A Color‐Shifting Near‐Infrared Fluorescent Aptamer–Fluorophore Module for Live‐Cell RNA Imaging

Fluorescent light‐up RNA aptamers (FLAPs) have become promising tools for visualizing RNAs in living cells. Specific binding of FLAPs to their non‐fluorescent cognate ligands results in a dramatic fluorescence increase, thereby allowing RNA imaging. Here, we present a color‐shifting aptamer‐fluorophore system, where the free dye is cyan fluorescent and the aptamer‐dye complex is near‐infrared (NIR) fluorescent. Unlike other reported FLAPs, this system enables ratiometric RNA imaging. To design the color‐shifting system, we synthesized a series of environmentally sensitive benzopyrylium‐coumarin hybrid fluorophores which exist in equilibrium between a cyan fluorescent spirocyclic form and a NIR fluorescent zwitterionic form. As an RNA tag, we evolved a 38‐nucleotide aptamer that selectively binds the zwitterionic forms with nanomolar affinity. We used this system as a light‐up RNA marker to image mRNAs in the NIR region and demonstrated its utility in ratiometric analysis of target RNAs expressed at different levels in single cells.