Molecular lanterns for multiplexed imaging of RNA dynamics - Project Summary RNAs play pivotal roles in diverse cellular processes ranging from memory formation to immune activity. Key questions remain, though, regarding the location, degradation, and dynamic interactions of multiple transcripts. This is due, in part, to a lack of robust methods to track RNAs in real time and in physiological settings. Conventional approaches rely on RNA tags coupled with fluorescent probes. Many of these tags are too large to use in conjunction with small RNAs. Additionally, the readouts require excitation light, which can damage biological samples upon repeated exposure. External light can also induce autofluorescence, precluding sensitive detection of low abundant targets. Capturing a broader set of RNAs—and the complete picture of their biological roles—requires new technologies. Our long-term goal is to develop enabling platforms for visualizing RNAs and their dynamics. The objective of this proposal is to build one foundational technology featuring ultra-small RNA tags that can assemble split luciferases. Signal production will only be observed when target transcripts (bearing the small tags) are present. Guided by strong preliminary data, our work involves (1) generating orthogonal RNA tags and complementary split luciferase binders, such that multiple transcripts can be visualized at once; (2) developing a platform for continuous, multiplexed imaging. This aim will make use of bioluminescent phasor, a method for rapid assignment of overlapping and often complex luciferase emission spectra. The experiments will establish optimized parameters for deploying combinations of the RNA tools in biologically relevant systems, and for continuously tracking their abundance and location. A third aim involves benchmarking the imaging probes and methodology against challenging targets, including endogenous and low-abundant RNAs, and collections of differentially expressed transcripts. Collectively, our work removes technical barriers to imaging RNAs and their dynamic functions, marking a significant advance over existing state-of-the-art approaches.
