The effects of PNA (peptide nucleic acid) bound to double-stranded (ds) DNA targets positioned downstream from phage T3 or T7 promoters in pBluescriptKS+ derived plasmids on transcription by RNA polymerases T3 or T7 have been studied. The dsDNA targets A10, 5'-A5GA4 or 5'-A2GA2GA4, and the corresponding PNAs T10, T5CT4 and T2CT2CT4 were used and the target-PNA strand displacement complexes were performed in low-salt buffer, since PNA does not bind efficiently to ds DNA in higher salt than 50 mM. It is shown that transcription elongation is arrested at the target site with PNA bound to the template strand, whereas only a marginal effect is observed with PNA bound to the non-template strand. With PNA T10, transcription arrest occurs at the first base of the PNA-binding site, while the arrest with the PNA T5CT4 takes place 2-3 nt inside the PNA binding site. In the case of PNA T2CT2CT4 the arrest is less efficient and occurs at the last 1-3 nt of the binding site. Transcription arrest was also shown for PNAs T6 and T8, although with a much lower efficiency. These results show that efficient transcription elongation arrest can be obtained by PNA targeting of the template DNA strand.