1H NMR and optical spectroscopic investigation of the sequence-dependent dimerization of a symmetrical cyanine dye in the DNA minor groove

A symmetrical cyanine dye was previously shown to bind as a cofacial dimer to alternating A-T sequences of duplex DNA. Indirect evidence suggested that dimerization of the dye occurred in the minor groove. H-1 NMR experiments reported here verify this model based on broadening and shifting of signals due to protons on carbon 2 of adenine and imino protons at the central five A-T pairs of the 11 base pair duplex: 5'-GCGTATATGCG-3'/3'-CGCATATACGC-5'. This binding mode is similar to that of distamycin A, even though the dye lacks the hydrogen-bonding groups used by distamycin for sequence-specific recognition. Surprisingly, the third base pair (G-C) was also implicated in the binding site. UV-vis experiments were used to compare the extent of dimerization of the dye for 11 different sequence variants. These experiments verified the importance of a G-C pair at the third position: replacing this pair with A-T suppressed dimerization. These results indicate that the dye binding site spans six base pairs: 5'-GTATAT-3'. The initial G-C pair seems to be important for widening the minor groove rather than for making important contacts with the dye molecules since inverting its orientation to C-G or replacing it with I-C still led to favorable dimerization of the dye.