Solution structure of a DNA complex with the fluorescent bis-intercalator TOTO modified on the benzothiazole ring

We have used two-dimensional 1H NMR spectroscopy to determine the solution structure of the DNA oligonucleotide d(5'-CGCTAGCG-3')2 complexed with the bis-intercalating dye 1,1'-(4,4,8,8-tetramethyl-4, 8-diazaundecamethylene)bis-4-[3-ethyl-2,3-dihydro(benzo-1, 3-thiazolyl)-2-methylidene]quinolinium tetraiodide (TOTOEt). The determination of the structure was based on a total relaxation matrix analysis of the NOESY cross-peak intensities. DQF-COSY spectra were used to obtain coupling constants for the deoxyribose ring protons. The coupling constants were transformed into angle estimates. The NOE-derived distance and dihedral restraints were applied in restrained molecular dynamics calculations. Twenty final structures each were generated for the TOTOEt complex from both A-form and B-form double-stranded (ds) DNA starting structures, giving a total of 40 final structures. Since many NOE contacts were observed between TOTOEt and dsDNA, the resulting structure has a fairly high resolution and allows determination of local features in the dsDNA structure after TOTOEt binding. The root-mean-square (rms) deviation of the coordinates for the 40 structures of the complex was 0.52 A. The local DNA structure is distorted in the complex. The helix is unwound by 80 degrees and has an overall helical repeat of 12 base pairs, caused by bis-intercalation of TOTOEt. The benzothiazole ring system is twisted relative to the quinoline in the uncomplexed TOTOEt molecule. The site selectivity of TOTOEt for the CTAG.CTAG site is explained by its ability to adapt to the base pair propeller twist of dsDNA to optimize stacking and the hydrophobic interaction between the thymidine methyl group and the benzothiazole ring. The polypropylene amine linker chain is located in the minor groove of dsDNA. The N-ethyl group on the benzothiazole of TOTOEt is placed in the major groove pointing toward the center of the oligonucleotide and the dyad symmetry axis of the complex. This orientation seems to make it feasible to create a TOTO analogue with a linker connecting the two chromophores in the major groove. The design of such an analogue and a macrocyclic analogue with a linker in both the major groove and the minor groove seems to be straightforward.