MOLECULAR DESIGN OF DNA-DIRECTED LIGANDS WITH SPECIFIC INTERACTIONS -SOLUTION NMR-STUDIES OF THE INTERACTION OF A M-HYDROXY ANALOG OF HOECHST-33258 WITH D(CGCGAATTCGCG)(2)

We have used one-dimensional (1D) and two-dimensional (2D) proton nucl ear magnetic resonance spectroscopy at 600 MHz for structural analysis of the complex formed between d(CGCGAATTCGCG)(2) and -methyl-4-pipera zinyl)benzimidazole(meta-Hoechst). This analogue differs from Hoechst 33258 only in the location of its meta rather than para phenolic hydro xyl group and was designed to introduce the possibility of intermolecu lar hydrogen bonding to PNA via the phenol. Complex formation was show n to be 1:1 at 25 degrees C in phosphate buffer in D2O by 1D NMR spect roscopic titration of a solution of d(CGCGAATTCGCG)(2) with meta-Hoech st. From 1D NMR spectroscopy the observed perturbations of the assigne d chemical shifts of the oligonucleotide observed on binding meta-Hoec hst 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 assign ments were made using the sequential assignment method and NOESY. Bind ing the unsymmetrical ligand lifted the C-2 upsilon symmetry of the DN A. Exchangeable hydrogens were assigned from NOESY data acquired in 85 % H2O/15% D2O medium for the complex and showed differences between th e Hoechst 33258 and meta-Hoechst complexes with d(CGCGAATTCGCG)(2). Th e location of meta-Hoechst in the minor-groove AATT region was triangu lated using 32 intermolecular NOEs determined for the complex. From th e intermolecular NOEs involving the aromatic C-H protons of the phenol ic 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 a nd 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 pK(a) of the N3H proton of meta-Hoechst in its bound state in this complex was measured as 6.1 by NMR spectrosco py, a value slightly elevated relative to estimates (similar to 5.2) o f the pK(a) of this proton for the free ligand. Molecular mechanics an d the distance restraints provided by the intermolecular NOEs were use d 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.