Spatially Resolved Sequential Activation of Allosteric DNA for <i>In Vivo</i> Dual-Target Detection within Mitochondria: A Strategy to Visualize of Drug-Resistant Neuroblastoma

Our previous study demonstrated that apurinic/apyrimidinic endonuclease 1 (APE1) and miR-514a are significantly overexpressed in the cytoplasm of drug-resistant neuroblastoma (NB) cells. Furthermore, we have developed a novel strategy for monitoring drug resistance in NB by targeting cytoplasmic APE1 and miR-514a. The overexpression of key enzymes in the mitochondrial base excision repair pathway, along with dysregulated miRNAs, is closely associated with chemotherapy resistance in tumors. Therefore, this study leverages cytochrome c (cyt c), located in the inner mitochondrial membrane as a targeting agent and the mitochondria-specific expression of 16S rRNA as a response switch to develop a spatially resolved, sequential activation system for an allosteric DNA nanomachine (AP-miR-tFNA), enabling <i>in viv</i>o detection of APE1 and miR-514a within mitochondria and facilitating molecular imaging of NB. AP-miR-tFNA sequentially responds to cyt c, 16S rRNA, miR-514a, and APE1, thereby undergoing a conformational change that efficiently achieves progressive dissociation of the fluorophore from the quencher through a sequential mechanism, ultimately generating a detectable fluorescence signal. Experimental results demonstrate that AP-miR-tFNA enables <i>in vivo</i> monitoring of drug resistance in NB, providing an innovative and dependable approach for monitoring therapeutic resistance in NB. In particular, AP-miR-tFNA enables <i>in situ</i> detection of APE1 and miR-514a within NB plasma exosomes, thereby allowing non-invasive differentiation between high-risk and low-to-intermediate-risk NB, as well as between drug-resistant NB and non-drug-resistant NB.