Effect of DNA target sequence on triplex formation by oligo-2′-deoxy- and 2′-O-methylribonucleotides

Abstract

The interactions of pyrimidine deoxyribo- or 2′-O- methylribo-psoralen-conjugated, triplex-forming oligo-nucleotides, psTFOs, with a 17-bp env-DNA whose purine tract is 5′-AGAGAGAAAAAAGAG-3′, or an 18-bp gag-DNA whose purine tract is 5′-AGG-GGGAAAGAAAAAA-3′, were studied over the pH range 6.0-7.5. The stability of the triplex formed by deoxy-env-psTFO containing 5-methylcytosines and thymines decreased with increasing pH (Tm = 56°C at pH 6.0; 27°C at pH 7.5). Replacement of 5-methylcytosines with 8-oxo-adenines reduced the pH dependence, but lowered triplex stability. A 2′-O-methyl-env-psTFO containing uracil and cytosine did not form a triplex at pH 7.5. Surprisingly, replacement of the cytosines in this oligomer with 5-methylcytosines dramatically increased triplex stability (Tm = 25°C at pH 7.5), and even greater stability was achieved by selective replacement of uracils with thymines (Tm = 37°C at pH 7.5). Substitution of the contiguous 5-methylcytosines of the deoxy-gag-psTFO with 8-oxo-adenines significantly reduced pH dependence and increased triplex stability. In contrast to the behavior of env-specific TFOs, triplexes formed by 2′-O-methyl-gag-psTFOs did not show enhanced stability. Replacement of the 3′-terminal phosphodiester of the TFO with a methylphosphonate group significantly increased the resistance of both deoxy- and 2′-O-methyl-TFOs to degradation by 3′-exonucleases, while maintaining triplex stability.