NMR-based model of a telomerase-inhibiting compound bound to G-quadruplex DNA
NMR-based model of a telomerase-inhibiting compound bound to G-quadruplex DNA
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DOI：
10.1021/bi981330n
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发表时间：
1998-09-08
期刊：
影响因子：
2.9
通讯作者：
Hurley, LH
中科院分区：
文献类型：
--
作者：
Fedoroff, OY;Salazar, M;Hurley, LH
已知人端粒DNA的单链（TTAGGG）（n）尾形成稳定的G-四链体结构。最佳的端粒酶活性需要引物的非折叠单链形式，并且已知G-四链体形式的稳定化会干扰端粒酶结合。我们已经通过使用引物延伸测定法鉴定了基于3，4，9，10-二萘嵌苯四羧酸二酰亚胺的配体作为人端粒酶的有效抑制剂，所述引物延伸测定法不使用基于PCR的端粒酶引物延伸产物扩增。使用与人端粒DNA相关的各种寡核苷酸将配体滴定到G-四链体的溶液中的一组NMR滴定显示配体与G-四链体的强和特异性结合。结合和游离DNA形式之间的交换速率在NMR时间尺度上是缓慢的，并且允许明确确定结合位点和结合模式。在5 '-TTAGGG序列的情况下，配体-DNA复合物由以尾对尾方式取向的两个四链体组成，其中配体夹在末端G4平面之间。较长的端粒序列，如TTAGGGTT、TTAGGGTTA和TAGGGTTA，通过穿线嵌入模式与GT步骤结合的配体形成1：1配体-四链体复合物。二维NOESY数据的基础上，后者复杂的模型已经推导出与现有的实验数据是一致的。这种端粒酶抑制剂结合到端粒四链体DNA的溶液结构的测定应有助于设计具有独特和新颖的作用机制的新抗癌剂。
The single-stranded (TTAGGG)(n) tail of human telomeric DNA is known to form stable G-quadruplex structures. Optimal telomerase activity requires the nonfolded single-stranded form of the primer, and stabilization of the G-quadruplex form is known to interfere with telomerase binding. We have identified 3,4,9,10-perylenetetracarboxylic diimide-based ligands as potent inhibitors of human telomerase by using a primer extension assay that does not use PCR-based amplification of the telomerase primer extension products. A set of NMR titrations of the ligand into solutions of G-quadruplexes using various oligonucleotides related to human telomeric DNA showed strong and specific binding of the ligand to the G-quadruplex. The exchange rate between bound and free DNA forms is slow on the NMR time scale and allows the unequivocal determination of the binding site and mode of binding. In the case of the 5'-TTAGGG sequence, the ligand-DNA complex consists of two quadruplexes oriented in a tail-to-tail manner with the ligand sandwiched between terminal G4 planes. Longer telomeric sequences, such as TTAGGGTT, TTAGGGTTA, and TAGGGTTA, form 1:1 ligand-quadruplex complexes with the ligand bound at the GT step by a threading intercalation mode. On the basis of 2D NOESY data, a model of the latter complex has been derived that is consistent with the available experimental data. The determination of the solution structure of this telomerase inhibitor bound to telomeric quadruplex DNA should help in the design of new anticancer agents with a unique and novel mechanism of action.