Multi-Responsive Tetrahedral DNA Frameworks for In-Situ Methyltransferase Imaging to Distinguish Living Chemoresistant Tumor Cells

Abstract

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). To address this challenge, we developed a multi-responsive tetrahedral DNA frameworks (Mr-TDF) that enables accurate distinguishing 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 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 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.