Ribosomal RNA-Selective Light-Up Fluorescent Probe for Rapidly Imaging the Nucleolus in Live Cells

RNA-based fluorescent probes are currently limited by their low selectivity toward RNA versus DNA, and low specificity to different RNA structures. Poor membrane permeability is another defect of existing fluorogenic RNA probes for intracellular imaging. In this work, a naphthalimide derivative, probe 1, was developed for the rapid and selective detection of intracellular rRNA (rRNA). Probe 1 exhibited a 32-fold fluorescent enhancement in response to rRNA binding and showed desirable selectivity for rRNA versus DNA and other nucleic acids in phosphate buffer at pH 7.2. Importantly, probe 1 displayed excellent permeability of the nucleolus, could be taken up in 1 min by four different cell lines, and may be the fastest nucleolus dye. The excellent selectivity of probe 1 toward rRNA is attributed to the specific interaction between the complicated 3D structures of rRNA, which was confirmed by quantum calculations using molecular docking simulations. An appropriate lipophilic balance in 1 with the hydrophilic amine group and hydrophobic naphthalimide, as well as its high water solubility, guarantees the high permeability of 1 in cell membranes and nucleolus pores, compared to other analogues (e.g., probes 2-8 in this work). Furthermore, enlarged confocal laser micro images of nucleoli and RNase digestion tests revealed that 1 remained highly selective toward rRNA, even for intracellular imaging. As a live cell probe, 1 also exhibited better photostability than the commercial RNA dye, SYTO RNA select.