title: Chromosome targeting at short polypurine sites by cationic triplex-forming oligonucleotides.
abstract: Triplex-forming oligonucleotides (TFOs) bind specifically to duplex DNA and provide a strategy for site-directed modification of genomic DNA. Recently we demonstrated TFO-mediated targeted gene knockout following systemic administration in animals. However, a limitation to this approach is the requirement for a polypurine tract (typically 15-30 base pairs (bp)) in the target DNA to afford high affinity third strand binding, thus restricting the number of sites available for effective targeting. To overcome this limitation, we have investigated the ability of chemically modified TFOs to target a short (10 bp) site in a chromosomal locus in mouse cells and induce site-specific mutations. We report that replacement of the phosphodiester backbone with cationic phosphoramidate linkages, either N,N-diethylethylenediamine or N,N-dimethylaminopropylamine, in a 10-nucleotide, psoralen-conjugated TFO confers substantial increases in binding affinity in vitro and is required to achieve targeted modification of a chromosomal reporter gene in mammalian cells. The triplex-directed, site-specific induction of mutagenesis in the chromosomal target was charge- and modification-dependent, with the activity of N,N-diethylethylenediamine > N,N-dimethylaminopropylamine phosphodiester, resulting in 10-, 6-, and <2-fold induction of target gene mutagenesis, respectively. Similarly, N,N-diethylethylenediamine and N,N-dimethylaminopropylamine TFOs were found to enhance targeting at a 16-bp G:C bp-rich target site in a chromatinized episomal target in monkey COS cells, although this longer site was also targetable by a phosphodiester TFO. These results indicate that replacement of phosphodiester bonds with positively charged N,N-diethylethylenediamine linkages enhances intracellular activity and allows targeting of relatively short polypurine sites, thereby substantially expanding the number of potential triplex target sites in the genome.
authors.affiliation: Department of Therapeutic Radiology and Genetics, Yale University School of Medicine, New Haven, Connecticut 06520-8040, USA.
authors.forename: K M
authors.initials: KM
authors.lastname: Vasquez
authors.forename: J M
authors.initials: JM
authors.lastname: Dagle
authors.forename: D L
authors.initials: DL
authors.lastname: Weeks
authors.forename: P M
authors.initials: PM
authors.lastname: Glazer
mesh_terms: D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002412:Cations; D002875:Chromosomes; D004247:DNA; D004252:DNA Mutational Analysis; D003959:Diamines / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005029:Ethylenediamines / Q000494:pharmacology; D005363:Ficusin / Q000737:chemistry / Q000494:pharmacology; D017930:Genes, Reporter; D016153:Genes, Suppressor; D005821:Genetic Techniques; D016678:Genome; D007202:Indicators and Reagents / Q000494:pharmacology; D008274:Magnesium / Q000494:pharmacology; D051379:Mice; D018345:Mice, Knockout; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D011188:Potassium / Q000494:pharmacology; D011485:Protein Binding; D011687:Purines / Q000737:chemistry*; D012343:RNA, Transfer / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid
chemical_list: D002412:Cations; D003959:Diamines; D005029:Ethylenediamines; D007202:Indicators and Reagents; D011687:Purines; C096275:supF tRNA; C051802:triplex DNA; C031931:N-(1-naphthyl)-N',N'-diethylethylenediamine; D004247:DNA; D012343:RNA, Transfer; D008274:Magnesium; C012928:3-dimethylaminopropylamine; D005363:Ficusin; D011188:Potassium