Conditional Deoxyribozyme–Nanoparticle Conjugates for miRNA-Triggered Gene Regulation

DNA–nanoparticle (NP) conjugates have been used to knockdown gene expression transiently and effectively, making them desirable tools for gene regulation therapy. Because DNA–NPs are constitutively active and are rapidly taken up by most cell types, they offer limited control in terms of tissue or cell type specificity. To take a step toward solving this issue, we incorporate toehold-mediated strand exchange, a versatile molecular programming modality, to switch the DNA–NPs from an inactive state to an active state in the presence of a specific RNA input. Because many transcripts are unique to cell subtype or disease state, this approach could one day lead to responsive nucleic acid therapeutics with enhanced specificity. As a proof of concept, we designed conditional deoxyribozyme–nanoparticles (conditional DzNPs) that knockdown tumor necrosis factor α (TNFα) mRNA upon miR-33 triggering. We demonstrate toehold-mediated strand exchange and restoration of TNFα DNAzyme activity in the presence of miR-33 trigger, with optimization of the preparation, configuration, and toehold length of conditional DzNPs. Our results indicate specific and strong ON/OFF response of conditional DzNPs to the miR-33 trigger in buffer. Furthermore, we demonstrate endogenous miR-33-triggered knockdown of TNFα mRNA in mouse macrophages, implying the potential of conditional gene regulation applications using these DzNPs.