MINOR-GROOVE RECOGNITION OF THE SELF-COMPLEMENTARY DUPLEX D(CGCGAATTCGCG)2 BY HOECHST-33258 - A HIGH-FIELD NMR-STUDY
MINOR-GROOVE RECOGNITION OF THE SELF-COMPLEMENTARY DUPLEX D(CGCGAATTCGCG)2 BY HOECHST-33258 - A HIGH-FIELD NMR-STUDY
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DOI：
10.1021/bi00496a005
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发表时间：
1990-11-06
期刊：
影响因子：
2.9
通讯作者：
SHARPLES, D
中科院分区：
文献类型：
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作者：
PARKINSON, JA;BARBER, J;SHARPLES, D
使用合成的自我互补寡核苷酸双链体 d(CGCGAATTCGCG)2 研究了荧光 DNA 染料 Hoechst 33258 的相互作用。分光荧光斯卡查德分析表明仅存在单一类别的结合位点并且1:1复合物具有(3.47±0.1)×的解离常数。 25°C 时 10-6 M C. 通过高场 1H NMR 光谱滴定证实了 1:1 复合物，并通过 ID 和 2D（NOESY、COSY）技术将结合位点定义为由 AATT 拉伸形成的小沟。由于寡核苷酸在配体结合上的二重对称性的提升，可以测量到大量的交叉峰并且发生共振加倍。由于这些特征，以及 NMR 时间尺度上缓慢交换的发生，DNA 两条链的许多单独共振可以归属于该复合物。该 NMR 光谱溶液研究的结果与之前同一配合物的 X 射线晶体学研究的结果进行了比较。根据为该复合物测量的核奥沃豪塞效应数据，借助分子图形构建了详细的三维模型。
The interaction of Hoechst 33258, a fluorescent DNA stain, has been studied by using the synthetic, self-complementary oligonucleotide duplex d(CGCGAATTCGCG)2. Spectrofluorometric Scatchard analysis indicated that there was only a single class of binding site and that the 1:1 complex had a dissociation constant of (3.47 .+-. 0.1) .times. 10-6 M at 25.degree. C. Spectroscopic titration by high-field 1H NMR confirmed the 1:1 complex and by means of ID and 2D (NOESY, COSY) techniques the binding site was defined as the minor groove formed by the AATT stretch. Plentiful cross-peaks were measurable and resonance doubling occurred because of the lifting of the diad symmetry of the oligonucleotide on ligand binding. Many individual resonances of both strands of the DNA could be assigned for the complex because of these features, along with the occurrence of slow exchange on the NMR time scale. The results of this NMR spectroscopic solution study were compared with those of previous X-ray crystallographic studies of the same complex. From nuclear Overhauser effect data measured for the complex, a detailed three-dimensional model was constructed with the aid of molecular graphics.