Antisense Oligonucleotide Releasing Cassette for Conditional Targeted RNA Degradation

Conventional single-stranded antisense oligonucleotides (ASOs) work by directly binding target mRNA, which limits their use to genes that need to be silenced without affecting healthy cells. To address this limitation and achieve conditional gene silencing specifically within target cells, we developed. This system activates its RNA-cleaving function only upon binding to a specific biomarker nucleic acid sequence followed by RNase H-dependent cleavage of the targeted RNA. Under cell-free conditions, ARC showed 3-fold greater RNA cleavage efficiency in the presence of the biomarker as compared to its absence. Intriguingly, in the ARC-treated GFP-expressing K562 cell line a 45% reduction in GFP fluorescence was achieved in the presence of the biomarker sequence, which was 5.3-fold greater than the reduction observed when it was absent. This ARC's conditional ASO release offers a new mechanism for safely targeting essential or vital genes exclusively in cells that produce specific RNA biomarkers. Our findings indicate that the future in the field of nucleic acid nanotechnology will critically depend on two key advancements: the development of appropriate intracellular delivery systems and the establishment of robust predictive tools for accurately modeling the thermodynamic behavior of complex nanostructures, including those incorporating diverse chemical modifications.