DNAzyme-Amplified Cascade Catalytic Hairpin Assembly Nanosystem for Sensitive MicroRNA Imaging in Living Cells

Sensitiveimaging of microRNAs (miRNAs) in living cells is significantfor accurate cancer clinical diagnosis and prognosis research studies,but it is challenged by inefficient intracellular delivery, instabilityof nucleic acid probes, and limited amplification efficiency. Herein,we engineered a DNAzyme-amplified cascade catalytic hairpin assembly(CHA)-based nanosystem (DCC) that overcomes these challenges and improvesthe imaging sensitivity. This enzyme-free amplification nanosystemis based on the sequential activation of DNAzyme amplification andCHA. MnO2 nanosheets were used as nanocarriers for thedelivery of nucleic acid probes, which can resist the degradationby nucleases and supply Mn2+ for the DNAzyme reaction.After entering into living cells, the MnO2 nanosheets canbe decomposed by intracellular glutathione (GSH) and release the loadednucleic acid probes. In the presence of target miRNA, the lockingstrand (L) was hybridized with target miRNA, and the DNAzyme was released,which then cleaved the substrate hairpin (H-1). This cleavagereaction resulted in the formation of a trigger sequence (TS) thatcan activate CHA and recover the fluorescence readout. Meanwhile,the DNAzyme was released from the cleaved H-1 and boundto other H-1 for new rounds of DNAzyme-based amplification.The TS was also released from CHA and involved in the new cycle ofCHA. By this DCC nanosystem, low-abundance target miRNA can activatemany DNAzyme and generate numerous TS for CHA, resulting in sensitiveand selective analysis of miRNAs with a limit of detection of 5.4pM, which is 18-fold lower than that of the traditional CHA system.This stable, sensitive, and selective nanosystem holds great potentialfor miRNA analysis, clinical diagnosis, and other related biomedicalapplications.

Automated summary

In this study, a DNAzyme-amplified cascade catalytic hairpin assembly (CHA)-based nanosystem (DCC) was engineered to improve the sensitive imaging of microRNAs (miRNAs) in living cells. This nanosystem utilized DNAzyme amplification and CHA activation for enzyme-free amplification. MnO2 nanosheets were used as carriers for nucleic acid probes, protecting them from degradation and providing Mn2+ for the DNAzyme reaction. The DCC nanosystem achieved sensitive and selective miRNA analysis with a limit of detection of 5.4 pM, 18 times lower than the traditional CHA system. This nanosystem holds great potential for miRNA analysis, clinical diagnosis, and other biomedical applications.