H-1-NMR STUDIES OF THE BIS-INTERCALATION OF A HOMODIMERIC OXAZOLE YELLOW-DYE IN DNA OLIGONUCLEOTIDES

We have used one and two dimensional 1H NMR spectroscopy to characteri ze the binding of a homodimeric oxazole yellow dye, dro-(benzo-1,3-oxa zole)-2-methylidene)-quinolinium tetraiodide (YOYO), to oligonucleotid es containing the (5'-CTAG-3')(2) and the (5'-CCGG-3')(2) binding site s in either different oligonucleotides or in the same oligonucleotide. YOYO bis-intercalates strongly in all the oligonucleotides used and b inds preferentially to a (5'-CTAG-3'), binding site in the oligonucleo tide d(CGCTAGCG)(2) ((1) under bar). YOYO also binds preferentially to a (5'-CCGG-3')(2) sequence in the oligonucleotide d(CGCCGGCG)(2) ((2) under bar) but slightly less favorably than to the (5'-CTAG-3')(2) se quence in (1) under bar. The binding of YOYO to the d(CGCTAGCCGGCG): d (CGCCGGCTAGCG) ((3) under bar) oligonucleotide, containing two prefere ntial binding sites, was also examined. YOYO forms mixtures of 1:1 and 1:2 complexes with oligonucleotide (3) under bar in ratios dependent on the relative amount of YOYO and the oligonucleotides in the sample. The binding of YOYO to the oligonucleotide (3) under bar occur sequen ce selective in the (5'-CTAG-3')(2) site and the (5'- CCGG-3')(2) site . We have also used two dimensional H-1 NMR spectroscopy to determine the solution structure of the DNA oligonucleotide d(5'-CGCTAGCG-3')(2) complexed with YOYO. The determination of the structure was based on a total relaxation matrix analysis of the NOESY cross peaks intensitie s. DQF-COSY spectra were used to obtain coupling constants for the deo xyribose ring protons. The coupling constants were transformed into an gle estimates. The NOE derived distance and dihedral restraints were a pplied in restrained molecular dynamics calculations. Twenty final str uctures each were generated far the YOYO-complex from both A-form and B-form dsDNA starting structures giving a total of 40 final structures . Since many NOE contacts were observed between YOYO and dsDNA the res ulting structure has a fairly high resolution and allows determination of local features in the dsDNA structure after YOYO binding. The root -mean-square (rms) deviation of the coordinates for the forty structur es of the complex was 0.39 Angstrom. The local DNA structure is distor ted in the complex. The helix is unwound by 106 degrees and has an ove rall helical repeat of 13 base pairs caused by the bis-intercalation o f YOYO. The polypropylene amine linker chain is located in the minor g roove of dsDNA. Even though the YOYO chromophore contains an oxygen at om instead of the larger sulphur atom in the corresponding compound, T OTO, the structures establish that YOYO require more space than TOTO i n the intercalation sites. This is probably caused by the more rigid a nd planar chromophores in YOYO compared to TOTO.