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J. Biol. Chem., Vol. 278, Issue 5, 3357-3362, January 31, 2003

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Transcription Dependence of Chromosomal Gene Targeting by Triplex-forming Oligonucleotides^*

Margaret A. Macris and Peter M. Glazer

From the Departments of Therapeutic Radiology and Genetics, Yale University School of Medicine, New Haven, Connecticut 06520-8040

Triplex-forming oligonucleotides (TFOs) recognize and bind to specific DNA sequences and have been used to modify gene function in cells. To study factors that might influence triplex formation at chromosomal sites in mammalian cells, we developed a restriction protection assay to detect triplex-directed psoralen crosslinks in genomic DNA prepared from TFO-transfected cells. Using this assay, we detected binding of a G-rich TFO to a chromosomal site even in the absence of transcription when high concentrations of the TFO were used for transfection. However, experimental induction of transcription at the target site, via an ecdysone-responsive promoter, resulted in substantial increases (3-fold or more) in target site crosslinking, especially at low TFO concentrations. When RNA polymerase activity was inhibited, even in the ecdysone-induced cells, the level of TFO binding was significantly decreased, indicating that transcription through the target region, and not just transcription factor binding, is necessary for the enhanced chromosomal targeting by TFOs. These findings provide evidence that physiologic activity at a chromosomal target site can influence its accessibility to TFOs and suggest that gene targeting by small molecules may be most effective at highly expressed chromosomal loci.

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