Interaction of the human NF-kappaB p52 transcription factor with DNA-PNA hybrids mimicking the NF-kappaB binding sites of the human immunodeficiency virus type 1 promoter.
We determined whether peptide nucleic acids (PNAs) are able to interact with NF-kappaB p52 transcription factor. The binding of NF-kappaB p52 to DNA-DNA, DNA-PNA, PNA-DNA, and PNA-PNA hybrid molecules carrying the NF-kappaB binding sites of human immunodeficiency type 1 long terminal repeat was studied by (i) biospecific interaction analysis (BIA) using surface plasmon resonance technology, (ii) electrophoretic mobility shift, (iii) DNase I footprinting, and (iv) UV cross-linking assays. Our results demonstrate that NF-kappaB p52 does not efficiently bind to PNA-PNA hybrids. However, a DNA-PNA hybrid molecule was found to be recognized by NF-kappaB p52, although the molecular complexes generated exhibited low stability. From the theoretical point of view, our results suggest that binding of NF-kappaB p52 protein to target DNA motifs is mainly due to contacts with bases; interactions with the DNA backbone are, however, important for stabilization of the protein-DNA complex. From the practical point of view, our results suggest that DNA-PNA hybrid can be recognized by NF-kappaB p52 protein, although with an efficiency lower than DNA-DNA NF-kappaB target molecules; therefore, our results should encourage studies on modified PNAs in order to develop potential agents for the decoy approach in gene therapy.
Mischiati
C
C
Department of Biochemistry, Ferrara University, 44100 Ferrara, Italy.
Borgatti
M
M
Bianchi
N
N
Rutigliano
C
C
Tomassetti
M
M
Feriotto
G
G
Gambari
R
R
D048868:Adaptor Proteins, Signal Transducing; D001665:Binding Sites; D002460:Cell Line; D003432:Cross-Linking Reagents; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000378:metabolism; D015316:Genetic Therapy; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D007700:Kinetics; D016328:NF-kappa B / Q000378:metabolism*; D009692:Nucleic Acid Heteroduplexes / Q000378:metabolism; D020135:Peptide Nucleic Acids / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D020349:Surface Plasmon Resonance; D015534:Trans-Activators; D014157:Transcription Factors; D014466:Ultraviolet Rays
D048868:Adaptor Proteins, Signal Transducing; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D016328:NF-kappa B; D009692:Nucleic Acid Heteroduplexes; C504649:PSIP1 protein, human; D020135:Peptide Nucleic Acids; D015534:Trans-Activators; D014157:Transcription Factors