Sequence Specificity of Dimeric Bisbenzimidazoles for DNA AT Sequences of Different Nucleotide Compositions, as Determined by Footprinting

Abstract

Objective: The study aimed to investigate the site-specificity of binding to DNA of three series of minor groove ligands – dimeric bisbenzimidazoles DB₂(n), DB₂P(n), and DB₂Py(n) – using DNAase I footprinting. The compounds consist of two bisbenzimidazole units linked by oligomethylene linkers of varying lengths (n), with structural modifications to enhance DNA-binding properties. Methods: The binding specificity of the compounds was determined using DNAase I footprinting. The DB₂(n) and DB₂P(n) series are analogs of Hoechst 33342, modified by removing hydrophobic ethoxyphenol cores and introducing hydrophilic aminomethylene groups. The DB₂Py(n) series incorporates a pyrrolcarboxamide group, a structural unit of the AT-specific antibiotic netropsin. The interaction of these compounds with DNA sequences was analyzed to identify their binding preferences. Results and Discussion: All studied compounds demonstrated specificity for AT-rich DNA sequences. The DB₂P(n) and DB₂(n) series exhibited increased affinity for (AATT)₃ and TTTT sequences. The DB₂Py(n) series showed high specificity to AT-rich regions, with a preference for the TTTT motif. None of the compounds interacted with sequences containing fewer than four AT base pairs. These findings highlight the influence of structural modifications on DNA-binding specificity and affinity. Conclusions: The study revealed that dimeric bisbenzimidazoles DB₂(n), DB₂P(n), and DB₂Py(n) exhibit distinct binding preferences for AT-rich DNA sequences, with DB₂Py(n) showing a pronounced affinity for the TTTT motif. The results demonstrate the potential of these compounds as tools for targeting specific DNA sequences, with implications for molecular biology and drug design.