The stability of DNA triplexes inside cells as studied by iodine-125 radioprinting

We studied the stability of a DNA tripler resulting from the binding of a 3 8 nt long purine motif triplex-forming oligonucleotide (TFO) to a covalentl y closed plasmid containing a target sequence from the human HPRT gene. Our in vitro experiments showed that the tripler formed at plasmid and TFO con centrations as low as 10(-9) M. Once formed, the triplex was remarkably sta ble and could withstand 10 min incubation at 65 degrees C. We next delivere d these TFO-plasmid complexes into cultured human cells. To monitor the TFO -plasmid complexes inside cells we applied a new technique that we call 'ra dioprinting'. Because the TFO was (125)l labeled, we could quantitatively m onitor the triplexes by measuring (125)l-induced DNA strand breaks in the t arget plasmid sequence. We found that the triplexes remain stable inside th e 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 comp lex 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 c ells.