Mechanism of Triplex DNA Formation ; And Its Application to Antisense

为了了解三链DNA形成的稳定性和特异性的机制，我们进行了实验和计算分析。我们在不同的位置合成了经过化学修饰的DNA，并测量了三链形成的热力学参数如解离常数和动力学参数如缔合速率常数。这些结果表明，化学修饰，特别是主链上的化学修饰，影响了三链形成的热力学和动力学数量。我们试图通过计算分析来研究这种效应的分子机制。我们对修饰后的DNA进行了量子化学计算，得到了DNA的部分原子电荷和构象。我们发现DNA的硫代修饰对电荷和构象有显著的影响。我们还使用了一种新的构象采样算法，对DNA进行了计算机模拟，计算了它的平均构象和涨落。化学修饰对DNA的平均构象和波动的影响取决于修饰的位置。这些结果表明，化学修饰可能通过改变DNA的结构和性质来影响三链DNA形成的稳定性和特异性。为了了解三链DNA在细胞中的生物学作用，我们对与三链DNA结合的MAZ蛋白进行了生化和功能分析。该蛋白作为转录因子，调节c-myc基因的表达。我们还检测了DNA的化学修饰对癌基因产物Myb结合和转录活性的影响。我们发现，这种修饰破坏了DNA的稳定性，并取消了Myb结合。目前对三链DNA的实验和计算研究为设计抑制基因表达的反义DNA奠定了基础。

In order to understand the mechanism of stability and specificity in triplex DNA formation, we performed experimental and computational analyses. We synthesized DNA with chemical modifications at different locations, and measured thermodynamic quantities such as dissociation constant and kinetic quantities such as association rate constant for the triplex formation. These results showed that the chemical modification, particularly at backbone, affected the thermodynamic and kinetic quantities of triplex formation. We attempted to investigate the molecular mechanism of that effect by computational analysis. We performed quantum-chemical calculations for the modified DNA to obtain partial atomic charges and conformation of DNA. We found that the phosphorothioation of DNA has significant effect on the charges and conformation. We also performed computer simulation of DNA to calculate its average conformation and fluctuation, by using a new algorithm for conformational sampling. The chemical modification affected the average conformation and fluctuation of DNA depending on the location of the modification. These results suggest that the chemical modification may affect the stability and specificity of triplex DNA formation through the changes in structure and property of DNA. In order to understand the biological role of triplex DNA in cell, we made biochemical and functional analyses of MAZ protein, which binds to triplex DNA. This protein acts as a transcription factor and regulates the expression of c-myc gene. We also examined the effect of chemical modification of DNA on the binding and transcriptional activities of an oncogene product, Myb. We found that the modification destabilized the DNA and abolished the Myb binding. The present experimental and computational studies of triplex DNA have laid a basis for the design of antisense DNA, which inhibits expression of genes.