Development of Genetically Encoded Near-Infrared Fluorescent Light-up Aptamers for Live-Cell RNA Imaging

Visualizing RNA localization and dynamics in living cells provides new insights into RNA biology. Genetically encoded fluorescent light-up RNA aptamers (FLAPs) that can specifically bind and activate the fluorescence of cell-permeable fluorogenic ligands represent emerging and exciting possibilities for real-time live-cell RNA imaging. Although several FLAPs have been developed so far, there is still a strong demand for FLAPs that fluoresce in the near-infrared (NIR) region where cellular autofluorescence and photocytotoxicity are low. In the first part, we presented the first color-shifting aptamer-fluorophore system that allows simultaneous imaging of both the NIR-fluorescent aptamer-dye complex and the cyan-fluorescent unbound dye. The ratiometric images (NIR/cyan) obtained from this color-shifting module are particularly useful for correcting differences in the probe’s cellular uptake, heterogeneous probe distribution, probe instability, and cell morphology variations. This novel system exploited the environmentally sensitive benzopyrylium-coumarin (BC) hybrid fluorophores, which exist in an equilibrium between a spirocyclic cyan-fluorescent form and a zwitterionic NIR-fluorescent form. First, we evolved a 38-nucleotide (nt) RNA aptamer (BeCA) that selectively binds the BC zwitterion with nanomolar affinity. Further variations in the BC fluorophore’s chemical structure enabled the BC to shift to the cyan spirocyclic form in the unbound state and provided an emission ratio change (cyan/NIR) as high as 15-fold upon aptamer binding. Using the BeCA-BC system, we not only imaged BeCA-tagged mRNAs fluorescing in the NIR region but also demonstrated its utility in the ratiometric analysis of target RNAs expressed at different levels in single cells using confocal microscopy. In the second part, we established a fluorescence-activated cell sorting (FACS)-based aptamer selection platform for spirocyclization-based fluorogenic probes. These fluorophores have shown great advantages such as high cell permeability, low ...