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The decoy approach against nuclear factor κB (NF-κB) is a useful tool to alter NF-κB dependent gene expression using synthetic oligonucleotides (ODNs) carrying NF-κB specific cis-elements. Unfortunately, ODNs are not stable and need to be be extensively modified to be used in vivo or ex vivo. We have previously evaluated the possible use of peptide nucleic acids (PNAs) as decoy molecules. The backbone of PNAs is composed of N-(2-aminoethyl)glycine units, rendering these molecules resistant to both nucleases and proteases. We found that the binding of NF-κB transcription factors to PNAs was either very low (binding to PNA–PNA hybrids) or exhibited low stability (binding to PNA–DNA hybrids). The main consideration of the present paper was to determine whether PNA–DNA chimeras mimicking NF-κB binding sites are capable of stable interactions with proteins belonging to the NF-κB family. Molecular modeling was employed for the design of PNA–DNA chimeras; prediction of molecular interactions between chimeras and NF-κB nuclear proteins were investigated by molecular dynamics simulations, and interactions between PNA–DNA chimeras and NF-κB proteins were studied by gel shifts. We found significant differences between the structure of duplex NF-κB PNA–DNA chimera and duplex NF-κB DNA–DNA. However, it was found that these differences do not prevent the duplex PNA–DNA chimera from binding to NF-κB transcription factors, being able to suppress the molecular interactions between HIV-1 LTR and p50, p52 and nuclear factors from B-lymphoid cells. Therefore, these results demonstrate that the designed NF-κB DNA–PNA chimeras could be used for a decoy approach in gene therapy. [ABSTRACT FROM AUTHOR]