Inhibiting Gene Expression with Peptide Nucleic Acid (PNA)−Peptide Conjugates That Target Chromosomal DNA

Abstract

Peptide nucleic acids (PNAs) are a nonionic DNA/RNA mimic that can recognize complementary sequences by Watson-Crick base-pairing. The neutral PNA backbone facilitates recognition of duplex DNA by strand invasion, suggesting that antigene PNAs (agPNAs) can be important tools for exploring the structure and function of chromosomal DNA inside cells. However, before agPNAs can enter wide use it will be necessary to develop straightforward strategies for introducing them into cells. Here we demonstrate that agPNA-peptide conjugates can target promoter DNA and block progesterone receptor (PR) gene expression inside cells. Thirty-six agPNA-peptide conjugates were synthesized and tested. We observed inhibition of gene expression using cationic peptides containing either arginine or lysine residues, with eight or more cationic amino acids being preferred. Both thirteen and nineteen base agPNA-peptide conjugates were inhibitory. Inhibition was observed in human cancer cell lines expressing either high or low levels of progesterone receptor. Modification of agPNA-peptide conjugates with hydrophobic amino acids or small molecule hydrophobic moities yielded improved potency. Inhibition by agPNAs did not require cationic lipid or any other additive, but adding agents to cell growth media that promote endosomal release caused modest increases in agPNA potency. These data demonstrate that chromosomal DNA is accessible to agPNA-peptide conjugates and that chemical modifications can improve potency.Peptide nucleic acids 1 (PNAs) are a class of DNA/RNA mimic with an uncharged amide backbone (1). PNAs hybridize to complementary sequences by Watson-Crick base-pairing and have an outstanding ability to invade double-stranded DNA (1-5). Reports have appeared suggesting that PNAs can also target duplex DNA inside cells (6)(7)(8). Recently, we reported that antigene PNAs (agPNAs) that target chromosomal DNA at transcription start sites inhibit gene expression (9). These data suggest that PNAs may be valuable tools for exploring promoter function and for controlling gene expression at the level of the chromosome.For our initial experiments with agPNAs, we delivered them into cultured human cells in complex with complementary DNA oligonucleotides and cationic lipid (9,10). This method is a variation of standard protocols for lipid-mediated transfection. The DNA binds to the PNA, the lipid binds to the DNA, and the PNA is transported into cells as cargo by the DNA/lipid complex.This method has worked well and can lead to potent inhibition of gene expression in the presence of nanomolar concentrations of agPNA (9,10). However, the combination of steps (annealing DNA and PNA, lipid transfection) is likely to discourage full exploitation of the substantial potential of agPNAs as tools for probing chromosomal DNA in cell culture. Animal * To whom correspondence should be addressed. Email: david.corey@utsouthwestern MATERIALS AND METHODS Synthesis of PNA-Peptide ConjugatesPNA-peptide conjugates were synthesized as previously describe...

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“…Recently, we have described the possibility to use LNA oligonucleotides to block transcription by hybridization to sequences within the transcribed part of a gene (Ge et al 2007). While we used a new type of construct, oligonucleotide-mediated blocking of transcription has been reported by several authors (Bailey et al 1998;Beane et al 2007;Cutrona et al 2003;Hu and Corey 2007). The precise mechanism underlying this block remains somewhat elusive.…”