The binding of peptide nucleic acids (PNAs) T10-LysNH2, T5CT4-LysNH2 and T2CT2CT4-LysNH2 to double-stranded DNA targets A10, A5GA4 and A2GA2GA4 was studied by nuclease S1 probing. It is found that the PNAs bind preferentially to their complementary targets, weaker to targets containing one mismatch and not to targets containing two mismatches. Using an RNA polymerase T3 in vitro transcription system, it is found that a PNA T10-LysNH2 bound downstream from the promoter causes transcription elongation arrest at the PNA binding site only when the PNA is bound to the template strand. Finally, it is shown that primer extension by Taq DNA polymerase on a single-stranded template is arrested at an occupied PNA T10 binding site. These results are discussed in relation to PNAs as potential anti-sense and anti-gene drugs.