Interaction of the Human NF-κB p52 Transcription Factor with DNA-PNA Hybrids Mimicking the NF-κB Binding Sites of the Human Immunodeficiency Virus Type 1 Promoter

Abstract

We determined whether peptide nucleic acids (PNAs) are able to interact with NF-B p52 transcription factor. The binding of NF-B p52 to DNA-DNA, DNA-PNA, PNA-DNA, and PNA-PNA hybrid molecules carrying the NF-B 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-B p52 does not efficiently bind to PNA-PNA hybrids. However, a DNA-PNA hybrid molecule was found to be recognized by NF-B p52, although the molecular complexes generated exhibited low stability. From the theoretical point of view, our results suggest that binding of NF-B 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-B p52 protein, although with an efficiency lower than DNA-DNA NF-B target molecules; therefore, our results should encourage studies on modified PNAs in order to develop potential agents for the decoy approach in gene therapy.It is well established that both constitutive and tissue-specific regulation of gene expression is operated at the transcriptional level by the interaction between nuclear proteins (transcription factors) and promoter regions containing DNA elements (transcription signals) that exhibit specific nucleotide sequences (1-4). Several reviews reporting the nucleotide sequences of transcription signals and the relative binding proteins have been published (5-7). The requirement of protein-

Transcription Factor Decoy Molecules Based on a Peptide Nucleic Acid (PNA)-DNA Chimera Mimicking Sp1 Binding Sites

“…In a recent study, we proposed PNAs as alternative reagents in experiments aimed at the control of gene expression involving the decoy approach (20). In PNAs, the pseudopeptide backbone is composed of N-(2-aminoethyl)glycine units (27)(28)(29)(30).…”

“…We demonstrated that NF-B p52 is able to bind to both DNA/DNA and DNA/PNA hybrids mimicking the NF-B target sites present in the HIV-1 LTR. On the contrary, low binding of NF-B p52 to PNA/PNA hybrids was found (20). We have also reported a conformational study to explain these binding data using a molecular dynamics approach.…”

“…Decoy molecules against NF-B inhibit the expression of NF-B regulated genes (MHC complex genes, IL2 receptor ␣, Igk, IL6, ␦ opioid receptor, preprogalanin, adhesion molecule-1) (20). More recently, dumbell DNA decoy elements against NF-B were demonstrated to be active in inhibiting ex vivo transcription driven by the long terminal repeat (LTR) of human immunodeficiency type-1 virus (HIV-1) (19).…”

“…One of the most effective decoy approaches so far described involves nuclear proteins belonging to the NF-B 1 superfamily (7)(8)(9)(13)(14)(15)(16)(17)(18)(19)(20).…”

The peptide nucleic acids (PNAs), powerful tools for molecular genetics and cytogenetics

“…This results in an attenuation of the authentic interactions of trans-factors with their cis-elements. 11 PNAs are able to interact with mRNA independently of the RNA secondary structure. Studies on the mechanisms of antisense activity have demonstrated that PNA inhibits expression differently than antisense oligonucleotides acting through RNase-H mediated degradation of the mRNA -oligonucleotide hybrid.…”