The potential pharmacologic benefits of using peptide nucleic acid (PNA) as an antisense agent are tempered by its incapacity to activate RNase H. The mixed backbone oligonucleotide (ON) (or gapmer) approach, in which a short internal window of RNAse H-competent residues is embedded within an RNase H-incompetent ON has not been applied previously to PNA because PNA and DNA hybridize to RNA with very different helical structures, creating structural perturbations at the two PNA-DNA junctions. It is demonstrated here for the first time that a short internal phosphodiester window within a PNA is sufficient to evoke the RNase H-dependent cleavage of a targeted RNA and to abrogate translation elongation in a well-characterized in vitro assay.