Rapid live-cell sequential multiplexed imaging with fluorogenic RNAs.

Abstract

Multiplexed detection of different cellular targets is an important goal in cell biology. However, multiplexed imaging in living cells is still extremely challenging. In terms of fluorescence imaging, significant spectral overlaps limit the number of distinguishable fluorophores that can be detected simultaneously. To solve this problem, in this project, we introduce a sequential imaging strategy termed “sequential Fluorogenic RNA Imaging-Enabled Sensor” (seqFRIES), to separately detect these spectrally overlapped fluorophores over time. Fluorogenic RNA aptamers are RNA strands that recognize and activate the fluorescence of otherwise non-fluorescent dye molecules. Here, we explored a list of reported fluorogenic RNA/dye pairs and identified five orthogonal ones. After genetically encoding all these fluorogenic RNAs together in cells, the corresponding dye molecules are added, imaged, and removed in a sequential manner. A very fast fluorescence activation and deactivation (in several minutes) was observed with these RNA/dye pairs. Taking advantages of such “image-and-wash” cycles, the multiplexing capacity of this imaging system is no longer limited by the excitation/emission spectral overlap of the fluorescent molecules. As a proof of concept, we show that the seqFRIES can perform robustly in both bacterial and mammalian cells, enabling multiplexed imaging of different RNAs, metabolites, and signaling molecules. We envision that this versatile multiplexed reporter system can be potentially adapted for imaging the cellular colocalization and dynamics of various target analytes, again inside living cells.