DNA molecular subtraction controlled hybridization chain reaction for high contrast detecting and selective killing...

miRNA-initiated hybridization chain reaction (HCR) has been explored for intracellular imaging and cancer immunomodulation. However, its specificity is often compromised because certain cancer-associated miRNAs are also expressed at low levels in normal cells, leading to insufficient contrast and off-target cytotoxicity. Herein, we report a DNA molecular subtraction-controlled hybridization chain reaction (SHCR) strategy for high-contrast detection and selective activation of immunogenic cytotoxicity in cancer cells. In this design, a partially complementary duplex (S1/S2) performs stoichiometric DNA molecular subtraction between two homologous miRNAs (miR-182 and miR-30a) prior to signal amplification. The residual miR-182 subsequently triggers HCR between fluorophore-labeled hairpins, generating FRET signals and double-stranded DNA. Owing to the differential expression pattern of miR-182 (upregulated) and miR-30a (downregulated) in MCF-7 cells relative to MCF-10A cells, SHCR markedly enhances imaging contrast and increases the fluorescence ratio difference from 1.5-fold to 3.5-fold in cells. The double-stranded DNA products further activate the cGAS-STING pathway, inducing selective immunogenic cytotoxicity in cancer cells while minimizing damage to normal cells. By integrating DNA molecular subtraction with enzyme-free amplification, SHCR establishes a differential-expression-responsive framework for high-contrast cancer cell discrimination and selective functional activation, providing a versatile strategy for precision nucleic acid-based theranostics.