DNA-Binding Properties of New Fluorescent AzaHx Amides: Methoxypyridylazabenzimidazolepyrroleimidazole/pyrrole.

ABSTRACT

DNA minor groove binding polyamides have been extensively developed to control abnormal gene expression. The establishment of novel, inherently fluorescent 2-(p-anisyl)benzimidazole (Hx) amides has provided an alternative path for studying DNA binding in cells by direct observation of cell localization. Because of the 21 antiparallel stacking homodimer binding mode of these molecules to DNA, modification of Hx amides to 2-(p-anisyl)-4-azabenzimidazole (AzaHx) amides has successfully extended the DNA-recognition repertoire from central CG [recognized by Hx-I (I=N-methylimidazole)] to central GC [recognized by AzaHx-P (P=N-methylpyrrole)] recognition. For potential targeting of two consecutive GG bases, modification of the AzaHx moiety to 2- and 3-pyridyl-aza-benzimidazole (Pyr-AzaHx) moieties was explored. The newly designed molecules are also small-sized, fluorescent amides with the Pyr-AzaHx moiety connected to two conventional five-membered heterocycles. Complementary biophysical methods were performed to investigate the DNA-binding properties of these molecules. The results showed that neither 3-Pyr-AzaHx nor 2-Pyr-AzaHx was able to mimic I-I=N-methylimidazole-N-methylimidazole to target GG dinucleotides specifically. Rather, 3-Pyr-AzaHx was found to function like AzaHx, f-I (f=formamide), or P-I as an antiparallel stacked dimer. 3-Pyr-AzaHx-PI (2) binds 5'-ACGCGT'-3' with improved binding affinity and high sequence specificity in comparison to its parent molecule AzaHx-PI (1). However, 2-Pyr-AzaHx is detrimental to DNA binding because of an unfavorable steric clash upon stacking in the minor groove.