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 in vitro 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 > 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.活细胞成像（Live-cell imaging）技术可在近生理条件下对细胞内蛋白质进行实时成像观测，捕捉其动态变化，为基础生命科学研究与医学应用均提供关键洞见。然而，传统基于抗体或基因编码的标记技术存在探针通透性差、伴随细胞毒性等局限，使得活细胞成像仍面临重大挑战。为解决上述问题，本研究开发并严格验证了一种新型基于适配体（aptamer）的荧光探针，用于活细胞内NEK9激酶的实时成像。首先，通过体外捕获式SELEX（指数富集配体系统进化技术），我们鉴定出一种可特异性结合NEK9的DNA适配体Apt-011。进一步，依托适配体尺寸小、免疫原性低且合成灵活性强的优势，我们构建了“信号开启”型NEK9特异性适配体荧光探针平台。该设计利用适配体的靶标诱导构象变化，使荧光基团-猝灭基团对发生物理分离；经验证，此荧光探针平台可在活细胞中成功可视化细胞内NEK9，且在工作浓度下无明显细胞毒性（细胞存活率＞95%），为研究NEK9相关信号通路提供了新契机。本研究不仅为激酶研究提供了一款可靠工具，还通过合理的适配体工程化策略，为攻克活细胞成像领域的关键瓶颈建立了一种可推广的技术方案。