Click-encoded rolling FISH for visualizing single-cell RNA polyadenylation and structures
Click-encoded rolling FISH for visualizing single-cell RNA polyadenylation and structures
复制标题
用于可视化单细胞 RNA 多腺苷酸化和结构的点击编码滚动 FISH
DOI：
10.1093/nar/gkz852
复制
发表时间：
2019-12-16
影响因子：
14.9
通讯作者：
Zhao, Yongxi
中科院分区：
文献类型：
--
作者：
Chen, Feng;Bai, Min;Zhao, Yongxi
RNA加工和结构的空间分辨可视化对于更好地研究单细胞RNA的功能和景观具有重要意义。然而，目前可用的RNA成像方法仅限于序列分析，无法识别RNA加工事件和结构。在这里，我们开发了点击编码的滚鱼(ClickerFISH)，用于可视化单细胞中的RNA多腺苷基化和结构。在ClickerFISH中，RNA 3‘聚腺苷酸化尾部、单链和双链区域被不同的可点击DNA条形码化学标记。然后，这些条形码启动DNA滚动扩增，为鱼类生成重复模板，以成像其亚细胞分布。与单分子FISH相结合，该策略还可以获得感兴趣的RNA的定量信息。最后，我们发现RNA Poly(A)拖尾和高阶结构在空间上是以细胞类型特定的风格组织的，具有细胞到细胞的异质性。我们还探索了它们在细胞周期阶段的时空格局，并揭示了高度动态的组织结构，特别是在S期。这一方法将有助于阐明RNA多聚腺苷的时空结构和结构。
Spatially resolved visualization of RNA processing and structures is important for better studying single-cell RNA function and landscape. However, currently available RNA imaging methods are limited to sequence analysis, and not capable of identifying RNA processing events and structures. Here, we developed click-encoded rolling FISH (ClickerFISH) for visualizing RNA polyadenylation and structures in single cells. In ClickerFISH, RNA 3' polyadenylation tails, single-stranded and duplex regions are chemically labeled with different clickable DNA barcodes. These barcodes then initiate DNA rolling amplification, generating repetitive templates for FISH to image their subcellular distributions. Combined with single-molecule FISH, the proposed strategy can also obtain quantitative information of RNA of interest. Finally, we found that RNA poly(A) tailing and higher-order structures are spatially organized in a cell type-specific style with cell-to-cell heterogeneity. We also explored their spatiotemporal patterns during cell cycle stages, and revealed the highly dynamic organization especially in S phase. This method will help clarify the spatiotemporal architecture of RNA polyadenylation and structures.