Structurally Ordered Cascade-Activatable Fluorescent RNA Probes for Multiplexed Imaging of Endogenous RNAs in Living Cells

Abstract Sequence-activated fluorescent RNA (FR) probes have emerged as valuable tools for visualizing endogenous RNAs in live cells without the need to genetically modify RNA transcripts. However, the lack of clear guidelines for assembling intramolecular dynamic structure ensembles and integrating multifunctional building blocks into interconnected architectures hinders the development of robust and scalable strategies for sensitive, multiplexed live-cell RNA imaging. Here, we report the development of cascade-activatable FR (CaFR) platform based on a novel, structurally ordered cascade activation strategy. Upon specific recognition of the target RNA, CaFR initiates cascading, domino-like conformational rearrangements that release and reconfigure the interlocked aptamers into their correctly folded, fluorogenic states. CaFR exhibits favourable properties including large dynamic range, high brightness, and excellent scalability, allowing for the robust imaging of endogenous RNAs in both live and fixed cells, even at the single-molecule level. Moreover, Pepper- and Clivia-based CaFR probes are bioorthogonal and enable the simultaneous visualization of two RNAs in live cells. Together, this study provides a generalized strategy to develop high-performance FR probes for the labeling and imaging of endogenous RNA in live cells, which will be useful for understanding the roles of RNA in complex biological processes.