Ultrasensitive Apurinic/Apyrimidinic Site-Specific Ratio Fluorescent Rotor for Real-Time Highly Selective Evaluation of mtDNA Oxidative Damage in Living Cells.

Abstract

The unrepaired apurinic/apyrimidinic site (AP site) in mitochondrial DNA (mtDNA) promotes misincorporation of nucleotides and further causes serious damage for the living organism. Thus, accurate quantitative detection of AP sites in mtDNA in a rapid, highly sensitive, and highly selective fashion is important for the real-time evaluation of mtDNA oxidative damage. In this study, a targeting mtDNA ultrasensitive AP site-specific fluorescent rotor (BTBM-CN2) was designed by the strategy of molecular conformation torsion adjustment ratio fluorescent signal. The specific recognition reaction is activated when it encountered AP sites in mtDNA within 20 s, and BTBM-CN2 presented a "turn-on" red fluorescence signal at 598 nm. Then, about 100 s later, BTBM-CN2 emitted a new green fluorescence signal at 480 nm, which is mainly due to the activation of the rate-limiting reaction. With increasing numbers of AP sites (1-40 in 1 × 105 bp of mtDNA), the fluorescence emission at 598 nm decreased gradually, and the new emission at 480 nm increased. Intracellular experiments indicated that BTBM-CN2 could detect AP sites in mtDNA in a rapid and quantitative fashion with high selectivity and ultrasensitivity. On the basis of the emergence of the fluorescence signal at 480 nm and its signal strength, the cell whose mtDNA was damaged could be screened by flow cytometry and its degree of damage could be evaluated in real time by comet assay. Hence, the rotor may have potential applications varying from accurate and ultrasensitive detection of AP sites to the real-time evaluation of the oxidative damage in living cells.