Effect of DNA target sequence on triplex formation by oligo-2'-deoxy- and 2'-O-methylribonucleotides.
The interactions of pyrimidine deoxyribo- or 2'-O-methylribo-psoralen-conjugated, triplex-forming oligonucleotides, 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 a deoxy-env-psTFO containing 5-methylcytosines and thymines decreased with increasing pH (T(m) = 56 degrees C at pH 6.0; 27 degrees 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 (T(m) = 25 degrees C at pH 7.5), and even greater stability was achieved by selective replacement of uracils with thymines (T(m) = 37 degrees 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.
Cassidy
Rachel A
RA
Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, 615 North Wolfe Street, Baltimore, MD 21205, USA.
Puri
Nitin
N
Miller
Paul S
PS
D001483:Base Sequence; D004247:DNA / Q000737:chemistry* / Q000235:genetics; D015686:Gene Products, env / Q000235:genetics; D015683:Gene Products, gag / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry* / Q000235:genetics; D009843:Oligoribonucleotides / Q000737:chemistry* / Q000235:genetics; D009943:Organophosphorus Compounds / Q000737:chemistry; D013696:Temperature
D015686:Gene Products, env; D015683:Gene Products, gag; D009838:Oligodeoxyribonucleotides; D009843:Oligoribonucleotides; D009943:Organophosphorus Compounds; C051802:triplex DNA; C032627:methylphosphonic acid; D004247:DNA