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17 The binding site was defined as the external 3′ G-quartet plane. Initially 200 ligand orientations were randomly centred on the G-quadruplex structure, charges were not considered and the complexes were minimized for 500 steps. The 75 lowest energy structures were then subjected to simulated annealing, then 500 steps of minimization were followed by molecular dynamics over a period of 10 ps. The energies of the resulting structures were then minimized.
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24 was purchased from Pierce, as a 1 mM solution in MES buffer.
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39
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33947721056
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note
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27 or DMSO. Samples were left equilibrating for half an hour before starting the experiment.
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44
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Radio-labelled TSG4 oligonucleotide (5′-GGGATTGGGATTGGGATTGGGTT-3′) was heated at 95 °C for 10 min and quickly cooled in ice, at a concentration of 12 μM. It was then incubated for 2 h at 30 °C in MES-KCl buffer (10 mM MES, pH 6.5, and 5 mM KCl) in the presence of different drug concentrations and with no drug. Complexes and structures formed after incubation were studied by native PAGE (15% polyacrylamide gel, TBE 0.5×, KCl 20 mM, run overnight at room temperature). In all electrophoresis runs, the G-quadruplex induction by PIPER (20 μM) was also reported as a useful standard to assign the electrophoresis bands to different DNA conformations. Percentage of ss-DNA represents the ratio between the intensity of the relative band on the electrophoresis gel and the total amount of DNA, obtained by Instant Imager (Packard).
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48
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33947731226
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