Visualizing NEK9 in action: aptamer-based fluorescent probes for real-time live-cell imaging

Live-cell imaging of intracellular proteins enables real-time observation of protein dynamics under near-physiological conditions, providing pivotal insights for both fundamental life science research and medical applications. However, due to limitations such as poor probe permeability and cytotoxicity associated with conventional antibody-based or genetically encoded labeling techniques, live-cell imaging remains a significant challenging. To address these limitations, here in this study, we developed and rigorously validated a novel aptamer-based fluorescent probe for real-time imaging of NEK9 kinase in living cells. First, through <i>in vitro</i> capture-SELEX, a DNA aptamer termed as Apt-011 which could selectively bind NEK9 was identified. Further, capitalizing on the small size, low immunogenicity, and synthetic flexibility of aptamers, we engineered a "signal-on" NEK9-specific aptamer-based fluorescent probe platform. This design leverages the aptamer’s target-­induced conformational change to physically separate the fluorophore-quencher pair, and it has been validated that this fluorescent probe platform could successfully visualize intracellular NEK9 in live-cells without obvious cytotoxicity (cell viability &gt; 95% at working concentrations), offering new opportunities to study NEK9-associated signaling pathways. This work not only provides a robust tool for kinase research but also establishes a generalizable strategy to overcome key bottlenecks in live-cell imaging through rational aptamer engineering.