieving high-resolution structures from initially poor data. In one case, reverting GU to GC base pairs also improved resolution. Our method facilitated the first structural determinations of the Long Interspersed Nuclear Element-1 and Olfactory Receptor family 4 subfamily K member 15 ribozymes, the 2'-deoxyguanosine-III riboswitch and the Broccoli RNA aptamer. The placement of GU base pairs within the first 5' helical stem of any given RNA species, or in one peripheral stem, is shown to be sufficient. These results offer a simple and effective approach for designing sequences or selecting sequences from homologous sequences, for high-resolution RNA structure determination.
X射线晶体学是一种能提供RNA结构原子水平见解的基础技术。然而，获得能衍射到高分辨率的RNA结构晶体具有挑战性。我们引入了一种简单策略，通过在RNA序列中用GU碱基对选择性取代沃森-克里克碱基对来提高RNA晶体的分辨率极限。我们的方法已成功为八个独特的RNA晶体生成了高分辨率结构。值得注意的是，六个实例在GC/AU碱基对替换为GU碱基对后显示出显著的分辨率提高，其中两个案例从最初较差的数据中获得了高分辨率结构。在一个案例中，将GU碱基对还原为GC碱基对也提高了分辨率。我们的方法促进了长散在核元件1和嗅觉受体家族4亚家族K成员15核酶、2'-脱氧鸟苷-III核糖开关和西兰花RNA适体的首次结构测定。结果表明，在任何给定RNA物种的第一个5'螺旋茎内或一个外围茎中放置GU碱基对就足够了。这些结果为设计序列或从同源序列中选择序列以进行高分辨率RNA结构测定提供了一种简单有效的方法。
