Molecular design of DNA-directed ligands with specific interactions: solution NMR studies of the interaction of a m-hydroxy analogue of Hoechst 33258 with d(CGCGAATTCGCG)2.
We have used one-dimensional (1D) and two-dimensional (2D) proton nuclear magnetic resonance spectroscopy at 600 MHz for structural analysis of the complex formed between d(CGCGAATTCGCG)2 and 2-[2-(3-hydroxyphenyl)-6-benzimidazoyl]-6-(1-methyl-4-piperazinyl) benzimidazole (meta-Hoechst). This analogue differs from Hoechst 33258 only in the location of its meta rather than para phenolic hydroxyl group and was designed to introduce the possibility of intermolecular hydrogen bonding to DNA via the phenol. Complex formation was shown to be 1:1 at 25 degrees C in phosphate buffer in D2O by 1D NMR spectroscopic titration of a solution of d(CGCGAATTCGCG)2 with meta-Hoechst. From 1D NMR spectroscopy the observed perturbations of the assigned chemical shifts of the oligonucleotide observed on binding meta-Hoechst could be used to locate the ligand in the central AATT stretch. By means of 2D NMR spectroscopic techniques, over 400 proton-proton NOEs were defined within the complex. DNA nonexchangeable resonance assignments were made using the sequential assignment method and NOESY. Binding the unsymmetrical ligand lifted the C2v symmetry of the DNA. Exchangeable hydrogens were assigned from NOESY data acquired in 85% H2O/15% D2O medium for the complex and showed differences between the Hoechst 33258 and meta-Hoechst complexes with d(CGCGAATTCGCG)2. The location of meta-Hoechst in the minor-groove AATT region was triangulated using 32 intermolecular NOEs determined for the complex. From the intermolecular NOEs involving the aromatic C-H protons of the phenolic ring of meta-Hoechst, it was clear that this region of the molecule did not rotate freely within the minor groove on the NMR time scale and was oriented with its hydroxyl group toward the floor of the minor groove, in line with the occurrence of the predicted hydrogen bonding between it and the DNA. The pKa of the N3H proton of meta-Hoechst in its bound state in this complex was measured as 6.1 by NMR spectroscopy, a value slightly elevated relative to estimates (approximately 5.2) of the pKa of this proton for the free ligand. Molecular mechanics and the distance restraints provided by the intermolecular NOEs were used in molecular modeling of the meta-Hoechst/d(CGCGAATTCGCG)2 complex, and the distances in the model were consistent with the formation of hydrogen bonds involving the m-OH group of meta-Hoechst and the DNA.
Parkinson
J A
JA
Department of Pharmacy, University of Manchester, U.K.
Ebrahimi
S E
SE
McKie
J H
JH
Douglas
K T
KT
D001483:Base Sequence; D001665:Binding Sites; D006690:Bisbenzimidazole / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D006860:Hydrogen Bonding; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism*; D012996:Solutions; D013696:Temperature; D013816:Thermodynamics
D009838:Oligodeoxyribonucleotides; D012996:Solutions; D004247:DNA; D006690:Bisbenzimidazole