Multiplex imaging in living cells with fluorogenic RNAs

Cellular processes involve the participation of multiple biological molecules. Multiplex detection methods are thus important for studying the relationships between different cellular targets in various biological processes and disease-related phenomena. However, multiplex fluorescence imaging in living cells is still challenging due to the spectral overlap of fluorophores. In this project, we will introduce a fluorogenic RNA-based strategy to overcome this challenge and allow live-cell imaging of multiple metabolite and RNA targets. Fluorogenic RNAs are aptamers that can selectively bind and activate the fluorescence signal of the corresponding dye molecules. A series of fluorogenic RNA/dye pairs have been developed. In this work, we have studied and identified several fluorogenic RNA/dye pairs that can function orthogonally. After genetically encoding these fluorogenic RNAs into cells, the dye molecules are sequentially added in several rounds of “image-wash” cycles. As a result, the multiplex imaging is no longer limited by the excitation or emission spectral overlap. We named this method as RNA Exchange Probes for Enhanced Analysis of Targets (REPEAT). In addition, by fusing different target-binding aptamers with each fluorogenic RNA, versatile sensors have been developed to allow us to image various metabolites, signaling molecules, antibiotics, and RNAs at the single-cell level. This modular REPEAT system can be readily adapted to image many other interesting cellular targets.