Computational protocol: The crystal structure of human telomeric DNA complexed with berberine: an interesting case of stacked ligand to G-tetrad ratio higher than 1:1

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Protocol publication

[…] The d[TAG3(T2AG3)3] sequence was purchased from Jena Bioscience (Jena, Germany), HPLC purified berberine chloride was purchased from Sigma-Aldrich Co. Ltd., USA. The purified oligonucleotide was dissolved to a final concentration of 4 mM, in a solution containing 20 mM potassium cacodylate buffer at pH 6.5 and 50 mM potassium chloride, and then heated to 368 K for 5 min. It was subsequently allowed to cool overnight to room temperature to induce the quadruplex formation.A search for crystallization conditions was performed using a home-made crystallization screen based on the one described by Campbell and Parkinson (). The DNA quadruplex–drug complex was crystallized at 296 K using the sitting drop vapour diffusion method. One microliter of the 4 mM solution of the human telomeric sequence d[TAG3(T2AG3)3] was mixed with 1 µL of a 2 mM solution of berberine in water, and with 2 µL of a solution containing the following: 1.8 M ammonium sulphate, 0.2 M KCl, 0.05 M Li2SO4 and 0.05 M potassium cacodylate with pH = 6.5. Drops were equilibrated against this latter solution. Yellow hexagonal crystals of the complex appeared after 2 weeks.Data collection on crystals of the quadruplex–drug complex was performed at 100 K, using as cryoprotectant a solution analogous to the crystallization solution but with an increased concentration of ammonium sulphate (1.9 M) and 30% glycerol. The berberine–quadruplex complex crystallizes in the space group P6, with unit cell parameters a = 65.620 Å and c = 41.81 Å.Data for the complex were collected at the X12 beamline (EMBL, DESY, Hamburg) to a maximum resolution of 2.4 Å, using a wavelength of 1.000 Å. Data were integrated using the program mosflm (,) (and scaled with SCALA from the CCP4 program package (). Unit cell parameters and data collection statistics are given in . The structure of the complex was solved by molecular replacement using the program Molrep (). Coordinates of the complex of the same DNA sequence with a tetra-substituted naphthalene diimide () (PDB code: 3CDM) were used as a search model, after deleting atoms belonging to ligand and solvent molecules.Fo-Fc electron density maps revealed a clear density for the drug molecules and they were introduced and refined at full occupancy. Two spherical densities were also present in the Fo-Fc electron density maps inside the G-quadruplex channel that could be interpreted as potassium ions. The model was refined using the program Refmac5 () from the CCP4 package (). Manual rebuilding of the model was performed using the program Coot (). The crystal packing analysis was performed with the program Mercury (). Coordinates of the complex have been deposited with the Protein Data Bank (PDB access number 3R6R). […]

Pipeline specifications

Software tools iMosflm, CCP4, Molrep, REFMAC5, Coot
Applications Small-angle scattering, Protein structure analysis
Organisms Dipturus trachyderma, Homo sapiens
Chemicals Berberine