BIS-INTERCALATION OF HOMODIMERIC THIAZOLE ORANGE DYES IN SELECTIVE BINDING-SITES OF DNA STUDIED BY H-1-NMR SPECTROSCOPY

The thiazole orange dye ydro-1,3-benzothiazol-2-ylidenemethyl)quinolin ium] tetraiodide (TOTO) binds to double stranded DNA (dsDNA) in a sequ ence-selective bis-intercalation. TOTO binds preferentially to oligonu cleotides containing a (5'-CTAG-3')(2) binding site. The preference of TOTO for the (5'-CTAG-3')(2), is caused by a delicate balance between various different binding contributions. In order to examine these co ntributions in further detail we have studied the binding to analogs o f the oligonucleotide 1 using the oligonucleotides d(CGCTAICG)(2) (3) (I = inosine, 2-desaminoguanosine) and d(CGCUAGCG)(2) (4) (U = uridine , 5-desmethylthymidine). In the modified (5'-CTAG-3')(2) binding sites examined in this work the preferential binding of TOTO was maintained to the (5'-CTAI-3')(2) and the (5'-CUAG-3')(2) sequences. However, th e selectivity in the binding to these sites is lower than that of the (5'-CTAG-3')(2) site. These results show that the binding constant is lowered when exchanging a thymidine for a uridine. This implies that t he methyl group on T4 in the (5'-CTAG-3')(2) of the 1-TOTO complex act ually contributes to the binding energy by van der Waals interaction. The lowering of the binding constant on going from the (5'-CTAG-3')(2) to the (5'-CTAI-3')(2) site is less and is not easily explained as du e to a change in a single contribution to the binding energy. It is pr obable that local changes between the conformations of the oligonucleo tides 1 and 3 cause this effect. We have used two-dimensional H-1 NMR spectroscopy to determine the solution structure of the 4-TOTO complex in order to compare it with the solution structure of the 1-TOTO comp lex. The determination of the structure was based on total relaxation matrix analysis of the NOESY cross peak intensities using the program MARDIGRAS. NOE-derived distance restraints were applied in restrained molecular dynamics calculations. Twenty final structures each were gen erated for the TOTO complex from both A-form and B-form dsDNA starting structures.