Crow Stephen D G, Bailly Christian, Garbay-Jaureguiberry Christiane, Roques Bernard, Shaw Barbara Ramsay, Waring Michael J
Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, UK.
Biochemistry. 2002 Jul 9;41(27):8672-82. doi: 10.1021/bi012207q.
The antitumor drug ditercalinium is a rare example of a noncovalent DNA-binding ligand that forms bisintercalation complexes via the major groove of the double helix. Previous structural studies have revealed that the two connected pyridocarbazolium chromophores intercalate into DNA with the positively charged bis(ethylpiperidinium) linking chain oriented to the wide groove side of the helix. Although the interaction of ditercalinium with short oligonucleotides containing 4-6 contiguous GC base pairs has been examined in detail by biophysical and theoretical approaches, the sequence preference for ditercalinium binding to long DNA fragments that offer a wide variety of binding sites has been investigated only superficially. Here we have investigated both sequence preferences and possible molecular determinants of selectivity in the binding of ditercalinium to DNA, primarily using methods based upon DNase I footprinting. A range of multisite DNA substrates, including several natural restriction fragments and different PCR-generated fragments containing unconventional bases (2,6-diaminopurine, inosine, uridine, 5-fluoro- and 5-methylcytosine, 7-deazaguanine, 7-deazaadenine, and N(7)-cyanoboranoguanine), have been employed to show that ditercalinium selectively recognizes certain GC-rich sequences in DNA and to identify some of the factors which affect its DNA-binding sequence selectivity. Specifically, the footprinting data have revealed that the 2-amino group on the purines or the 5-methyl group on the pyrimidines is not essential for the formation of ditercalinium-DNA complexes whereas the major groove-oriented N(7) of guanine does appear as a key element in the molecular recognition process. The loss of N(7) at guanines but not adenines is sufficient to practically abolish sequence-selective binding of ditercalinium to DNA. Thus, as expected for a major groove binding drug, the N(7) of guanine is normally required for effective complex formation with GC base pairs, but interestingly the substitution of the N(7) with a relatively bulky cyanoborane group does not markedly affect the sequence recognition process. Therefore, the hydrogen bond accepting capability at N(7) of guanines is not sufficient to explain the GC-selective drug-DNA association, and the implications of these findings are considered.
抗肿瘤药物双喹啉是一种罕见的非共价DNA结合配体，它通过双螺旋的大沟形成双插入复合物。先前的结构研究表明，两个相连的吡啶并咔唑发色团插入DNA，带正电荷的双（乙基哌啶鎓）连接链朝向螺旋的宽沟侧。尽管已经通过生物物理和理论方法详细研究了双喹啉与含有4-6个连续GC碱基对的短寡核苷酸的相互作用，但对于双喹啉与提供多种结合位点的长DNA片段结合的序列偏好仅进行了表面研究。在这里，我们主要使用基于DNase I足迹法的方法，研究了双喹啉与DNA结合的序列偏好和可能的选择性分子决定因素。一系列多位点DNA底物，包括几个天然限制片段和不同的PCR生成片段，含有非常规碱基（2,6-二氨基嘌呤、次黄嘌呤、尿嘧啶、5-氟和5-甲基胞嘧啶、7-脱氮鸟嘌呤、7-脱氮腺嘌呤和N(7)-氰基硼烷鸟嘌呤），已被用于表明双喹啉选择性识别DNA中某些富含GC的序列，并确定一些影响其DNA结合序列选择性的因素。具体而言，足迹数据表明，嘌呤上的2-氨基或嘧啶上的5-甲基对于双喹啉-DNA复合物的形成不是必需的，而鸟嘌呤面向大沟的N(7)在分子识别过程中似乎是一个关键元素。鸟嘌呤而非腺嘌呤的N(7)缺失足以几乎消除双喹啉与DNA的序列选择性结合。因此，正如对大沟结合药物的预期，鸟嘌呤的N(7)通常是与GC碱基对有效形成复合物所必需的，但有趣的是，用相对较大的氰基硼烷基团取代N(7)不会明显影响序列识别过程。因此，鸟嘌呤N(7)的氢键接受能力不足以解释GC选择性药物-DNA结合，并且考虑了这些发现的意义。
