Pyrrole (P) and imidazole (I) containing N-formamido-triheterocylic polyamides capable of binding in the minor-groove of DNA can regulate gene expression and potentially treat a variety of diseases. Recent work has shown that the order of heterocycles in the central or 'core' region of these polyamides, where the partnering heterocycles are stacked, plays a significant role in the binding affinity that these compounds exhibit when interacting with their cognate DNA sequences. In order to extend the size of base-pair recognition that these stacked heterocycle pairs can “read”, 18 tetraamide polyamides (each containing an N-terminal pyrrole or N-terminal imidazole moiety and a C-terminal dimethylamino functionality) were synthesized. The DNA binding properties of these compounds were investigated using molecular biology and biophysical chemistry methods. DNaseI footprinting of each compound with its cognate DNA sequence revealed that the 'core' arrangement of heterocycles was an influential factor in the compounds' ability to bind DNA. The order established for the N-formamido-triheterocyles (IP>PP>>PI~II) appears to be followed in the tetraamide series although overall binding affinity appears lower. Biophysical analyses (Tm, CD, ITC and SPR) are underway and initial results indicate that PIPI is a superior binder than the other tetraamides and has increased selectivity compared to the other compounds in the series. This compound is also more sequence selective than f-IPI (the best triheterocyclic polyamide); however, the binding affinity is reduced. More in-depth biophysical studies will determine if a broader pattern of binding exists with these compounds.