DNA RECOGNITION BY ALTERNATE STRAND TRIPLE-HELIX FORMATION - AFFINITIES OF OLIGONUCLEOTIDES FOR A SITE IN THE HUMAN P53 GENE

Duplex DNA recognition by oligonucleotide-directed triple helix format ion is generally limited to homopurine target domains. Various approac hes have been suggested for the relief of this constraint. Artificial DNA sequences have previously been used to show that adjacent homopuri ne domains on opposite DNA strands can be simultaneously recognized by oligonucleotide probes that switch triple helix recognition motifs be tween domains. Using assays of electrophoretic mobility and chemical p rotection, we have explored in detail whether such strategies are of b enefit 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 mode st but statistically significant enhancement in affinity can be achiev ed for this sequence by designing an oligonucleotide that simultaneous ly recognizes all three purine domains, (ii) correction of a pyrimidin e interruption in one purine domain does not dramatically alter this r esult, (iii) the relative energetic and structural contributions attri butable to recognition of each purine domain can be assessed using pro bes with combinations of specific and nonspecific nucleotide sequences , and (iv) probe affinity is not correlated with the apparent number o f base triplets for certain complexes. These data suggest that unfavor able free energy changes may be associated with alternation between tr iple helix motifs using existing strategies. In contrast to artificial DNA sequences optimized for this purpose, a substantial affinity enha ncement was not observed using alternate strand DNA recognition at thi s natural target sequence. We therefore conclude that such enhancement is sequence dependent.