Domain-crosslinked DNA polymer spherical nucleic acid nanomachines for amplified miRNA imaging in living cells

This study reports a robust DNA polymer spherical nucleic acid (PSNA) nanomachine based on domain-crosslinked self-assembly for highly selective and ultrasensitive imaging of microRNAs (miRNAs) in living cells. The nanomachine is constructed from DNA micelles formed via self-assembly of amphiphilic monomers followed by radical-initiated polymerization. This design effectively overcomes key limitations of conventional spherical nucleic acids, including poor biostability, inefficient cellular uptake, and nonspecific signal leakage, by providing a covalently stabilized architecture with high DNA loading capacity. A target-recognizing lock-walking strand and a fluorophore-labeled track strand containing the substrate sequence are immobilized on two distinct PSNA particles, respectively. Upon specific activation by endogenous miRNA-21 within tumor cells, the DNA walker is released and initiates a cyclic walking process that binds to the substrate strands and triggers a fuel-driven strand displacement cycle, leading to significant signal amplification. This walking mode is multi-legged and spatially confined. It enables high-speed reaction kinetics, substantially enhances fluorescence signals, and improves imaging clarity. The resulting PSNA nanomachine exhibits excellent stability and amplification capability, permitting ultrasensitive miRNA detection under low-background conditions. Moreover, this platform has been successfully applied for in situ imaging of miRNA in various cell lines, demonstrating strong potential for early clinical diagnostics and real-time monitoring of therapeutic responses.