Imaging of endogenous RNA in live cells using sequence-activated fluorescent RNA probes

Abstract

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.