STRUCTURE AND INTERACTIONS OF DNA

我们有几个广泛的目标，所有的重点都是是否以及在多大程度上 最近邻碱基对之间的堆叠力有助于选择性 DNA的物理和生物特性。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构成 特殊设计，多拷贝，简单重复的合成插入物 聚合质粒DNA，在TM中很容易检测到移位 对堆积能量的微小变化作出响应。微量热法 还将对这些构造进行测量，以尝试 测量磁区热容对相邻频率的依赖关系。这个 该项目的进一步目标将是调查选定的序列 自然序列和合成序列中显示异常的结构域 热稳定性；检查堆积能量与 用单晶衍射法测定局部螺旋参数； 探索修复模式对堆积能量的依赖关系 碱基对不匹配，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 堆积能量的测量将在特定的邻居上进行， 以重复或片状形式类似地组装在合成插入物中， 为了确定这些区域是否表现出异常的稳定性， 归因于双工中的隔壁邻居和外部交互。在一个 相关研究，敏感的热稳定性研究将在 含多拷贝串联的再结合重组质粒-双链 带有特定错对的合成插入物的重复。的目标是 这些研究是为了测量选定的错配对的能量以及 局部序列环境的影响。这些热力学研究 将伴随着测序研究以确定其性质 在大肠杆菌细胞转化后，这些插入片段中的错配校正。

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.