Direct evidence of mitochondrial G-quadruplex DNA by using fluorescent anti-cancer agents

G-quadruplex (G4) is a promising target for anti-cancer treatment. In this paper, we provide the first evidence supporting the presence of G4 in the mitochondrial DNA (mtDNA) of live cells. The molecular engineering of a fluorescent G4 ligand, 3,6-bis(1-methyl-4-vinylpyridinium) carbazole diiodide (BMVC), can change its major cellular localization from the nucleus to the mitochondria in cancer cells, while remaining primarily in the cytoplasm of normal cells. A number of BMVC derivatives with sufficient mitochondrial uptake can induce cancer cell death without damaging normal cells. Fluorescence studies of these anti-cancer agents in live cells and in isolated mitochondria from HeLa cells have demonstrated that their major target is mtDNA. 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. This work underlines the importance of fluorescence in the monitoring of drug-target interactions in cells and illustrates the emerging development of drugs in which mtDNA G4 is the primary target.


Absorption and fluorescence
Absorption spectra were taken on a ultraviolet-visible spectrophotometer (HELIOS α, Thermo Fisher Scientific, USA), and fluorescence spectra were recorded on a spectrofluorometer (LS-55, PerkinElmer, USA) with a 2 nm band-pass in a 1 cm cell length of quartz cuvette at room temperature.

Circular dichroism
The CD spectra were averaged 10 scans on a J-815 spectropolarimeter (Jasco, Japan), with a 2 nm bandwidth at 50 nm/min scan speed, and a 0.2 nm step resolution. The CD spectra were measured under N2 over the range of 210-350 nm to ascertain the G4 10 structures. The melting curves were recorded at a set temperature controlled by a peltier thermal coupler chamber (PFD-425S/15, Jasco, Japan).

NMR spectroscopy
Experiments were performed using Bruker AVIII 800 MHz spectrometer equipped with a cryoprobe at 25 °C. The one-dimensional imino proton NMR spectra were measured in H2O/D2O (90%/10%) using a WATERGATE pulsed sequence for solvent suppression. The DNA samples were prepared at a strand concentration of 100 μM and specific salt condition with an internal reference of DSS (0.1 mM sodium 4,4-dimethyl-4-silapentane-1-sulfonate).

Polyacrylamide gel electrophoresis (PAGE)
The PAGE was conducted using 20% Polyacrylamide gels. Electrophoresis of the gels was carried out at 25 mA for 3 h at 4 o C. They were then photographed under ultraviolet light at 254 nm using a digital camera.

Cyclosporin A (CsA) test
For this experiment, HeLa cells were seeded in 12-well plated with coverslip incubated with 1 μM Cyclosporin A (CsA, Sigma, USA) for 24 h to block the mitochondria inner membrane permeability transition pore. After discarded medium, 5 μM BMVC-12C-P was added to incubate with cells for 4 h. Finally, cells were stained with Mitotracker Red and observed by Leica TC5 SP8 confocal microscopy.

Figure S1
Absorption and fluorescence of 12 μM BMVC-12C-P and its complexes with 6 μM each of LD12, calf thymus, mt6363, mt9438, mt8095, mt1015, and mt16250 in 150 mM K + solution. The absorption maximum of BMVC-12C-P at ~450 nm is red-shifted to ~465 nm upon interaction with duplex DNA and further red-shifted to ~475 nm in the presence of G4s. Of importance is that the fluorescence of BMVC-12C-P increases ~70 times upon interaction with duplex DNA and 20-50 times in the presence of G4s.

Figure S2
The mean fluorescence intensity of 1 μM BMVC-12C-P incubated with various cancer and normal cell lines for 24 h measured by flow cytometry. The CD spectra of 4 μM mt377, mt714, mt1015, mt8095, mt10252, mt12086, mt15653, mt16250 (a-h) annealed in 10 mM Tris buffer, after the addition of 150 mM K + for 1 h, and overnight.

Figure S7
Aromatic and methyl NMR spectra of 100 μM mt6363 annealed in 10 mM Tris buffer, after the addition of 150 mM K + for 1 h, and overnight. The histograms of the fluorescence decay time of 0.2 μM o-BMVC-12C-P upon interaction with mt1015 G4, mt9438 G4, HT23 G4, and LD12 duplex DNA.

Figure S11
RT-PCR was used to evaluate the suppression of ND3 and COX I expression by TMPyP4. HeLa cells were incubated with TMPyP4 for 3 d. Here β-actin and a non-G4 forming gene sequence were used as controls.
23 Table S1 Primers and sequences of mitochondria DNA studied in RT-PCR and PCR stop assay.