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Understanding the dynamic interactions of ligands to DNA is important in DNA-based nanotechnologies. By structurally tracking the dissociation of Hoechst 33258-bound DNA (d(CGCAAATTTGCG) 2 ) complex ( H-DNA ) with T-jump 2D-IR spectroscopy, the ligand is found to strongly disturb the stability of the three C:G base pairs adjacent to A:T the binding site, with the broken base pairs being more than triple at 100 ns. The strong stabilization effect of the ligand on DNA duplex makes this observation quite striking, which dramatically increases the melting temperature and dissociation time. MD simulations demonstrate an important role of hydration water and counter cations in maintaining the separation of terminal base pairs. The hydrogen bonds between the ligand and thymine carbonyls are crucial in stabilizing  H-DNA , whose breaking signal appearing prior to the complete dissociation. Thermodynamic analysis informs us that  H-DNA  association is a concerted process, where  H  cooperates with DNA single strands in forming  H-DNA .

DNA minor-groove binder Hoechst 33258 destabilizes base-pairing adjacent to its binding site