Spatial confinement-enhanced CHA system based on tetrahedral DNA nanostructures for sensitive in situ mRNA analysis

Sensitive and efficient detection of tumor-related mRNA is essential for the early diagnosis of cancer yet remains challenging due to low biomarker abundance and methodological limitations. Here, we developed a three-dimensional DNA nanomachine termed T-CHA, which couples catalytic hairpin assembly (CHA) with a rigid tetrahedral DNA nanostructure (TDN). This design enables target mRNAs, such as UBE2C overexpressed in hepatocellular carcinoma (HCC), to trigger a localized, cascading amplification reaction. We demonstrated that T-CHA quantitatively detected UBE2C mRNA with a limit of detection of 8.53 fM within 30 min and accurately distinguished HCC cells by expression level (LM3 > HepG2 > LO2). Applied to clinical tissue sections, T-CHA exhibited more favorable performance compared with the conventional fluorescence in situ hybridization (FISH) protocol used in this study in visualizing heterogeneous UBE2C expression at single-cell resolution. Furthermore, by modifying the recognition sequence, T-CHA achieved highly sensitive detection of miR-21, demonstrating the modular adaptability of the platform for different RNA targets as an initial proof of concept. By combining the structural precision of TDN with the signal amplification of CHA, T-CHA provides a robust and precise platform for the molecular diagnosis of HCC, highlighting its potential for further development toward diagnostic applications.