Thermodynamic Origins of Sequence-Recognition in Ligand-DNA Interactions

Graves，大卫E.MCB-0092177本研究的长期目标是获得对小分子与核酸的序列选择性相互作用的关键方面的生物物理见解。迄今为止研究的模型系统涉及序列特异性DNA结合剂，放线菌素D。该化合物通过在相邻的G和C碱基对之间插入吩恶嗪生色团，在d（GpC）步骤显示出对DNA结合的显著偏好。 伴随着这种嵌入结合的是两个环状五肽侧链与DNA小沟底部的相互作用。 该实验室最近已经证明了该插层位点侧翼的碱基在指导复合物形成的热力学机制中起着重要作用。 如果5 '-侧翼序列是T（-TGCA-），则结合焓的变化已被测量为-2.5 kcal/mol。相比之下，当5 '-侧翼碱基是C（-CGCA-）时，发现结合焓的变化为-5.8kcal/mol，比其他侧翼序列更有利近3 kcal/mol。5历史上，放线菌素D与DNA的结合据报道是焓驱动的，如大的正（有利）结合焓和接近零或-1千卡/摩尔的结合焓所示。本实验室最近的这些结果表明，放线菌素D的热力学结合特性的寡核苷酸与专门设计的单一结合位点是相当不同的，这取决于放线菌素D结合位点侧翼的碱基序列。基于在与复合物形成相关的焓和熵分量中观察到的变化，将探索放线菌素D-DNA复合物的热力学结合机制与结构特征之间的联系。 具体地，将通过高分辨率NMR方法检查与-TGCA-双链体和-CGCA-双链体形成的放线菌素D复合物的结构，以辨别复合物的结构特征的差异，这可以解释放线菌素D与CGCA-双链体结合的增强的焓贡献。自互补脱氧寡核苷酸是偶然发现的ofsequence选择性相互作用放线菌素D的单链DNA。DNA碱基序列对放线菌素D-单链DNA相互作用的能量学和结构特性的影响正在研究中。这些观察可能是相当大的好处，在深入了解蛋白质如何结合单链DNA的序列选择性的方式，并扩大到单链DNA相互作用的生物学作用。放线菌素D与双链DNA的体内相互作用的特征在于高亲和力和缓慢解离，并且已经被认为通过阻断RNA聚合酶沿着DNA模板的前进来实现其特异性抑制转录的生物学功能。随着最近发现放线菌素D对单链DNA具有高亲和力，可能会对这一拟议机制增加额外的警告，包括药物可能与开放复合物内的单链DNA结合，和/或抑制单链DNA的再退火。

Graves, David E.MCB-0092177The long-term objective of this research is to gain biophysical insight into the key aspects of sequence selective interactions of small molecules with nucleic acids. The model system that has been studied thus far involves the sequence-specific DNA binding agent, actinomycin D. This compounds shows a marked preference for DNA binding at the d(GpC) step through intercalation of the phenoxazone chromophore between the adjacent G and C base pairs. Concomitant with this intercalative binding is the interactions of the two cyclic pentapeptide sidechains with the floor of the DNA minor groove. This laboratory has recently demonstrated the bases that flank this intercalation site play a major role in directing the thermodynamic mechanism for complex formation. If the 5'-flanking sequence is T (-TGCA-), the change in binding enthalpy has been measured to be -2.5 kcal/mol. In contrast, when the 5'-flanking base is C (-CGCA-), the change in binding enthalpy is found to be -5.8 kcal/mol, nearly 3 kcal/mol more favorable than the other flanking sequences. 5 Historically, the binding of actinomycin D to DNA has been reported to be enthalpy driven as signified by large positive (favorable) binding enthalpies and binding enthalpies of near zero or -1 kcal/mol. These recent results from this laboratory indicate that the thermodynamic binding properties of actinomycin D to oligonucleotides with specifically designed single binding sites are quite varied, depending on the sequence of bases flanking the actinomycin D binding site. Based on the variances observed in the enthalpy and entropy components associated with complex formation, linkages between thermodynamic binding mechanism(s) and structural features of the actinomycin D-DNA complexeswil be explored. Specifically the structures of the actinomycin D complexes formed with the -TGCA- duplex and the -CGCA- duplex will be examined by high-resolution NMR methods to discern differences in the structural features of the complex that may explain the enhanced enthalpy contribution toward actinomycin D binding to the CGCA- duplex.The use of non-self complimentary deoxyoligonucleotides was serendipitous toward the finding ofsequence selective interactions of actinomycin D to single-strand DNA. The influence of DNA base sequence on the energetics and structural properties of actinomycin D-single strand DNA interactions is being examined. These observations could be of considerable benefit in gaining insight into how proteins bind single-strand DNA in a sequence-selective manner and expanded to biological roles of single-strand DNA interactions. The in vivo interactions of actinomycin D to double stranded DNA are characterized by high affinity and slow dissociation and have been suggested to carry out their biological function of specifically inhibiting transcription through blocking the progression of the RNA polymerase along the DNA template. With the recent findings of a high affinity of actinomycin D for single-stranded DNA, additional caveats may be added to this proposed mechanism, including the possible binding of the drug to single-strand DNA within the open complex, and/or to inhibiting reannealing of the single-strand DNA.