No abstract

Minor-groove binding hairpin polyamides (PAs) bind specific DNA sequences. Synthetic modifications can improve PA-DNA binding affinity and include flexible modules, such as β-alanine (β) motifs to replace pyrroles (Py), and increasing compound charge using N-terminal cationic substituents. To better understand the variations in kinetics and affinities caused by these modifications on PA-DNA interactions, a comprehensive set of PAs with different numbers and positions of β and different types of N-cationic groups was systematically designed and synthesized to bind their cognate sequence, the λB motif. The λB motif is also a strong binding promoter site of the major groove targeting transcription factor PU.1. The PA binding affinities and kinetics were evaluated using a spectrum of powerful biophysical methods: thermal melting, biosensor surface plasmon resonance and circular dichroism. The results show that β inserts affect PA-DNA interactions in a number and position dependent manner. Specifically, a β replacement between two imidazole heterocycles (ImβIm) generally strengthens binding. In addition, N-terminal cationic groups can accelerate the association between PA and DNA, but the bulky size of TMG can cause steric hindrance and unfavourable repulsive electrostatic interactions in some PAs. The future design of stronger binding PA requires careful combination of βs and cationic substituents.

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 2:1 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.