The binding of PNA (peptide nucleic acid) T2CT2CT4-LysNH2 to the double-stranded DNA target 5'-A2GA2GA4 was studied by KMnO4 and dimethylsulfate (DMS) probing. It is found that upon sequence-specific strand displacement binding of the PNA to the dsDNA target concomitant protection of the N-7 of guanines within the target takes place. It is furthermore shown that the binding of this PNA is more efficient at pH 5.5 than at pH 6.5 and very inefficient at pH 7.5. These results clearly indicate that C+G Hoogsteen base pairing is present and important for binding and that the strand displacement complex therefore involves a PNA.DNA-PNA triplex.