Mitochondria‐Targeting Abasic Site‐Reactive Probe (mTAP) Enables the Manipulation of Mitochondrial DNA Levels

Abstract

Abstract Mitochondrial DNA (mtDNA) encodes essential genes for mitochondrial and cellular functions and acts as a cell signaling molecule in innate immune and inflammatory responses. Defects in mtDNA are implicated in a range of mitochondrial disorders and human diseases. Currently, no chemical strategy exists to prevent mtDNA loss under genotoxic stress. To address this, we developed a mitochondria‐targeting probe (mTAP) that selectively reacts with key mtDNA repair intermediates–abasic (AP) sites. We confirmed that mTAP forms oxime conjugates exclusively with mitochondrial AP sites without conjugation with nuclear AP sites. Upon mTAP conjugation, DNA substrates containing AP sites were resistant to cleavage by AP endonuclease (APE1) and mitochondrial extracts. This conjugation significantly reduced the DNA‐binding affinity of APE1 without affecting the DNA‐binding activity of a mtDNA‐packaging factor, mitochondrial transcription factor A (TFAM). Importantly, cellular experiments demonstrated that mTAP treatment alleviated the decrease in mtDNA and transcription product levels induced by mitochondrial AP site damage. Functional assays also demonstrated that mTAP treatment did not compromise mtDNA replication activity or increase the overall mtDNA damage level. These findings highlight the potential of mTAP as a valuable chemical tool to modulate mtDNA levels under genotoxic stress.