“Repaired and Activated” DNAzyme-RCA Circuit Enables One-Pot and Label-Free Detection of O⁶-Methylguanine DNA Methyltransferase in Clinical Tissues

O⁶-Methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme that catalyzes the reversal of O⁶-alkylguanine lesions, with an essential role in tumor resistance to alkylating chemotherapeutic agents. Herein, we develop a "repaired and activated" DNAzyme-RCA circuit for label-free and one-pot detection of MGMT in cells and tissues. The presence of MGMT can catalyze the demethylation of O⁶MeG-caged circular DNAzyme probe (O⁶MeG-cDZ), restoring its catalytic activity to specifically cleave adenine ribonucleotide (rA)-bearing substrate probe to produce a trigger sequence with a 2',3'-cyclic phosphate at its 3'-end. Subsequently, the resulting trigger can serve as a primer to initiate rolling circle amplification (RCA) upon healing its 3'-end by T4 polynucleotide kinase (T4 PNK), generating a large number of long G-quadruplex sequences. The G-quadruplex sequences can incorporate with thioflavin T (ThT) to produce a dramatically amplified fluorescence signal. Notably, O⁶MeG-cDZ integrates both a DNAzyme sequence and an RCA template sequence to achieve cleavage-and-amplification detection, efficiently eliminating nonspecific amplification. This method enables one-pot, isothermal, and label-free detection of MGMT down to 8.17 × 10^-9 ng/μL and even quantification of MGMT at the single-cell level. Moreover, it can be employed for screening of MGMT inhibitors and precise discrimination of MGMT expression in breast cancer tissues and healthy counterparts, providing a novel paradigm for DNA repair enzyme-related clinical diagnosis and drug discovery.