TOPOLOGICAL ANALYSIS OF LOCAL STRUCTURE IN CLOSED DNA

拓扑和几何分析将应用于闭合双链DNA 为了预测在程度和类型上的差异的影响 在内部形成局部结构元素时的超级卷曲 双工的特定区域。特别是，扭曲和扭曲的意志 对几种可能的DNA结构模型进行计算，包括 不同维度的超螺旋交织在一起。从数学上讲，这是 涉及通信概念的进一步发展和使用 浮出水面。类似的技术将被应用于计算 特殊的局部结构，如十字架，既完美又包含 各种具体缺陷。数学处理将被推广 进一步确定链环的最可能构型 各种几何结构的DNA分子。为了测试测试结果， 理论分析，将合成短的寡核苷酸序列 通过化学方法克隆到适当构建的质粒DNA中。这个 这个项目的本金利息顺序是回文，来自 它可以作为扭转应力的结果而产生十字形 伴随着拓扑链接数的有序减少。这个 与双工关联的拓扑链接数的临界值 结构转换将通过解决以下问题来确定 双向凝胶电泳法中的拓扑异构体。结果是 环境条件的系统变化将被确定， 包括温度、离子强度、离子组成和pH。这个 回文中引入特定缺陷的后果 序列将被确定。拓扑和几何计算 将用于两种方式：(1)分析自由能的变化 与特定的局部结构元素的形成有关，以及(2) 为了预测对DNA全局性质的影响， 局部复式结构中的拓扑约束变化。这个 在这里获得的结果，以及计划稍后对克隆的扩展 寡核苷酸，将提供重要的热力学信息 二级和三级结构的预测。

Topological and geometric analysis will be applied to closed duplex DNA in order to predict the effects of variations in extent and type of supercoiling upon the formation of local structural elements within specific regions of the duplex. In particular, the twist and writhe will be calculated for several possible DNA structural models, including interwound superhelices of various dimensions. Mathematically this involves the further development and use of the concept of a correspondence surface. Similar techniques will be applied to calculate the twist of special local structures, such as cruciforms, both perfect and containing various specific defects. The mathematical treatment will be extended further to determine the most probable configuration for catenated circular DNA molecules of various geometries. In order to test the results of the theoretical analyses, short oligonucleotide sequences will be synthesized chemically and cloned into appropriately constructed plasmid DNAs. The sequence of principal interest in this project is the palindrome, from which a cruciform can arise as a consequence of the torsional stress which accompanies ordered reduction in the topological linking number. The critical value of the topological linking number associated with duplex structural transistions will be determined by means of resolution of topoisomers in two dimensional gel electrophoresis. The results of systematic variation in the environmental conditions will be ascertained, including temperature, ionic strength, ion composition, and pH. The consequences of the introduction of specific defects in palindromic sequences will be determined. The topological and geometric calculations will be used in two ways: (1) to analyze the free energy changes associated with the formation of specific local structural elements and (2) to predict the effect upon the global properties of the DNA of topologically constrained alterations in local duplex structure. The results obtained here, along with a projected later extention to cloned oligoribonucleotides, will provide vital thermodynamic information for the prediction of secondary and tertiary structures.