Zhu, L.; Min, L.; Zhu, C.-s.; Wu, X.; Li, W.; Ma, J.; Lei, Y.; Gao, C.; Qiu, X.; Liu, C.

MicroRNAs (miRNAs) are vital post-transcriptional regulators that govern key cellular processes such as proliferation, migration, and apoptosis. Current loss-of-function approaches, including chemically modified antisense oligonucleotides (ASOs), face significant challenges, including high costs, limited scalability, and off-target effects. To overcome these limitations, we developed \"miRNA Locker\", a novel miRNA inhibition platform created using the Overlapped Oligo Assembly (OOA) method. This innovative platform constructs highly stable dumbbell-shaped single-stranded DNA structures, offering improved target specificity, scalability, and cost-effectiveness. Using miR-214 as a proof-of-concept target, we demonstrated that miRNA Lockers effectively bind Argonaute-miRNA complexes, reduce miRNA levels, and induce downstream changes in gene expression and cellular phenotypes, surpassing the performance of commercial antagomirs. Furthermore, applying miRNA Lockers to miR-654 validated the regulatory role of miR-654 in modulating RNF8 expression and promoting epithelial-mesenchymal transition (EMT) in lung cancer cells. Our results highlight the potential of miRNA Lockers as a versatile tool for studying miRNA function and advancing miRNA-based therapies.