Olivas W M, Maher L J
Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha 68198-6805.
Biochemistry. 1994 Feb 1;33(4):983-91. doi: 10.1021/bi00170a017.
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.
通过寡核苷酸定向三链体形成进行双链DNA识别通常仅限于同型嘌呤靶结构域。人们已经提出了各种方法来缓解这一限制。此前已使用人工DNA序列表明，寡核苷酸探针可以同时识别相反DNA链上相邻的同型嘌呤结构域，这些探针在不同结构域之间切换三链体识别基序。通过电泳迁移率测定和化学保护分析，我们详细探究了这些策略在设计针对人类p53基因天然DNA序列的高亲和力探针方面是否有益。该靶位点在相反的DNA链上包含三个相邻的富含嘌呤的结构域。我们的结果表明：（i）通过设计同时识别所有三个嘌呤结构域的寡核苷酸，可以使该序列的亲和力适度但在统计学上有显著提高；（ii）一个嘌呤结构域中嘧啶中断的校正不会显著改变这一结果；（iii）可以使用具有特异性和非特异性核苷酸序列组合的探针来评估识别每个嘌呤结构域所产生的相对能量和结构贡献；（iv）对于某些复合物，探针亲和力与碱基三联体的表观数量无关。这些数据表明，使用现有策略，三链体基序之间的交替可能与不利的自由能变化相关。与为此目的优化的人工DNA序列不同，在这个天然靶序列上使用交替链DNA识别未观察到亲和力的显著提高。因此，我们得出结论，这种提高是序列依赖性的。
