Defining the peptide nucleic acids (PNA) length requirement for PNA binding-induced transcription and gene expression

Induction of gene expression has great potential in the treatment of many human diseases. Peptide nucleic acid (PNA) as a novel DNA-binding reagent provides an ideal system to induce gene-specific expression. In our recent studies, we have demonstrated that PNA bound to double-stranded DNA targets and, therefore, generated single-stranded D-loops and induced transcription of target genes both in vitro and in vivo. Most importantly, we have demonstrated that treatment of cultured human cells with PNAs led to expression of an endogenous target gene. Therefore, the study of the molecular mechanism of PNA binding-induced gene expression will have great implications for the gene therapy of many human diseases. In the current study, we have investigated the PNA length requirement for PNA binding-induced transcription initiation. Using a series of PNAs with different lengths, we have determined that PNAs with lengths of 16∼18 nt induce very high levels of transcription in a HeLa nuclear extract in vitro transcription system. Transfection of the PNA-bound GFP reporter gene plasmid into human normal fibroblast (NF) cells led to a similar result. Gel-mobility shift assays revealed very strong binding affinities of these PNAs. DNA footprinting analysis further demonstrated the specificity of PNAs binding to the targets. These results lead to important understanding of the molecular mechanism of transcription initiation and highly valuable information in PNA design, especially for PNA binding-induced, gene-specific expression.