Targeted Conversion from the Nucleus to the Nucleolus of Nu-red by Shortening Its Conjugated Chain

The nucleolus plays a pivotal role in the biosynthesis of rRNA and the assembly of ribosomal subunits, as well as in the regulation of mitosis, the control of cell cycle progression, and the response to cellular stress. Therefore, it is highly significant to develop probes that specifically target the nucleolus for investigating its pathophysiological functions. However, the current molecular design predominantly focuses on an elegant start-from-scratch approach, which probably contradicts the expedited creation of nucleolar probes. This study highlights a targeted conversion strategy for rapidly constructing a novel nucleolus-targeted probe called Nuo-green by shortening the conjugated chain of Nu-red, an established nucleus-targeted probe. Specifically, through merely reducing one double bond in the nuclear probe Nu-red, its size is thereby decreased to facilitate its robust binding to RNA, ultimately attaining nucleolar targeting. Nuo-green not only exhibits a robust RNA affinity with a binding constant (<i>K</i>_a) of 5.3 × 10⁷ M^-1 but also presents long excitation and emission wavelengths (λ_ex/λ_em = 515/575 nm), remarkable permeability across cellular membranes and nuclear pores, broad applicability across diverse cell types, and two-photon excitability. Leveraging these advantages, Nuo-green has been effectively utilized for visualizing the disappearance and reconstruction of nucleoli during mitosis, tracking the morphological changes of nucleoli during apoptosis, mapping the three-dimensional fluorescence imaging of nucleoli, and observing nuclear and nucleolar contraction in either fixed brain tissues or the brain of a living mouse using a photothrombosis stroke model.