Stimuli-Responsive Fusion of Binary-Star Intelligent DNA System for the Signal-On Amplification Detection of Cancer-Related miRNA

While extensive efforts have been devoted toward multifunctional DNA assembly-based systems for imaging intracellular miRNAs, challenges remain in clinical translation due to their inherently vulnerable degradability and poor assay sensitivity. In this contribution, based on the hydrophobic aggregation of cholesterols, we demonstrate a binary-star intelligent DNA system (essentially dual-DNA nanosphere probes, DDNS) for fluorescently imaging intracellular miRNA-224, a well-known cancer-related biomarker. This system is made of DNA nanosphere-1 (DNS-1) and DNA nanosphere-2 (DNS-2), which are assembled separately from two cholesterol-modified DNA hairpin probes, one of which is designed for identifying target miRNA and the other is employed to trigger a target-recycled strand displacement nonenzymatic amplification (T-SDA) reaction. Utilizing the DDNS system, while the assay sensitivity is improved by 100 times and target miRNA-224 is detected down to 25.7 pM, the linear response range is 100 times widened, and interference from nontarget miRNA family members is approximately 100% suppressed. Moreover, the nuclease resistance of the DDNS system is enhanced by 95 times, so that no substantial degradation occurs even after 24 h of serum incubation. More importantly, the DDNS system efficiently enters living cells without the assistance of external reagents, and its ability to fluorescently distinguish cancerous cells from healthy cells is comparable to a commercial transfection agent-mediated counterpart system, but without compromising cell viability or circumventing common safety concerns, which is also validated by blind cell imaging. As a proof-of-concept, the DDNS system presents a paradigm for the advancement and application of structural DNA nanotechnology in the field of molecular biosensing and precision medicine.