Multiplexed Sequential Imaging in Living Cells with Orthogonal Fluorogenic RNA Aptamer/Dye Pairs

Abstract Single-cell detection of multiple target analytes is an important goal in cell biology. However, due to the spectral overlap of common fluorophores, multiplexed fluorescence imaging beyond two-to-three targets inside living cells remains a technical challenge. Herein, we introduce a multiplexed imaging strategy that enables live-cell target detection via sequential rounds of imaging-and-stripping process, which is named as “sequential Fluorogenic RNA Imaging-Enabled Sensor” (seqFRIES). In seqFRIES, multiple orthogonal fluorogenic RNA aptamers are genetically encoded inside cells, and then the corresponding cell membrane permeable dye molecules are added, imaged, and rapidly removed in consecutive detection cycles. As a proof-of-concept, we have identified in this study five in vitro orthogonal fluorogenic RNA aptamer/dye pairs (>10-fold higher fluorescence signals), four of which can be used for highly orthogonal and multiplexed imaging in living bacterial and mammalian cells. After further optimizing the cellular fluorescence activation and deactivation kinetics of these RNA/dye pairs, the whole four-color semi-quantitative seqFRIES process can now be completed in ∼20 min. Meanwhile, seqFRIES-mediated simultaneous detection of two critical signaling molecules, guanosine tetraphosphate and cyclic diguanylate, was also achieved within individual living cells. We expect our validation of this new seqFRIES concept here will facilitate the further development and potential broad usage of these orthogonal fluorogenic RNA/dye pairs for highly multiplexed and dynamic cellular imaging and cell biology studies.