The aberrant expression of genes has been associated with a wide range of diseases. The transcription factors (TFs) implicated in the misregulation of genes are prime targets of drug therapeutics. However, targeting TFs has been especially challenging due to the lack of targeting sites. An alternative approach is targeting their cognate sequences. In that respect, small molecules that can bind specific DNA sequences to function as potent TF inhibitors are paramount in targeted gene therapies and biotechnology. Diamidine compounds can bind selectively to AT base pairs in the minor groove of AT-rich DNA sequences. Multiple reports have demonstrated that some diamidines are excellent inhibitors of TFs. Thiophene-benzimidazole-phenyl diamidine and its analog, thiophene-indole-phenyl diamidine, bind the minor groove of d(CGCGAATTCGCG)₂ in a strong and selective manner with a K_D = 1.5 nM and 6.5 nM, respectively. Another AT-specific binder, benzimidazole-diphenyl diamidine, binds similarly to the thiophene-based diamidines with a K_D = 1.0 nM. A combination of high-resolution biophysical methods, including but not limited to X-ray crystallography, are used to describe their binding affinities, TF inhibition properties, structures, mechanism of AT recognition, and the analysis of the structural properties of their complexes with B-DNA.