A DNA tetrahedron-based light-controlled intramolecular CHA for spatiotemporal imaging of miRNA in living cells with high sensitivity and imaging contrast

miRNA is an effective disease diagnostic biomarker. Developing efficient miRNA in living cells analysis methods, holds significant importance for disease diagnosis and understanding disease progression. Strategies combining DNA nanostructures and enzyme-free amplification have been developed for highly sensitive and rapid imaging of miRNA. However, due to nonspecific amplification of enzyme-free amplification inherent in the probe assembly process and during transfection, these strategies suffer from significant background signals and reduced sensitivity, limiting their further application. To overcome these limitations, we have developed a DNA tetrahedral-based and photocontrolled intramolecular CHA strategy (TCHA-P) to achieve spatiotemporal, highly sensitive, and accurate imaging of miRNA in living cells. In this strategy, the DNA tetrahedron not only serves as a transfection vector but also enhances the local concentration of reactant probes, boosting the reaction kinetics and sensitivity of CHA. Additionally, the light-controlled group (PC-linker) can trigger CHA at selected times and locations, enhancing the spatiotemporal precision of miRNA imaging and ensuring high sensitivity and accuracy by eliminating nonspecific amplification during the assembly and transfection processes. Using miR-10b as a proof-of-concept model, the proposed strategy demonstrated higher signal-to-noise ratio compared to traditional CHA, with a detection limit of 28 pM. Living cell imaging experiments showed that the strategy could effectively differentiate the expression of miR-10b between normal breast cells and breast cancer cells, providing high imaging contrast. The strategy holds promise for broadening the path of nucleic acid amplification-based imaging and is expected to become an effective tool in nucleic acid-based disease diagnosis, treatment, and drug development.