Orientation preferences of hairpin pyrrole-imidazole polyamides toward CGG site.

Hairpin pyrrole-imidazole (Py-Im) polyamides are promising medium-sized molecules that bind sequence-specifically to the minor groove of B-form DNA. Here, we synthesized a series of hairpin Py-Im polyamides and explored their binding affinities and orientation preferences to methylated DNA with the CGG target sequence. Thermal denaturation assays revealed that the five hairpin Py-Im polyamides, which were anticipated to recognize CGG in a forward orientation, bind to nontarget DNA, GGC, in a reverse orientation. Therefore, we designed five Py-Im polyamides that could recognize CGG in a reverse orientation. We found that the two Py-Im polyamides containing Im/β pairs preferentially bound to CGG in a reverse orientation. The reverse binding Py-Im polyamide successfully inhibited TET1 binding on the methylated DNA. Taken together, this study illustrated the importance of designing reverse binding Py-Im polyamides for the target sequence, CGG, which paved the way for Py-Im polyamides that can be used with otherwise difficult to access DNA with CG sequences.