美国斯坦福大学Rhiju Das、Wah Chiu等研究人员合作利用冷冻电镜实现了对RNA三维结构的快速测定。相关论文于2020年7月2日发表在《自然—方法学》上。

研究人员表明，冷冻电镜能够常规地解析单独RNA系统的图谱，并且这些图谱与多维化学图谱和Rosetta DRRAFTER计算模型互补时可以实现亚纳米级分辨率的坐标估计。

该杂交“ Ribosolve”流程可在11个以往未知的119至338个核苷酸无蛋白质RNA结构中检测并修改同源性和构象重排：全长四膜虫核酶、带有和不带有底物的hc16连接酶、全长霍乱弧菌和核梭状芽胞杆菌带或不带甘氨酸的甘氨酸核糖开关适配体、带和不带S-腺苷甲硫氨酸的分枝杆菌SAM-IV核糖开关，以及带或不带AMP的计算机设计ATP-TTR-3适配体。

仿真基准、盲测、补偿性诱变、跨RNA同源性以及内部对照表明Ribosolve可以准确解析RNA分子的整体结构，但不能解析原子细节。这些测试为今后的RNA结构研究提供了指导原则。

据介绍，生物学上重要的RNA分子的发现和设计超越了三维结构表征。

附：英文原文

Title: Accelerated cryo-EM-guided determination of three-dimensional RNA-only structures

Author: Kalli Kappel, Kaiming Zhang, Zhaoming Su, Andrew M. Watkins, Wipapat Kladwang, Shanshan Li, Grigore Pintilie, Ved V. Topkar, Ramya Rangan, Ivan N. Zheludev, Joseph D. Yesselman, Wah Chiu, Rhiju Das

Issue&Volume: 2020-07-02

Abstract: The discovery and design of biologically important RNA molecules is outpacing three-dimensional structural characterization. Here, we demonstrate that cryo-electron microscopy can routinely resolve maps of RNA-only systems and that these maps enable subnanometer-resolution coordinate estimation when complemented with multidimensional chemical mapping and Rosetta DRRAFTER computational modeling. This hybrid ‘Ribosolve’ pipeline detects and falsifies homologies and conformational rearrangements in 11 previously unknown 119- to 338-nucleotide protein-free RNA structures: full-length Tetrahymena ribozyme, hc16 ligase with and without substrate, full-length Vibrio cholerae and Fusobacterium nucleatum glycine riboswitch aptamers with and without glycine, Mycobacterium SAM-IV riboswitch with and without S-adenosylmethionine, and the computer-designed ATP-TTR-3 aptamer with and without AMP. Simulation benchmarks, blind challenges, compensatory mutagenesis, cross-RNA homologies and internal controls demonstrate that Ribosolve can accurately resolve the global architectures of RNA molecules but does not resolve atomic details. These tests offer guidelines for making inferences in future RNA structural studies with similarly accelerated throughput.

DOI: 10.1038/s41592-020-0878-9

Source: https://www.nature.com/articles/s41592-020-0878-9