In Situ Genetically Cascaded Amplification for Imaging RNA Subcellular Locations

In situ amplification methods, such as hybridization chain reaction, are valuable tools for mapping the spatial distribution and subcellular location of target analytes. However, the live-cell applications of these methods are still limited due to challenges in the probe delivery, degradation, and cytotoxicity. Herein, we report a novel genetically encoded in situ amplification method to noninvasively image the subcellular location of RNA targets in living cells. In our system, a fluorogenic RNA reporter, Broccoli, was split into two nonfluorescent fragments and conjugated to the end of two RNA hairpin strands. The binding of one target RNA can then trigger a cascaded hybridization between these hairpin pairs and thus activate multiple Broccoli fluorescence signals. We have shown that such an in situ amplified strategy can be used for the sensitive detection and location imaging of various RNA targets in living bacterial and mammalian cells. This new design principle provides an effective and versatile platform for tracking various intracellular analytes.