Duplex DNA recognition by oligonucleotide-directed triple helix formation is generally limited to homopurine target domains. Various approaches have been suggested for the relief of this constraint. Artificial DNA sequences have previously been used to show that adjacent homopurine domains on opposite DNA strands can be simultaneously recognized by oligonucleotide probes that switch triple helix recognition motifs between domains. Using assays of electrophoretic mobility and chemical protection, we have explored in detail whether such strategies are of benefit in designing high-affinity probes for a natural DNA sequence in the human p53 gene. This target site contains three adjacent, purine-rich domains on opposite DNA strands. Our results show that (i) a modest but statistically significant enhancement in affinity can be achieved for this sequence by designing an oligonucleotide that simultaneously recognizes all three purine domains, (ii) correction of a pyrimidine interruption in one purine domain does not dramatically alter this result, (iii) the relative energetic and structural contributions attributable to recognition of each purine domain can be assessed using probes with combinations of specific and nonspecific nucleotide sequences, and (iv) probe affinity is not correlated with the apparent number of base triplets for certain complexes. These data suggest that unfavorable free energy changes may be associated with alternation between triple helix motifs using existing strategies. In contrast to artificial DNA sequences optimized for this purpose, a substantial affinity enhancement was not observed using alternate strand DNA recognition at this natural target sequence. We therefore conclude that such enhancement is sequence dependent.
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