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Mitochondria-Directed Fluorescent Probe for the Detection of Hydrogen Peroxide near Mitochondrial DNA
Ying Wen1, Keyin Liu1, Huiran Yang1
1Department of Chemistry and Collaborative Innovation Center of Chemistry for Energy Materials, Fudan University , Shanghai 200433, China.
Abstract:
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.
Insights
Researchers developed pep3-NP1, a novel mitochondria-targeted probe, to detect hydrogen peroxide (H2O2) near mitochondrial DNA (mtDNA). This tool enables precise monitoring of oxidative stress in mitochondria, crucial for protecting mtDNA.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Imaging
Background:
- Mitochondrial DNA (mtDNA) is highly susceptible to oxidative damage compared to nuclear DNA (nDNA).
- Hydrogen peroxide (H2O2) is a key reactive oxygen species implicated in cellular damage.
- Accurate detection of H2O2 near mtDNA is essential for understanding and preventing mtDNA damage.
Purpose of the Study:
- To design and synthesize a mitochondria-targeted fluorescence probe for detecting H2O2 specifically around mtDNA.
- To evaluate the probe's ability to localize within mitochondria and bind to mtDNA.
- To establish the probe's sensitivity and utility for real-time H2O2 monitoring in live cells.
Main Methods:
- Synthesis of pep3-NP1, a probe incorporating a DNA-binding peptide, H2O2 reporter, and a mitochondria-targeting styryl dye.
- Confocal microscopy to assess subcellular localization of pep3-NP1 using MitoTracker as a control.
- Fluorescence spectroscopy to analyze the probe's response to H2O2, including changes in emission spectra and quantum yield.
- Determination of H2O2 detection limits in vitro and in cultured cells.
Main Results:
- Pep3-NP1 successfully accumulated in the mitochondria of live cells, co-localizing with mtDNA.
- The probe exhibited a 'turn-on' fluorescence response upon binding to DNA, with increased red fluorescence.
- In the presence of H2O2, pep3-NP1 emitted green fluorescence, allowing for ratiometric detection.
- The probe demonstrated detection limits of 2.9 μM in vitro and 5.0 μM in cultured cells for H2O2.
Conclusions:
- Pep3-NP1 is an effective mitochondria-targeted probe for labeling mtDNA and detecting localized H2O2.
- The probe's ratiometric fluorescence response provides a reliable method for quantifying H2O2 levels near mtDNA.
- This development offers a valuable tool for studying oxidative stress and protecting mtDNA from damage.
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