Physical and structural basis for the strong interactions of the -ImPy- central pairing motif in the polyamide f-ImPyIm

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 center dot T) CGCG( A center dot T) over ( G center dot C)CGCG( G center dot 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 ( Delta Cp) contribute differently to formation of the 2:1 complexes for each triamide. Experimental and theoretical determinations of Delta Cp for binding of f- ImPyIm to CGCG were in good agreement (- 142 and - 177 cal mol(-1) K-1, respectively). H-1 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.