RCA-mediated tandem assembly of DNA molecular probes on lipid particles surface for efficient detection and imaging of intracellular miRNA.

Aberrant microRNA (miRNA) expression is frequently implicated in various cancers, making the monitoring of intracellular miRNA levels a promising strategy for cancer diagnosis and therapy. However, detecting miRNA with high precision and sensitivity at the cellular level remains challenging due to its small size and low abundance. In this study, we attached hydrophobic cholesterol molecules to hydrophilic DNA chains to self-assemble into cholesterol-DNA micelles. The products of rolling ring amplification were linked to the surface of cholesterol-DNA, and two hairpins (H1 and H2) used for hybridization chain reaction (HCR) were simultaneously tethered to the branch, ultimately forming the assembled nanoprobe (RC-HCR) with signal amplification for detecting and imaging miRNA in living cells. This design significantly increased the concentration of HCR hairpins and also shortened their physical distance, thereby enhancing kinetics and signal amplification. Moreover, we demonstrated that the lipid particles could be assembled by simply stirring in a buffered solution, allowing the system to enter cells naturally. Using miR-21 as the model target, we found that the RC-HCR probe had a detection limit of 1 fM and a wide quantitative range (1 fM to 80 nM) at 37 °C within 0.5 h. In addition, RC-HCR exhibited high selectivity for miRNA detection and could accurately identify wild-type miR-21 from its mutants and other miRNAs. Furthermore, we showed that RC-HCR could efficiently image miR-21 in living cells. Collectively, our strategy provides a valuable nanoprobe for detecting and imaging miRNAs in live cells, highlighting a novel tool for early clinical diagnosis.