MnO₂-Nanosheet-Powered Protective Janus DNA Nanomachines Supporting Robust RNA Imaging

Both biomarker and probe degradations cause serious false assay results. However, protecting a target or a target and a probe simultaneously has rarely been explored. Herein, MnO₂-nanosheet-powered target- and probe-protective Janus DNA nanomachines are reported. It is formed in living cells by an RNA-responsive assembly of two chemically modified DNA partzymes and one substrate probe. MnO₂ nanosheets are used to facilitate the cellular uptake of DNA reagents and generate Mn²⁺, which are indispensable DNAzyme cofactors for efficient catalytic cleavage. We find that DNA partzymes with modified sugar moieties (e.g., LNA or ones with 2'-O-methylation) protect the RNA of RNA-DNA hybrids from RNase H degradation. LNA blocks RNase H recruitment on the hybrid best because of its 2'-O, 4'-C methylene bridge structure. In contrast, modifications at DNA phosphate moieties fail to protect the RNA. RNA protection can exclude target-degradation-induced false negative results. In addition, the phosphorothioate-modified substrate probe is known to resist nuclease degradation, which minimizes false positive interference. Compared to canonical DNA systems without chemical modifications, the protective Janus nanomachine avoids false results and supports robust RNA imaging.