Wadkins R M, Jares-Erijman E A, Klement R, Rüdiger A, Jovin T M
Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
J Mol Biol. 1996 Sep 13;262(1):53-68. doi: 10.1006/jmbi.1996.0498.
We have studied the sequence specificity in the binding of the potent antitumor drug actinomycin D (AMD) to single-stranded DNA (ssDNA) by fluorescence and NMR spectroscopy and by molecular modeling. The significant absorption and emission changes accompanying the interaction of the fluorescent derivative 7-amino-AMD with DNAs varying in length and base composition were used to calculate affinity constants for the drug-DNA complexes. The guanine-containing trinucleotide sequences AGT, AGA, and TGT embedded within 25-base oligonucleotides, constituted favorable binding sites. In contrast, the sequence TGA did not bind the drug appreciably. Among the DNAs studied, the highest affinity was for the tetranucleotide sequence TAGT. The binding was length dependent, an oligonucleotide of at least 14 bases being required for effective complex formation (Ka > 10(4) M1=). AMD also bound to poly(d(AGT)). Gel electrophoresis confirmed that the complex was formed between the drug and individual unstructured DNA strands. The 1H NMR spectra of oligonucleotides containing the TAGT site and their complexes with AMD provided further insight into the mode(s) of interaction. A comparison of the measured chemical shifts with those estimated from ring-current calculations provided strong evidence for a hemi-intercalation of AMD between the A and G purine bases with a preference for one of two possible relative orientations. The latter were modeled as complexes with the sequence T3AGT3 and refined by force field calculations with the AMBER program. The biological implications for this novel form of interaction of AMD with single-stranded DNA are discussed.
我们通过荧光光谱、核磁共振光谱以及分子建模，研究了强效抗肿瘤药物放线菌素D（AMD）与单链DNA（ssDNA）结合的序列特异性。利用荧光衍生物7-氨基-AMD与长度和碱基组成各异的DNA相互作用时伴随的显著吸收和发射变化，来计算药物-DNA复合物的亲和常数。嵌入25个碱基的寡核苷酸中的含鸟嘌呤三核苷酸序列AGT、AGA和TGT构成了有利的结合位点。相比之下，序列TGA与药物的结合并不明显。在所研究的DNA中，对四核苷酸序列TAGT的亲和力最高。结合具有长度依赖性，有效形成复合物（Ka>10(4) M-1）需要至少14个碱基的寡核苷酸。AMD也与聚（d(AGT)）结合。凝胶电泳证实药物与单个无结构的DNA链之间形成了复合物。含有TAGT位点的寡核苷酸及其与AMD的复合物的1H NMR光谱进一步深入了解了相互作用模式。将测得的化学位移与通过环电流计算估计的化学位移进行比较，为AMD在A和G嘌呤碱基之间半嵌入提供了有力证据，且对两种可能的相对取向之一有偏好。后者被建模为与序列T3AGT3的复合物，并通过使用AMBER程序的力场计算进行优化。讨论了AMD与单链DNA这种新型相互作用形式的生物学意义。
