The stability of DNA triplexes inside cells as studied by iodine-125 radioprinting.
We studied the stability of a DNA triplex resulting from the binding of a 38 nt long purine motif triplex-forming oligonucleotide (TFO) to a covalently closed plasmid containing a target sequence from the human HPRT gene. Our in vitro experiments showed that the triplex formed at plasmid and TFO concentrations as low as 10(-9)M. Once formed, the triplex was remarkably stable and could withstand 10 min incubation at 65 degrees C. We next delivered these TFO-plasmid complexes into cultured human cells. To monitor the TFO-plasmid complexes inside cells we applied a new technique that we call 'radioprinting'. Because the TFO was(125)I labeled, we could quantitatively monitor the triplexes by measuring(125)I-induced DNA strand breaks in the target plasmid sequence. We found that the triplexes remain stable inside the cells for at least 48 h. Based on these findings we propose using TFO for indirect labeling of intact plasmid DNA. As a demonstration, we show that the intracellular distribution of a fluorescein-labeled TFO was different when it was liposome-delivered into cultured human cells alone or in a complex with the plasmid. In the latter case, the fluorescence was detected in nearly all the cells while detection of the plasmid by use of a marker gene (beta-galactosidase) revealed expression of the gene in only half of the cells.
Sedelnikova
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OA
Department of Nuclear Medicine, Warren G. Magnuson Clinical Center, National Institutes of Health, Bethesda, MD 20892, USA.
Panyutin
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Luu
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Neumann
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RD
D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D007457:Iodine Radioisotopes; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010957:Plasmids
D007457:Iodine Radioisotopes; D009838:Oligodeoxyribonucleotides; D004247:DNA; D007041:Hypoxanthine Phosphoribosyltransferase