Thermodynamic Origins of Sequence-Recognition in Ligand-DNA Interactions

这项研究的长期目标是深入了解小分子与核酸序列选择性相互作用的关键方面。到目前为止所研究的模型体系包括序列特异的DNA结合剂放线菌素D。该化合物通过插入相邻的G和C碱基对之间的苯恶唑酮发色团，在d(GPC)步显示出明显的DNA结合偏好。伴随着这种插层结合的是两个环状五肽侧链与DNA小沟底部的相互作用。这个实验室最近证明了这个插层位置两侧的碱基在指导络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-)，则结合热变为-2.5kcal/mol。相反，当5‘-侧翼碱基为C(-CGCA-)时，结合热的变化为-5.8kcal/mol，比其他侧翼序列有利近3kcal/mol。5历史上，放线菌素D与DNA的结合被认为是由大的正(有利)结合热和接近零或-1kcal/mol的结合热驱动的。本实验室的这些最新结果表明，放线菌素D与具有特殊设计的单一结合位点的寡核苷酸的热力学结合性质有很大的不同，这取决于放线菌素D结合位点两侧的碱基序列。根据观察到的与络合物形成有关的焓和熵分量的变化，探索了放线菌素D-DNA络合物的热力学结合机理(S)与结构特征之间的联系。具体地说，将用高分辨核磁共振方法研究放线菌素D与-TGCA-双链和-CGCA-双链形成的放线菌素D复合体的结构，以辨别该复合体结构特征的差异，以解释放线菌素D与CGCA-双链结合的热效应增强。非自互补脱氧寡核苷酸的使用偶然发现了放线菌素D与单链DNA的序列选择性相互作用。DNA碱基序列对放线菌素D-单链DNA相互作用的能量和结构性质的影响正在研究中。这些观察结果可能对深入了解蛋白质如何以序列选择性的方式结合单链DNA并扩展到单链DNA相互作用的生物学作用有相当大的好处。放线菌素D与双链DNA的体内相互作用具有高亲和力和缓慢解离的特点，并被认为通过阻断RNA聚合酶沿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的高亲和力，可能会对这一拟议的机制增加额外的警告，包括药物可能与开放复合体中的单链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.