DNA tetrahedron-assisted catalytic hairpin assembly enables robust in situ miRNA detection and imaging in living cells and clinical samples

Isothermal, enzyme-free amplification techniques, such as conventional catalytic hairpin assembly (C-CHA), have garnered significant interest for miRNA analysis. Nonetheless, key challenges remain, including inefficient cellular uptake of DNA probes, limited intracellular stability of nucleic acids, and unwanted signal leakage from amplification products. To address these issues, we developed a DNA tetrahedron-assisted catalytic hairpin assembly (DTN-CHA) system for the intracellular detection and imaging of miR-21. In this strategy, C-CHA probes are anchored onto DNA tetrahedra through sticky-end hybridization, resulting in three tetrahedral nanoprobes (THP 1 , THP 2 , and THP 3 ) that effectively shield the hairpin probes from nuclease-mediated degradation. Upon recognition of the target miR-21, DTN-CHA is triggered among these nanoprobes, producing a compact, high-molecular-weight DNA nanostructure that amplifies the fluorescence signal. This integrated platform not only improves cellular penetration and intracellular stability but also offers outstanding sensitivity and specificity. The superior performance of the DTN-CHA system was verified in vitro, in living cells, and in clinical serum samples. Collectively, these results underscore its potential for cancer diagnosis and broader biomedical applications. Future research may extend its use to other biomarkers, as well as refine the design for in vivo imaging, real-time monitoring, and personalized diagnostics. • DNA tetrahedra ensure high stability and reduce signal leakage in CHA detection. • Enzyme-free, isothermal DTN-CHA amplifies miRNA signals rapidly and specifically. • Steric protection of hairpin probes boosts nuclease resistance and accuracy in cells. • Versatile platform enables in situ imaging of miR-21 in diverse cell lines and serum. • Diagnostic potential validated by distinguishing cancer patients from healthy donors.