Mechanism of Triplex DNA Formation ; And Its Application to Antisense

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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.