Transcription dependence of chromosomal gene targeting by triplex-forming oligonucleotides.
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.
Macris
Margaret A
MA
Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, Connecticut 06520-8040, USA.
Glazer
Peter M
PM
D001483:Base Sequence; D002460:Cell Line; D002877:Chromosomes, Human / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors; D011564:Furocoumarins; D018390:Gene Targeting / Q000379:methods*; D006801:Humans; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry*; D010957:Plasmids; D015183:Restriction Mapping; D014158:Transcription, Genetic; D014162:Transfection
D011564:Furocoumarins; D009841:Oligonucleotides; D012321:DNA-Directed RNA Polymerases