Recognition of ATGA sequences by the unfused aromatic dication DB293 forming stacked dimers in the DNA minor groove
Recognition of ATGA sequences by the unfused aromatic dication DB293 forming stacked dimers in the DNA minor groove
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DOI：
10.1021/bi0108453
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发表时间：
2001-08-21
期刊：
影响因子：
2.9
通讯作者：
Wilson, WD
中科院分区：
文献类型：
--
作者：
Bailly, C;Tardy, C;Wilson, WD
呋喃咪胺和相关的二胺类药物代表了一系列有希望的药物，这些药物对广泛存在的寄生虫，特别是卡氏肺孢子虫病原体有效。在这个系列中，苯基呋喃苯并咪唑二胺衍生物DB293最近被鉴定为第一个能够在含GC序列的DNA小槽中形成堆叠二聚体的未融合芳香族化合物。在这里，我们提供了DB293的DNA序列识别特性的详细的生化和生物物理特性。利用DNase I、Fe-II-EDTA和一个蒽醌光核酸酶的3种互补足迹技术，在不同的DNA限制片段中定位了DB293的结合位点。通过DNase I足迹法鉴定了两类位点：(I)包含连续A-T对的4/5bp序列，如5‘-AAAA和5’-ATTA；(Ii)包含基序5‘-ATGA(.)5’-TCAT的序列。特别是，包含两个相邻ATGA基序的13个碱基序列为DB293提供了一个高度优先的识别位点。定量足迹分析表明，与富含AT的位点相比，5‘-ATGA位点的占有率更高。DB293与其他DNA片段的优先结合也被观察到，并通过Fe-II-EDTA体系产生的羟基足迹以及使用探针蒽醌-2-磺酸(AQS)的光足迹方法独立地证实了DB293的结合。此外，这种光敏试剂显示了DB293特异的增强切割位点的存在。正如DNase I和AQS探针独立显示的那样，这种分子，而不是其他次要的沟槽结合物，如netropsin，会在某些结合位点附近诱导DNA的特定局部结构变化。通过熔融温度实验和表面等离子体共振(SPR)进一步研究了DB293对ATGA序列的识别作用。不同发夹状寡核苷酸的使用表明，DB293可以通过形成1：1的药物-DNA复合体与AT位点相互作用，但结合更强，并协同地与含有ATGA的序列形成2：1的药物-DNA复合体。DB293与ATGA序列强结合，没有明显的上下文依赖性，但对靶序列的方向高度敏感。通过颠倒5‘-ATGA-3’-ATGA序列，取消了2：1 DB293/DNA复合体的形成，表明方向性在药物-DNA识别过程中起着重要的作用。同样，A[T-->；G]GA序列中的一个突变对DB293的二聚体相互作用非常不利。从互补的足迹和SPR数据中，5‘-ATGA序列被鉴定为DB293高度青睐的二聚体结合位点。这些数据为描绘二胺型小槽结合的识别码提供了线索，并最终指导针对遗传物质特定部位的基因调控分子的合理设计。
Furamidine and related diamidines represent a promising series of drugs active against widespread parasites, in particular the Pneumocystic carinii pathogen. In this series, the phenylfuranbenzimidazole diamidine derivative DB293 was recently identified as the first unfused aromatic dication capable of forming stacked dimers in the DNA minor groove of GC-containing sequences. Here we present a detailed biochemical and biophysical characterization of the DNA sequence recognition properties of DB293. Three complementary footprinting techniques using DNase I, Fe-II-EDTA, and an anthraquinone photonuclease were employed to locate binding sites for DB293 in different DNA restriction fragments. Two categories of sites were identified by DNase I footprinting: (i) 4/5 bp sequences containing contiguous A-T pairs, such as 5 ' -AAAA and 5 ' -ATTA; and (ii) sequences including the motif 5 ' -ATGA(.)5 ' -TCAT. In particular, a 13-bp sequence including two contiguous ATGA motifs provided a highly preferential recognition site for DB293. Quantitative footprinting analysis revealed better occupancy of the 5 ' -ATGA site compared to the AT-rich sites. Preferential binding of DB293 to ATGA sites was also observed with other DNA fragments and was confirmed independently by means of hydroxyl radical footprinting generated by the Fe-II-EDTA system, as well as by a photofootprinting approach using the probe anthraquinone-2-sulfonate (AQS). In addition, this photosensitive reagent revealed the presence of sites of enhanced cutting specific to DB293. This molecule, but not other minor groove binders such as netropsin, induces specific local structural changes in DNA near certain binding sites, as independently shown by DNase I and the AQS probe. Recognition of the ATGA sequence by DB293 was investigated further using melting temperature experiments and surface plasmon resonance (SPR). The use of different hairpin oligonucleotides showed that DB293 can interact with AT sites via the formation of 1:1 drug-DNA complexes but binds much more strongly, and cooperatively, to ATGA-containing sequences to form 2:1 drug-DNA complexes. DB293 binds strongly to ATGA sequences with no significant context dependence but is highly sensitive to the orientation of the target sequence. The formation of 2:1 DB293/DNA complexes is abolished by reversing the sequence 5 ' -ATGA-3 ' -ATGA, indicating that directionality plays an important role in the drug-DNA recognition process. Similarly, a single mutation in the A[T -->G]GA sequence is very detrimental to the dimer interactions of DB293. From the complementary footprinting and SPR data, the 5 ' -ATGA sequence is identified as being a highly favored dimer binding site for DB293. The data provide clues for delineating a recognition code for diamidine-type minor groove binding, agents, and ultimately to guide the rational design of gene regulatory molecules targeted to specific sites of the genetic material.