Controllable autocatalytic cleavage-mediated fluorescence recovery for homogeneous sensing of alkyladenine DNA glycosylase from human cancer cells

Abstract

DNA alkylation and oxidation are two most common forms of cytotoxic damage with the characteristics of mutagenic and carcinogenic. Human alkyladenine DNA glycosylase (hAAG) is the only glycosylase known to repair a wide variety of alkylative and oxidative DNA lesions. However, few approaches are capable of real-time monitoring hAAG activity. Methods: Herein, we develop a facile fluorescent strategy for homogeneous and sensitive sensing of hAAG activity based on the controllable autocatalytic cleavage-mediated fluorescence recovery. The presence of hAAG enables the cleavage of hairpin probe 1 (HP1) at the damaged 2'-deoxyinosine site by AP endonuclease 1 (APE1), forming a DNA duplex. The trigger 1 built in the resultant DNA duplex may hybridize with hairpin probe 2 (HP2) to induce the T7 exonuclease (T7 exo)-catalyzed recycling cleavage of HP2 (Cycle I) to release trigger 2. The trigger 2 can further hybridize with the signal probe (a fluorophore (FAM) and a quencher (BHQ1) modified at its 5' and 3' ends) to induce the subsequent recycling cleavage of signal probes (Cycle II) to liberate FAM molecules. Through two-recycling autocatalytic cleavage processes, large amounts of fluorophore molecules (i.e., FAM) are liberated from the FAM-BHQ1 fluorescence resonance energy transfer (FRET) pair, leading to the amplified fluorescence recovery. Results: Taking advantage of the high accuracy of in vivo DNA repair mechanism, the high specificity of T7 exo-catalyzed mononucleotides hydrolysis, and the high efficiency of autocatalytic recycling amplification, this strategy exhibits high sensitivity with a detection limit of 4.9 × 10-6 U/μL and a large dynamic range of 4 orders of magnitude from 1 × 10-5 to 0.1 U/μL, and it can further accurately evaluate the enzyme kinetic parameters, screen the potential inhibitors, and even quantify the hAAG activity from 1 cancer cell. Conclusion: The proposed strategy can provide a facile and universal platform for the monitoring of DNA damage-related repair enzymes, holding great potential for DNA repair-related biochemical research, clinical diagnosis, drug discovery, and cancer therapy.