RNA相分离相关的动态结构研究

我们报道了在Mg2+的存在下，含有20CAG重复序列的RNA分子凝聚成三种不同形态的液滴。利用高光谱受激拉曼成像技术，我们发现RNA在凝聚过程中，碱基进行堆积，从而丧失了标准碱基对结构。随着RNA/Mg2+的增加，RNA液滴先变大后收缩，最终，呈中空囊泡状结构。值得注意的是，无论是大液滴还是囊泡状液滴，RNA液滴边缘的碱基堆积结构非常明显，这是RNA液滴硬化的原因。这一机制可能与含有RNA无膜细胞器的衰老过程有关。为了研究CAG重复序列RNA的相分离，利用中科院精密测量科学与技术创新研究院的激光共聚焦显微镜对相分离液滴进行成像，采集了不同条件下CAG重复序列的相分离图像，并利用液滴融合和该仪器自带的插件--荧光猝灭恢复实验研究液滴的流动性。为了研究相分离过程中CAG重复序列RNA的结构变化，我们对RNA稀溶液和相分离液滴分别进行结构解析。利用的是华中科技大学分析测试中心的激光共聚焦自发拉曼光谱进行结构信息的采集。为了研究相分离液滴中RNA的结构分布，我们利用受激拉曼光谱成像技术对相分离液滴进行结构成像。通过成像图像表明，RNA在液滴中的结构分布具有非均一性。本部分的数据在华中科技大学国家光电实验室王平教授课题组自行搭建的高光谱受激拉曼光谱成像设备上完成。

We report that RNA molecules containing 20 CAG repeats condense into droplets with three distinct morphologies in the presence of Mg²⁺. Using hyperspectral stimulated Raman imaging, we found that during condensation, RNA bases undergo stacking, resulting in the loss of standard base-paired structures. As the RNA/Mg²⁺ ratio increases, RNA droplets first grow larger and then shrink, eventually forming hollow vesicle-like structures. Notably, the base stacking structures at the edges of RNA droplets—both large droplets and vesicle-like ones—are clearly observable, which accounts for the stiffening of RNA droplets. This mechanism may be related to the aging process of RNA-containing membrane-less organelles. To study the phase separation of CAG repeat-containing RNA, we imaged phase-separated droplets using a laser confocal microscope from the Innovation Academy for Precision Measurement Science and Technology, Chinese Academy of Sciences, collected phase separation images of CAG repeats under different conditions, and investigated the fluidity of the droplets via droplet fusion assays and fluorescence recovery after photobleaching (FRAP) experiments using the instrument's built-in plugin. To study the structural changes of CAG repeat-containing RNA during phase separation, we performed structural characterization of dilute RNA solutions and phase-separated droplets separately. Structural information was collected using laser confocal spontaneous Raman spectroscopy at the Analytical and Testing Center of Huazhong University of Science and Technology. To study the structural distribution of RNA in phase-separated droplets, we performed structural imaging of phase-separated droplets using stimulated Raman spectroscopic imaging. The imaging results demonstrate that the structural distribution of RNA within droplets is heterogeneous. The data in this section were collected using a home-built hyperspectral stimulated Raman spectroscopic imaging system in the research group of Professor Wang Ping at the National Laboratory of Optoelectronics, Huazhong University of Science and Technology.

搜集汇总

背景与挑战

背景概述

该数据集聚焦于RNA相分离的动态结构研究，通过高光谱受激拉曼成像等技术，揭示了含有20CAG重复序列的RNA在Mg2+作用下形成液滴过程中的结构变化，包括碱基堆积导致标准结构丧失、液滴形态演变及硬化机制，可能与无膜细胞器衰老相关。数据集包含实验图像和结构数据，适用于分析化学领域的研究。