Mitochondria-targeting abasic site-reactive probe (mTAP) enables the manipulation of mitochondrial DNA repair and turnover

Mitochondrial DNA (mtDNA) encodes essential protein subunits in the oxidative phosphorylation system and a set of tRNAs and rRNAs, making it critical for mitochondrial and cellular functions. Defects in mtDNA are implicated in a range of mitochondrial disorders and diseases, including cancer and neurodegeneration. In addition, mtDNA plays a role in the innate immune response and inflammation, as it is released into the cytoplasm and bloodstream. mtDNA is particularly susceptible to damage by endogenous and exogenous factors, and its stability is maintained through redundancy, repair, and turnover mechanisms. However, there is currently no chemical strategy to manipulate the mtDNA repair and turnover. To address this gap, we developed a mitochondria-targeting, water-soluble probe named mTAP, which selectively reacts with key mtDNA repair intermediates – abasic (AP) sites. Using a sensitive mass spectrometry approach, we confirmed that mTAP forms oxime conjugates exclusively with mitochondrial AP sites, without producing conjugates with nuclear AP sites. Upon mTAP conjugation, DNA substrates containing AP sites become resistant to cleavage by AP endonuclease (APE1) and mitochondrial extracts from HeLa cells. Biochemical assays showed that mTAP conjugation significantly reduced APE1’s binding affinity for AP sites, without disrupting the DNA-binding activity of mitochondrial transcription factor A, as confirmed through fluorescence polarization assays. Importantly, cellular studies demonstrated that mTAP treatment alleviated the decrease in mtDNA copy number and transcription levels induced by mitochondrial AP site damage. These findings highlight the potential of mTAP as a valuable chemical tool to modulate mitochondrial DNA repair and degradation processes.