Actively targeted and dual-stimuli-responsive branch-shaped system for simultaneous microRNAs imaging in living cells

Designing a multifunctional nucleic acid platform with the ability to identify multiple biomarkers is crucial for accurate molecular diagnostics. Here we reported a dual-target branch-shaped nucleic acid probing (DBNP) system and its targeted delivery for simultaneous analysis of intracellular microRNAs (miRNAs). The system consisted of a cross-shaped DNA scaffold equipped with four hairpin strands and an aptamer strand. The four hairpin strands constituted two catalytic hairpin assembly (CHA) reactions, enabling the system to simultaneously recognize and amplify two kinds of miRNAs. We demonstrated that the cross-shaped DNA scaffold structurally offered a spatial-confinement effect, well-known in increasing the intramolecular collision of the probes and accelerating the CHA reactions, which allows us to resolve multiplex miRNAs profiling with high efficiency by using DBNP system. In addition, because of the inherent character of aptamer capable of specifically binding with cell-membrane receptor, targeted delivery of DBNP system into the unique receptor-positive cells can be realized to detect intracellular miRNAs. The dual miRNA-mediated stimuli-response, operating in parallel with a receptor-specific recognition of target cells, synergistically enhances the accuracy of cell discrimination and cancer diagnosis. This innovative approach holds great promise for advancing the development of cuttingedge signal amplification technologies and biosensors utilized in cancer diagnostics.