Tumor microenvironment-responsive dynamic self-assembly of DNAzyme nanowires for high-contrast imaging of multiple messenger RNAs and chemotherapy.

Messenger RNAs (mRNAs) are a category of protein-coding RNA, and their dysregulation is closely implicated in diverse cancers. Static DNA nanodevices have been engineered for the imaging and therapy, but they rely on the pre-assembly of DNA components and are limited by the low imaging contrast. Herein, we demonstrate the tumor microenvironment-responsive dynamic self-assembly of DNAzyme nanowires for high-contrast imaging of multiple mRNAs and chemotherapy. We design a cruciform DNA module (C-model) as a basic structural unit of DNAzyme nanowire, which consists of two DNAzyme strands and two substrate strands with the chemotherapeutic drug doxorubicin (DOX) being inserted into the C-models by inlaying between guanine and cytosine. Once approaching tumor cells, C-models with two split i-motif spontaneously assemble into a DNAzyme nanowire through protonation-driven Hoogsteen interaction in response to extracellular acidic microenvironments. Upon the internalization, intracellular TK1 mRNA and GalNAc-T mRNA hybridize with the overhangs of DNAzyme strands and substrate strands to form a stable DNAzyme/substrate/target three-way junction, inducing the cleavage of substrates by DNAzymes and the recovery of Cy5/Texas Red fluorescence. AS1411 aptamer-functionalized DOX-nanowires can selectively endocytose into cancer cells via AS1411-mediated recognition of nucleolin overexpressed on the cancer cell surface. Notably, the dynamic self-assembly of DNAzyme nanowires is characterized by efficient intracellular delivery and intrinsic resistance to nucleases, facilitating the high-contrast imaging of mRNAs. This method can accurately measure TK1 mRNA and GalNAc-T mRNA in living cells and discriminate multiple mRNAs in breast cancer tissues and their normal counterparts, providing a powerful platform for clinical diagnosis.