Nucleic Acids Research, 2003, Vol. 31, No. 14 4099-4108
© 2003 Oxford University Press
Effect of DNA target sequence on triplex formation by oligo-2'-deoxy- and 2'-O-methylribonucleotides
Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, 615 North Wolfe Street, Baltimore, MD 21205, USA
*To whom correspondence should be addressed. Tel: +1 410 955 3489; Fax: +1 410 955 2926; Email: pmiller{at}jhsph.edu
Present address:
Nitin Puri, Ambion, Inc., 2130 Woodward Street, Austin, TX 78744, USA
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 (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.