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. 1997 Feb 1;25(3):633–640. doi: 10.1093/nar/25.3.633

Potassium-resistant triple helix formation and improved intracellular gene targeting by oligodeoxyribonucleotides containing 7-deazaxanthine.

A F Faruqi 1, S H Krawczyk 1, M D Matteucci 1, P M Glazer 1
PMCID: PMC146453  PMID: 9016606

Abstract

Triple helix formation by purine-rich oligonucleotides in the anti-parallel motif is inhibited by physiological concentrations of potassium. Substitution with 7-deazaxanthine (c7X) has been suggested as a strategy to overcome this effect. We have tested this by examining triple helix formation both in vitro and in vivo by a series of triple helix-forming oligonucleotides (TFOs) containing guanine plus either adenine, thymine, or c7X. The TFOs were conjugated to psoralen at the 5'end and were designed to bind to a portion of the supF mutation reporter gene. Using in vitro gel mobility shift assays, we found that triplex formation by the c7X-substituted TFOs was relatively resistant to the presence of 140 mM K+. The c7X-containing TFOs were also superior in gene targeting experiments in mammalian cells, yielding 4- to 5-fold higher mutation frequencies in a shuttle vector-based mutagenesis assay designed to detect mutations induced by third strand-directed psoralen adducts. When the phosphodiester backbone was replaced by a phosphorothioate one, the in vitro binding of the c7X-TFOs was not affected, but the efficiency of in vivo triple helix formation was reduced. These results indicate the utility of the c7X substitution for in vivo gene targeting experiments, and they show that the feasibility of the triplex anti-gene strategy can be significantly enhanced by advances in nucleotide chemistry.

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