Vashisht Gopal Y N, Van Dyke Michael W
Department of Molecular and Cellular Oncology, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030-4009, USA.
Biochemistry. 2003 Jun 10;42(22):6891-903. doi: 10.1021/bi027373s.
Development of sequence-specific DNA-binding drugs is an important pharmacological goal, given the fact that numerous existing DNA-directed chemotherapeutic drugs rely on the strength and selectivity of their DNA interactions for therapeutic activity. Among the DNA-binding antibiotics, hairpin polyamides represent the only class of small molecules that can practically bind any predetermined DNA sequence. DNA recognition by these ligands depends on their side-by-side amino acid pairings in the DNA minor groove. Extensive studies have revealed that these molecules show extremely high affinity for sequence-directed, minor groove interaction. However, the specificity of such interactions in the presence of a large selection of sequences such as the human genome is not known. We used the combinatorial selection method restriction endonuclease protection, selection, and amplification (REPSA) to determine the DNA binding specificity of two hairpin polyamides, ImPyPyPy-gamma-PyPyPyPy-beta-Dp and ImPyPyPy-gamma-ImPyPyPy-beta-Dp, in the presence of more than 134 million different sequences. These were verified by restriction endonuclease protection assays and DNase I footprinting analysis. Our data showed that both hairpin polyamides preferentially selected DNA sequences having consensus recognition sites as defined by the Dervan pairing rules. These consensus sequences were rather degenerate, as expected, given that the stacked pyrrole-pyrrole amino acid pairs present in both polyamides are unable to discriminate between A.T and T.A base pairs. However, no individual sequence within these degenerate consensus sequences was preferentially selected by REPSA, indicating that these hairpin polyamides are truly consensus-specific DNA-binding ligands. We also discovered a preference for overlapping consensus binding sites among the sequences selected by the hairpin polyamide ImPyPyPy-gamma-PyPyPyPy-beta-Dp, and confirmed by DNase I footprinting that these complex sites provide higher binding affinity. These data suggest that multiple hairpin polyamides can cooperatively bind to their highest-affinity sites.
鉴于众多现有的DNA导向化疗药物依赖于其与DNA相互作用的强度和选择性来发挥治疗活性，开发序列特异性DNA结合药物是一个重要的药理学目标。在DNA结合抗生素中，发夹型聚酰胺是唯一一类能够实际结合任何预定DNA序列的小分子。这些配体对DNA的识别取决于它们在DNA小沟中并排的氨基酸配对。广泛的研究表明，这些分子对序列导向的小沟相互作用表现出极高的亲和力。然而，在存在大量序列（如人类基因组）的情况下，这种相互作用的特异性尚不清楚。我们使用组合选择方法——限制性内切酶保护、选择和扩增（REPSA），在超过1.34亿种不同序列存在的情况下，确定了两种发夹型聚酰胺ImPyPyPy-γ-PyPyPyPy-β-Dp和ImPyPyPy-γ-ImPyPyPy-β-Dp的DNA结合特异性。这些通过限制性内切酶保护试验和DNase I足迹分析得到了验证。我们的数据表明，两种发夹型聚酰胺都优先选择具有由Dervan配对规则定义的共有识别位点的DNA序列。正如预期的那样，这些共有序列相当简并，因为两种聚酰胺中存在的堆叠吡咯-吡咯氨基酸对无法区分A.T和T.A碱基对。然而，REPSA并没有优先选择这些简并共有序列中的任何单个序列，这表明这些发夹型聚酰胺是真正的共有特异性DNA结合配体。我们还发现发夹型聚酰胺ImPyPyPy-γ-PyPyPyPy-β-Dp选择的序列之间倾向于重叠共有结合位点，并通过DNase I足迹证实这些复杂位点提供了更高的结合亲和力。这些数据表明，多个发夹型聚酰胺可以协同结合到它们的最高亲和力位点。
