Direct evidence of mitochondrial G-quadruplex DNA by using fluorescent anti-cancer agents.
Bottom Line: In this paper, we provide the first evidence supporting the presence of G4 in the mitochondrial DNA (mtDNA) of live cells.In this study, we use fluorescence lifetime imaging microscopy to verify the existence of mtDNA G4s in live cells.Bioactivity studies indicate that interactions between these anti-cancer agents and mtDNA G4 can suppress mitochondrial gene expression.
Affiliation: Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei 10617, Taiwan.Show MeSH
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Mentions: In this work, we first demonstrated the effectiveness of FLIM as a tool for the visualization of G4 localization in live cells. The main target of o-BMVC is the mitochondria of CL1–0 cancer cells; therefore, we used lipofectamine for the delivery of o-BMVC into the nucleus (27). We applied a discrete time model to divide the image into two temporal regions, which made it possible to detect fluorescent spots with a longer decay time (≥2.4 ns) in the nucleus of live cells (Figure 1A). At the same time, fluorescent spots with an extended decay time (≥3.0 ns) were rarely observed in the nucleus of live cells (Figure 1B). It should be noted that a large number of fluorescent spots with extended decay time (≥3.0 ns) could be clearly observed in the nucleus of live cells, when lipofectamine was used to deliver o-BMVC and ssDNA of thrombin binding aptamer (TBA) (23) simultaneously (Figure 1C). In addition, DAPI was used to stain the cell nuclei to confirm the delivery of TBA into the nucleus by lipofectamine (Figure 1D). Figure 1E shows the overlay between Figure 1C and D. This finding unambiguously demonstrates that the bright fluorescent spots shown in Figure 1C can be attributed to the presence of exogenous TBA G4s in the nucleus of live cells. This conclusion is based on the fact that the fluorescence decay time of o-BMVC is ∼4.0 ns upon interacting with TBA G4s in vitro (23). Moreover, the structural conversion of TBA from ssDNA to G4 in the presence of potassium cations in the nucleus further supports the formation of endogenous G4 in live cells (Figure 1A).
Affiliation: Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei 10617, Taiwan.