STRUCTURE AND INTERACTIONS OF DNA

我们有几个广泛的目标，都集中在是否以及在多大程度上 最近邻碱基对之间的堆积力有助于选择性地 DNA的物理和生物学特性。 最直接、最具体的 目标是量化堆积相互作用的自由能， 十个唯一的邻居对。 堆积能的灵敏测量 将由急剧亚转变的熔化温度制成， 专门设计的，多拷贝，串联重复合成插入， 多聚质粒DNA，其显示出容易检测到的Tm变化， 响应于堆叠能量的微小变化。 微量热 还将对这些结构进行测量， 测量频域相关性对相邻频率的依赖性。 的 本项目的进一步目标将是研究选定的序列 天然和合成序列中的结构域， 热稳定性;检查堆叠能与 用单晶衍射分析确定局部螺旋参数; 探索修复模式对堆叠能量的依赖性， 不匹配的碱基对，以及突变模式和进化 所选序列元素的模式。 相似的热稳定性 将在特定的相邻物上进行堆积能的测量， 类似地以重复或束的形式组装在合成插入物中， 以确定这些纤维束是否表现出异常稳定性， 归因于双工中的下一个邻居和更远的交互。 中 相关的研究，敏感的热稳定性研究将进行 含有多拷贝串联体的重组质粒双链体 具有特定错配的合成插入物的重复。 的目标 这些研究是为了测量选定的错配对的能量学， 本地序列环境的影响。 这些热力学研究 将伴随测序研究，以确定 在E. coli细胞。

We have several broad goals and all focus on whether and to what extent stacking forces between nearest neighbor base pairs contribute to selective physical and biological properties of DNA. The most immediate, specific goal is the quantification of free energies of stacking interactions for the ten unique neighbor pairs. Sensitive measurements of stacking energies will be made from melting temperatures of sharp subtransitions for specially designed, multicopy, tandemly repeating synthetic inserts in polymeric plasmid DNAs, that exhibit readily detectable shifts in Tm in response to small variations in stacking energy. Microcalorimetric measurements will also be performed on these constructs in an attempt to measure the dependence of domain enthalpies on neighbor frequencies. The further goals of this project will then be to investigate selected sequence domains in both natural and synthetic sequences that exhibit anomalous thermal stabilities; to examine the relationship of stacking energies to local helical parameters determined by single crystal diffraction analysis; to explore for a dependence on stacking energies of patterns of repair of mismatched base pairs, as well as of mutation patterns and evolutionary patterns of selected sequence elements. Similar thermal stability measurements of stacking energies will be made on specific neighbors, similarly assembled in synthetic inserts in the form of repeats or tracts, to determine whether such tracts exhibit anomalous stabilities that can be attributed to next neighbor and beyond interactions in the duplex. In a related study, sensitive thermal stability studies will be conducted on reassociated recombinant plasmid-duplexes containing multicopy tandem repeats of synthetic inserts with specific mispairs. The objective of these studies is to measure the energetics of selected mispairs as well as the effects of the local sequence environment. These thermodynamic studies will be accompanied by sequencing studies to determine the nature of mispair correction in these inserts after transformation of E. coli cells.