Abstract
A comprehensive understanding of RNA biology requires methods to visualize transcripts and their interactions within physiological environments. However, few technologies can report on multiple RNAs continuously and in living systems. We are addressing this void with bioluminescent probes. Bioluminescence does not require excitation light and can enable sensitive, noninvasive readouts in a variety of settings. Here, we report a panel of structured RNA tags and luciferase fragments (RNA lanterns) for multiplexed RNA detection. The lanterns emit few photons on their own but assemble and emit light when brought into proximity by the RNA tags. Three orthogonal tag-lantern combinations were designed based on known RNA-binding proteins that recognize distinct targets. The probes were optimized in vitro for selective transcript detection. We further applied the tags and lanterns in cultured cells, achieving multitarget imaging with good subcellular resolution. Collectively, the probes expand the toolkit for RNA imaging and will facilitate efforts to trace RNA targets in living systems.