Multi-responsive tetrahedral DNA frameworks for in situ methyltransferase imaging to distinguish living chemoresistant tumor cells.

Chemoresistance, a primary contributor to approximately 90% of cancer-related deaths, stems from the tumor cells' ability to endure chemotherapy-induced DNA methylation damage via aberrant upregulation of DNA repair enzymes, such as O6-methylguanine-DNA methyltransferase (MGMT). Herein, we report a multi-responsive tetrahedral DNA framework (Mr-TDF) that enables accurate distinction of chemoresistant tumor cells through in situ and highly sensitive imaging of MGMT activity in living cells. The Mr-TDF incorporates four identical multi-responsive DNAzyme probes (Mr) anchored on a DNA tetrahedral scaffold, with each probe integrating three key modules: MGMT recognition, DNAzyme activation and fluorescent signal output. Upon MGMT-mediated demethylation of the O6-methylguanine lesion on the Mr probe, the DNAzyme's catalytic activity is specifically reactivated, resulting in rA-site cleavage and subsequent fluorescence signal generation. Leveraging the spatial confinement effect of the DNA tetrahedral scaffold, the Mr-TDF achieves highly sensitive and rapid monitoring of MGMT activity, producing fluorescence signals more than three times stronger in chemoresistant tumor cells compared to those of chemosensitive counterparts. This study establishes a robust platform for probing epigenetic dynamics in living chemoresistant cells and offers new avenues for mechanistic investigations and early diagnosis of chemoresistance.