Department of Chemistry, Georgetown University, Washington, DC, USA.
Department of Biochemistry and Molecular & Cellular Biology, Georgetown University, Washington, DC, USA.
Nat Commun. 2023 Feb 16;14(1):867. doi: 10.1038/s41467-023-36531-y.
Visualization of RNAs in live cells is critical to understand biology of RNA dynamics and function in the complex cellular environment. Detection of RNAs with a fluorescent marker frequently involves genetically fusing an RNA aptamer tag to the RNA of interest, which binds to small molecules that are added to live cells and have fluorescent properties. Engineering efforts aim to improve performance and add versatile features. Current efforts focus on adding multiplexing capabilities to tag and visualize multiple RNAs simultaneously in the same cell. Here, we present the fluorescence lifetime-based platform Riboglow-FLIM. Our system requires a smaller tag and has superior cell contrast when compared with intensity-based detection. Because our RNA tags are derived from a large bacterial riboswitch sequence family, the riboswitch variants add versatility for using multiple tags simultaneously. Indeed, we demonstrate visualization of two RNAs simultaneously with orthogonal lifetime-based tags.
在活细胞中可视化 RNA 对于理解 RNA 动态和在复杂细胞环境中的功能的生物学至关重要。使用荧光标记物检测 RNA 通常涉及将 RNA 适体标签基因融合到感兴趣的 RNA 上，该标签与添加到活细胞中的小分子结合，具有荧光特性。工程努力旨在提高性能并添加多功能特性。目前的努力集中在添加多路复用功能，以同时在同一细胞中标记和可视化多个 RNA。在这里，我们提出了基于荧光寿命的平台 Riboglow-FLIM。与基于强度的检测相比，我们的系统需要更小的标签并且具有更好的细胞对比度。因为我们的 RNA 标签源自一个大型细菌核糖开关序列家族，所以核糖开关变体为同时使用多个标签添加了多功能性。实际上，我们证明了使用正交寿命标签同时可视化两个 RNA。
