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J. Biol. Chem., Vol. 276, Issue 42, 38536-38541, October 19, 2001
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§,
, and**
From the 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 Departments of Therapeutic Radiology and
Genetics, Yale University School of Medicine,
New Haven, Connecticut 06520-8040 and the ¶ Department
of Pediatrics and the Department of Biochemistry, University of
Iowa, Iowa City, Iowa 52242
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.
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