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 are reported, providing a molecular tool for decoding ion-based signaling networks in cancer biology.
Abstract
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.
For subcellular imaging, activating probes by specific species in targeted organelles is an attractive strategy to eliminate nonspecific signals during transport. The characteristic pH (∼4.5-5.0) of lysosomes enables selective probe activation within lysosomes. In this work, a pH-activated K+ nanosensor for lysosomal K+ imaging was designed by capturing Y-typed DNA probe on linker modified gold nanoparticle. The DNA probe could be conveniently prepared by assembling dual fluorophores-labelled G-quadruplex (G4) strand, Sgc8c modified i-motif structure and capturing DNA sequence. The folding capability of G4 in the presence of K+ was inhibited at neutral pH, which prevented fluorescence resonance energy transfer (FRET) between two fluorophores labelled at both ends of the G4. Upon targeted delivery into lysosomes, the conformational change of the i-motif structure was activated by acidic pH, leading to G4 folding and thus K+-dependent FRET signal. Sgc8c modification enhanced target-cell-specific internalization for nanosensor delivery. Both in vitro experiments and in situ lysosomal K+ imaging along with external pharmacological stimuli demonstrated the excellent performance of the proposed K+ nanosensor, which expanded the application of DNA-based probes in subcellular imaging field.
Guangming Li, Ying An, Jiahui Sun et al.· Biosensors & bioelectronics· 0 citations
RNA demethylase plays a crucial role in the dynamic regulation of m6A modification and has been identified as promising biomarker and therapeutic target. However, real-time monitoring of its activity in living cells remains challenging due to poor probe stability, and lack of tumor selectivity. Herein, we develop a cancer-targeting, endogenous-activated DNA nanosponge (DNS) platform for cancer-selective FTO imaging. The DNS is constructed via rolling circle amplification and loaded with FTO-responsive DNAzyme probes (FDz) and photosensitizer zinc phthalocyanine (ZnPc) to form FDz-ZnPc@DNS. The FDz are initially silenced by m6A modification and conjugated to DNS via glutathione (GSH)-cleavable disulfide linkage. Upon folate receptor-mediated internalization of FDz-ZnPc@DNS into cancer cells, intracellular GSH triggers the release of FDz. Subsequent FTO-mediated demethylation reactivates the DNAzyme, leading to amplified fluorescence recovery for FTO imaging. Concurrently, under 660 nm laser illumination, ZnPc generates cytotoxic singlet oxygen to destroy cancer cells/tissues via photodynamic therapy. Both in vitro and in vivo studies validate the feasibility of this nanoplatform for cancer-selective imaging and effective treatment. This work presents a robust and versatile nanoplatform that merges targeted delivery and endogenous activation for precise cancer imaging and therapy, with the potential for adaptation to diverse sensing and therapeutic payloads through modular reprogramming.
Yunhong Han, Weijie Tong, Fei Ma et al.· Small· 0 citations
Magnesium plays crucial roles in many biological processes and the stabilization of biomolecules, including DNA, RNA, and proteins. Despite significant progress, however, our understanding of how cells regulate Mg2+ homeostasis and transport remains incomplete. One of the goals is to develop approaches for detecting Mg2+ dynamics with high spatial and temporal resolution. Herein, we describe the development of MagFR, a ratiometric Mg2+ sensor based on Pepper and Clivia fluorescent RNAs. In this sensor, Pepper fluorescence is highly Mg2+ dependent, and Clivia fluorescence acts as the normalizer. MagFR has favourable properties, including a large dynamic range, pH insensitivity, and high selectivity, allowing robust detection of Mg2+ in both live bacterial and mammalian cells. By targeting MagFR to distinct subcellular compartments in mammalian cells, we observed that the free cytoplasmic Mg2+ concentration is slightly higher than that in the nucleoplasm. We demonstrated that mammalian cells maintain Mg2+ homeostasis even under conditions of elevated extracellular Mg2+, revealing the robustness of intracellular regulatory mechanisms governing Mg2+ balance. MagFR also enabled real-time detection of cellular Mg2+ dynamics following ATP depletion. Overall, this study offers a robust and versatile tool for imaging Mg2+ dynamics in cells, which will be useful for elucidating the functionality and mechanism of Mg2+ homeostasis underlying diverse cellular processes.
Xin Xie, Bibi Zhang, Ming-Yan Liu et al.· ACS Sensors· 0 citations
A DNA nanodevice that anchors to cell membranes and releases chemotherapy only when three specific cancer markers are detected simultaneously is developed, establishing a versatile platform for logic‑controlled, multimarker‑guided cancer theranostics.
Meiqin Zhang, Ying-Sheng Cheng, Nan Chen et al.· Nature Communications· 0 citations
DNA-based nanomachines hold great promise for intracellular miRNA sensing and cancer diagnosis. However, conventional approaches often suffer from insufficient sensitivity and specificity, which limits their applicability in clinical diagnostics. Herein, we report an APE1-powered AND-gated DNA walker with autocatalytic amplification (AWA) for the imaging of dual intracellular miRNAs. In this platform, system activation requires the simultaneous presence of miRNA-10b and miRNA-155, which triggers the release of the walking strand. APE1 then drives autonomous walker movement and releases initiators that activate the downstream autocatalytic circuit, resulting in efficient signal amplification. Benefiting from the autocatalytic amplification circuit, the AWA system achieved a low detection limit of 1.62 pM. Moreover, owing to the AND-gated recognition mechanism, the platform accurately discriminated cancer cells based on their distinct miRNA coexpression profiles, and further distinguished cancerous from normal breast tissues in clinical samples. Overall, the AWA system provides a powerful tool for intracellular miRNA analysis and shows significant potential for early cancer diagnosis.
Liuting Mo, Danting He, Yi Zeng et al.· Analytical Chemistry· 0 citations
Exosomal microRNA methylation represents a critical epigenetic modification closely implicated in cancer progression. However, its sensitive detection continues to pose substantial challenges, owing to the inherent coupling between charge accumulation and steric restriction during signal amplification. Here, we report a light-regulated iontronic biosensor for sensitive detection of exosomal m5C-modified microRNA-21 (m5C-miR-21) by regulating the charge-steric trade-off during hydrogel formation. The DNAzyme selectively recognizes and cleaves m5C-miR-21, releasing a trigger strand that initiates the assembly of HP/AuNPs/CaCO3 probes onto anodic aluminum oxide (AAO) nanochannels. Upon ultraviolet irradiation, o-nitrobenzaldehyde generates H+, which induces CaCO3 decomposition and Ca2+ release. The released Ca2+ subsequently cross-links sodium alginate to form a negatively charged hydrogel in situ on the nanochannel surface. The resulting hydrogel increases local charge density and promotes ion enrichment, while excessive gel growth imposes steric resistance to ion transport. By regulating the irradiation process, the charge-steric trade-off between these two effects is optimized to maximize the ionic current response. When the charge-steric trade-off is properly optimized, the biosensor achieved a linear detection range from 0.1 fM to 0.1 nM with a detection limit of 32 aM. The platform further demonstrated reliable performance in cell lysates and plasma samples, enabling discrimination between colorectal cancer samples and controls. This strategy provides an effective route for iontronic analysis of nucleic acid methylation through light-controlled regulation of charge-steric interplay in nanochannels.
Baojing Jiang, Tianyi Lu, Yanlei Li et al.· Analytical Chemistry· 0 citations
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