Revealing the Regulatory Interplay of NHE1 mRNA and Na⁺ in Cancer Cells Using a DNA Nanosensor

Cellular signaling networks are orchestrated by complex interactions between gene expression and ion flux, yet tools for simultaneously visualizing these events in living cells remain limited. Herein, we report two orthogonal gold-nanoparticle-based DNA nanosensors that enable simultaneous imaging of sodium/hydrogen exchanger 1 (NHE1) mRNA and Na⁺ dynamics in hepatocellular carcinoma cells. The sensing mechanism relies on proximity-dependent fluorescence quenching. For mRNA detection, Cy3-labeled reporter strands hybridize to DNA-functionalized gold nanoparticles (AuNPs), holding the fluorophore close to the quenching surface. Target binding triggers strand displacement, releasing Cy3, and restoring emission. For Na⁺ detection, a Cy5-labeled substrate strand hybridizes with a Na⁺-specific DNAzyme anchored on AuNPs. Na⁺ activates the DNAzyme, cleaving the substrate at a defined site and liberating the Cy5 fluorophore. This dual-sensor system enables the quantitative monitoring of both analytes in living cells. Using this platform, we directly visualize that NHE1 mRNA downregulation suppresses intracellular Na⁺ accumulation, establishing a regulatory link between gene expression and ion homeostasis. Notably, mRNA-targeted hybridization inhibits cancer-cell migration in a dose-dependent manner, revealing a potential therapeutic mechanism. This approach provides a molecular tool for decoding ion-based signaling networks in cancer biology.