Three-Dimensional CHA-HCR System Using DNA Nanospheres for Sensitive and Rapid Imaging of miRNA in Live Cells and Tissues

Isothermal, enzyme-free amplification techniques, suchas the hybridizationchain reaction (HCR) and catalytic hairpin assembly (CHA), have gainedincreasing attention for miRNA analysis. However, current methodologicalchallenges, including slow kinetics, low amplification efficiency,difficulties in efficient cellular internalization of DNA probes,and concerns regarding the intracellular stability of nucleic acids,need to be addressed. To this end, we propose a novel strategy forsensitive miRNA detection based on a three-dimensional (3D) CHA-HCRsystem. This system comprises two DNA nanospheres, named DS-13 andDS-24, which are functionalized with CHA and HCR hairpins. TargetmiR-21 initiates CHA between the two nanospheres, thereby activatingdownstream HCR and bringing cyanine 3 (Cy3) and cyanine 5 (Cy5) intoproximity. The 3D CHA-HCR process leads to the formation of largeDNA aggregates and the generation of fluorescence resonance energytransfer signals. In this strategy, the employment of a cascaded reactionand spatial confinement effect improve sensitivity and kinetics, whilethe use of DNA nanocarriers facilitates cellular delivery and protectsnucleic acid probes. The experimental results in vitro, in livingcells, and in clinical tissue samples demonstrated the desirable sensingperformance. Collectively, this approach holds promise as a valuabletool for cancer diagnosis and biomedical research.