The polyamide f-ImPyIm has a higher affinity for its cognate DNA than either the parent analogue, distamycin A (10-fold), or the structural isomer, f-PyImIm (250-fold), has for its respective cognate DNA sequence. These findings have led to the formulation of a two-letter polyamide language in which the -ImPy- central pairings associate more strongly with Watson-Crick DNA than -PyPy-, -PyIm-, and -ImIm-. Herein, we further characterize f-ImPyIm and f-PyImIm, and we report thermodynamic and structural differences between -ImPy- (f-ImPyIm) and -PyIm- (f-PyImIm) central pairings. DNase I footprinting studies confirmed that f-ImPyIm is a stronger binder than distamycin A and f-PyImIm and that f-ImPyIm preferentially binds CGCG over multiple competing sequences. The difference in the binding of f-ImPyIm and f-PyImIm to their cognate sequences was supported by the Na(+)-dependent nature of DNA melting studies, in which significantly higher Na(+) concentrations were needed to match the ability of f-ImPyIm to stabilize CGCG with that of f-PyImIm stabilizing CCGG. The selectivity of f-ImPyIm beyond the four-base CGCG recognition site was tested by circular dichroism and isothermal titration microcalorimetry, which shows that f-ImPyIm has marginal selectivity for (A.T)CGCG(A.T) over (G.C)CGCG(G.C). In addition, changes adjacent to this 6 bp binding site do not affect f-ImPyIm affinity. Calorimetric studies revealed that binding of f-ImPyIm, f-PyImIm, and distamycin A to their respective hairpin cognate sequences is exothermic; however, changes in enthalpy, entropy, and heat capacity (DeltaC(p)) contribute differently to formation of the 2:1 complexes for each triamide. Experimental and theoretical determinations of DeltaC(p) for binding of f-ImPyIm to CGCG were in good agreement (-142 and -177 cal mol(-)(1) K(-)(1), respectively). (1)H NMR of f-ImPyIm and f-PyImIm complexed with their respective cognate DNAs confirmed positively cooperative formation of distinct 2:1 complexes. The NMR results also showed that these triamides bind in the DNA minor groove and that the oligonucleotide retains the B-form conformation. Using minimal distance restraints from the NMR experiments, molecular modeling and dynamics were used to illustrate the structural complementarity between f-ImPyIm and CGCG. Collectively, the NMR and ITC experiments show that formation of the 2:1 f-ImPyIm-CGCG complex achieves a structure more ordered and more thermodynamically favored than the structure of the 2:1 f-PyImIm-CCGG complex.