DNA-binding properties of new fluorescent AzaHx-amides: Methoxy-pyridyl-aza-benzimidazole-pyrrole-imidazole/pyrrole

摘要

DNA minor groove binding polyamides have been extensively developed for use as a molecular gene surgery tool to control abnormal gene expression. Establishment of the novel, inherently fluorescent Hx-amides has provided an alternative path for studying DNA binding in cells by direct observation of cell localization. Because of the 2:1 antiparallel stacking homodimer binding mode of these molecules to DNA, modification of Hx-amides to AzaHx-amides has successfully extended the DNA recognition repertoire, from central CG (recognized by Hx-I) to central GC (recognized by AzaHx-P) recognition. In order to potentially target two consecutive GG bases, new modifications from AzaHx to 3-Pyr-AzaHx and 2-Pyr-AzaHx moiety, were developed. The newly designed molecules are also small-sized, fluorescent amides with Pyr-AzaHx connected to two conventional five-member heterocycles. Complementary biophysical methods such as thermal melting, DNase I footprinting, surface plasmon resonance (SPR) and circular dichroism were carried out to investigate their DNA binding properties. The results showed that neither 3-Pyr-AzaHx nor 2-Pyr-AzaHx was able to mimic I-I to specifically target GG. Rather, 3-Pyr-AzaHx functions like AzaHx or f-I or P-I as antiparallel stacked dimer. 3-Pyr-AzaHx-PI (>2) binds 5’-ACGCGT’−3’ with higher binding affinity and high sequence specificity when compared 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.