Recently, Associate Researcher Qiao Qinglong and Researcher Xu Zhaochao, from the Dalian Institute of Chemical Physics, Chinese Academy of Sciences, along with their team, have developed a "buffered fluorescent probe" named Nu-AN that can specifically and reversibly bind to RNA, enabling stable imaging of the nucleolus in living cells. This innovation allows imaging of the dynamic contour of the nucleolus and visualizing specific morphologies induced by drugs in living cells, providing a visual tool for screening nucleolar stress agents. The related research findings have been published in "Advanced Science".
The nucleolus, the largest membrane-less organelle within the cell nucleus, has attracted widespread attention due to its diverse physiological and pathological functions. Serving primarily as the center for ribosome biogenesis, the nucleolus also exhibits non-ribosomal functions. Its morphology, including shape, size, and number within the cell nucleus, reflects physiological activities and is associated with various human diseases. Moreover, nucleolar size correlates strongly with cell lifespan, with smaller nucleoli being indicative of cell longevity. Under nucleolar stress conditions, changes in nucleolar morphology are particularly pronounced, providing a potential approach for disease diagnosis and drug screening. However, fluorescence imaging of nucleolar morphology still faces challenges.
In earlier work, the team developed a "buffered fluorescent probe" named LD-FG for dynamic recognition of lipid droplets within cells, effectively addressing the issue of dye photostability during long-term dynamic super-resolution imaging. Inspired by this strategy, the team incorporated a pyrrole ring into a naphthalimide moiety, developing the reversible RNA-binding nucleolar "buffered fluorescent probe" Nu-AN. The study revealed that the incorporation of the pyrrole ring into the naphthalimide fluorophore maintained planarity while retaining the characteristics of twisted intramolecular charge transfer, while also exhibiting the characteristic fluorescence quenching of traditional naphthalimide dyes via hydrogen bonding. In living cells, Nu-AN can effectively and reversibly stain the nucleolus, while avoiding mislocalization in mitochondria or lysosomes, common issues with commercial RNA probes. This reversibility of binding not only minimizes structural perturbation to the nucleolus, preserving its activity, but also forms a "dye pool" outside the nucleolus. When the probe within the nucleolus undergoes photobleaching, it can rapidly recruit new probes from the "dye pool," ensuring the photostability of nucleolar imaging.
The reversible binding and selective fluorescence activation of Nu-AN with RNA ensure specific and stable fluorescence imaging of the nucleolus within living cells. Leveraging Nu-AN for visualizing nucleolar morphology, the team observed specific changes induced by two drugs, demonstrating the highly dynamic and organized nature of the nucleolus as a membrane-less organelle.
Nu-AN holds promise for providing tools for studying live-cell nucleolar morphology in the field of nucleolar biology. Additionally, the design of this probe opens up new avenues for the development of probes for other membrane-less organelles within cells. In the future, the development of various probes for membrane-less organelles will fill the gap in visualizing these organelles and enhance our understanding of cell biology.
Related Paper Information: https://doi.org/10.1002/advs.202309743
