| Abstract
| - DNA minor groove ligands provide a paradigm for double-stranded DNA recognition, wherecommon structural motifs provide a crescent shape that matches the helix turn. Since minor groove ligandsare useful in medicine, new ligands with improved binding properties based on the structural informationabout DNA−ligand complexes could be useful in developing new drugs. Here, two new synthetic analoguesof AT specific Hoechst 33258 5-(4-methylpiperazin-1-yl)-2-[2‘-(3,4-dimethoxyphenyl)-5‘-benzimidazolyl]benzimidazole (DMA) and 5-(4-methylpiperazin-1-yl)-2-[2‘{2‘ ‘-(4-hydroxy-3-methoxyphenyl)-5‘ ‘-benzimidazolyl}-5‘-benzimidazolyl] benzimidazole (TBZ) were evaluated for their DNA binding properties.Both analogues are bisubstituted on the phenyl ring. DMA contains two ortho positioned methoxy groups,and TBZ contains a phenolic group at C-4 and a methoxy group at C-3. Fluorescence yield upon DNAbinding increased 100-fold for TBZ and 16-fold for DMA. Like the parent compound, the new ligandsshowed low affinity to GC-rich (K ≈ 4 × 107 M-1) relative to AT-rich sequences (K ≈ 5 × 108 M-1),and fluorescence lifetime and anisotropy studies suggest two distinct DNA−ligand complexes. Bindingstudies indicate expanded sequence recognition for TBZ (8−10 AT base pairs) and tighter binding (ΔTmof 23 °C for d (GA5T5C). Finally, EMSA and equilibrium binding titration studies indicate that TBZpreferentially binds highly hydrated duplex domains with altered A-tract conformations d (GA4T4C)2 (K= 3.55 × 109 M-1) and alters its structure over d (GT4A4C)2 (K = 3.3 × 108 M-1) sequences. AlteredDNA structure and higher fluorescence output for the bound fluorophore are consistent with adaptivebinding and a constrained final complex. Therefore, the new ligands provide increased sequence andstructure selective recognition and enhanced fluorescence upon minor groove binding, features that canbe useful for further development as probes for chromatin structure stability.
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