Zheng Haifeng, Liu Xiyu, Liu Luhui, Hu Jiarui, Chen Xianjun
Optogenetics & Synthetic Biology Interdisciplinary Research Center, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Mei Long Road, Shanghai 200237, China.
Shanghai Frontiers Science Center of Optogenetic Techniques for Cell Metabolism, School of Pharmacy, East China University of Science and Technology, 130 Mei Long Road, Shanghai 200237, China.
Nucleic Acids Res. 2025 Jan 11;53(2). doi: 10.1093/nar/gkae1209.
RNA performs a remarkable range of functions, such as RNA processing, chromosome maintenance and dosage compensation. Technologies that robustly and specifically image RNA in its native state are highly desirable, as these technologies can help researchers clarify the localization and functionality of diverse RNAs. Here, we describe the development of a sequence-activated fluorescent RNA (SaFR) technique. In SaFR, in the absence of target RNA, the structure of fluorogenic RNA is disrupted by the invader sequence, and the ability to activate the Pepper's cognate fluorophores is lost as a result. In the presence of target RNA, SaFR undergoes conformational reorganization and transforms into the fluorogenic conformation of Pepper, enabling the activation of fluorophores to produce fluorescent signals. SaFR exhibits favourable properties, such as large dynamic ranges, high specificity and fast fluorescence generation. Further studies showed that exogenous or endogenous RNAs can be tracked in live and fixed cells through SaFR. We further demonstrated the usefulness of SaFR in monitoring the assembly and disassembly of stress granules in real-time. Overall, this study offers a robust and versatile tool for labelling and imaging endogenous RNA in cells, which will be useful for clarifying the functionality and molecular mechanism of RNA.
RNA具有一系列非凡的功能，如RNA加工、染色体维持和剂量补偿。能够在天然状态下对RNA进行稳健且特异性成像的技术非常受欢迎，因为这些技术可以帮助研究人员阐明各种RNA的定位和功能。在此，我们描述了一种序列激活荧光RNA（SaFR）技术的开发。在SaFR中，在没有靶RNA的情况下，荧光RNA的结构被入侵序列破坏，从而失去激活辣椒同源荧光团的能力。在靶RNA存在的情况下，SaFR经历构象重组并转变为辣椒的荧光构象，从而能够激活荧光团产生荧光信号。SaFR具有良好的特性，如大动态范围、高特异性和快速荧光产生。进一步的研究表明，通过SaFR可以在活细胞和固定细胞中追踪外源或内源RNA。我们进一步证明了SaFR在实时监测应激颗粒的组装和拆卸方面的实用性。总体而言，这项研究为细胞内源性RNA的标记和成像提供了一种强大且通用的工具，这将有助于阐明RNA的功能和分子机制。
