Mitochondria-Directed Fluorescent Probe for the Detection of Hydrogen Peroxide near Mitochondrial DNA

It is important to detect hydrogen peroxide (H₂O₂) near mitochondrial DNA (mtDNA) because mtDNA is more prone to oxidative attack than nuclear DNA (nDNA). In this study, a mitochondria-targeted fluorescence probe, pep3-NP1, has been designed and synthesized. The probe contains a DNA-binding peptide, a H₂O₂ fluorescence reporter, and a positively charged red emissive styryl dye to facilitate accumulation in mitochondria. Due to groove binding of the peptide with DNA, the styryl dye of pep3-NP1 intercalated into the bases of DNA, leading to an increase in red fluorescence intensity (centered at 646 nm) and quantum yield. In this case, pep3-NP1 was a turn-on probe for labeling DNA. Subcellular locations of pep3-NP1 and MitoTracker suggested that pep3-NP1 mostly accumulated in the mitochondria of live cells. Namely, as an intracellular DNA marker, pep3-NP1 bound to mtDNA. In the presence of H₂O₂, pep3-NP1 emitted green fluorescence (centered at 555 nm). Thus, the ratio of green with red fluorescence of pep3-NP1 was suitable to reflect the change of the H₂O₂ level near mtDNA in living cells. The detecting limit for H₂O₂ was estimated at 2.9 and 5.0 μM in vitro and in cultured cells, respectively. The development of pep3-NP1 could help in studies to protect mtDNA from oxidative stress.