Illuminating RNA through multiplexing in live mammalian cells.

Abstract

Subcellular localization of RNAs play a central role in biology. Intense efforts are underway to develop tools for RNA fluorescence detection, but a common limitation is reliance on fluorescence intensity. Here, we present Riboglow-FLIM to illuminate localizations of RNAs in live mammalian cells. Riboglow-FLIM builds upon the Riboglow platform, where a fluorescent probe binds a genetically encoded RNA tag, leading to increasing fluorescence intensity and lifetime of the probe upon RNA binding. As a foundation for a fluorescence lifetime sensor, we demonstrate that lifetime detection leads to robust cellular contrast. We find that cellular contrast for FLIM measurements are superior in direct comparison with fluorescence intensity. Importantly, the RNA tag in Riboglow-FLIM is derived from a phylogenetically diverse family of RNA sequences. We then explore if variations in the RNA tag sequence lead to distinguishable lifetime signals. We demonstrate that lifetime changes observed for RNA tag variants were robust, establishing the RNA tag element of the Riboglow platform as a variable to achieve sensor orthogonality. As a proof-of-concept, we use a model system to demonstrate orthogonality of two RNA tags to visualize two different RNAs with distinct subcellular localizations simultaneously (the long non-coding RNA NORAD and an mRNA). Importantly, by using lifetime as a readout, we are visualizing two RNAs simultaneously with a single channel of detection. Lifetime microscopy image analysis was robust by diverse image processing workflows, including phasor approach and multiexponential reconvolution fitting. We are expanding the available Riboglow-FLIM RNA tags by exploring phylogenetic diversity and structure/function relationships of the RNA sequence that binds the probe ligand. Current efforts are underway to add additional capabilities, including expanding the platform to visualize RNAs in complex model systems like tumor spheroids and developing a split-Riboglow sensor to visualize mRNA splicing.