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.