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Papers In Press, published online ahead of print August 14, 2001
Therapeutic Radiology and Genetics, Yale University School of Medicine, New Haven, CT 06536
Corresponding Author: peter.glazer{at}yale.edu
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 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-diethyl-ethylenediamine or N,N-dimethyl-aminopropylamine, 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-diethyl-ethylenediamine > N,N-dimethyl-aminopropylamine >> phosphodiester, resulting in 10-fold, 6-fold, and <2-fold induction of target gene mutagenesis, respectively. Similarly, DEED and DMAP 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-diethyl-ethylenediamine 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.
J. Biol. Chem, 10.1074/jbc.M101797200
Submitted on February 27, 2001
Revised on August 9, 2001
Accepted on August 13, 2001
Chromosome targeting at short polypurine sites by cationic triplex-forming oligonucleotides
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