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Abstract

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 background staining, and strong photostability in background-free labeling of various cellular targets in living cells. Unlike the traditional affinity-oriented aptamer selection method SELEX, this new platform offered the possibility of aptamer evolution directed by both brightness and binding affinity, two key parameters of FLAPs. The FACS-based aptamer selection against the fluorogenic and NIR-fluorescent silicon rhodamine (SiR) and the subsequent in vitro activity screening successfully revealed three SiR-binding sequence families. Among them, a 103-nt variant L2-1 showed the highest binding affinity (KD = 25 nM, turn-on = 4.4-fold) and a 53-nt variant L2-2-3 possessed highest fluorescence turn-on (KD = 192 nM, turn-on = 5.5-fold) towards SiR. The two NIR FLAPs developed in this thesis provide valuable tools for live-cell RNA imaging and inspire the development of the next-generation of spirocyclization-based FLAPs.