THERMODYNAMIC STUDIES OF DNA INTRAMOLECULAR STRUCTURES

在分析理论得以发展、测试和实施之前， 构建基因的物理图谱，并准确预测 二级结构从一级序列单独，数据库 围绕DNA的序列依赖稳定性， 发夹和内环的形成和序列依赖性 三链体的形成必须大大扩展。 这项建议的目标是扩大热力学的基础， 围绕这些DNA分子内序列依赖性的知识 结构. 热变性和差示扫描量热法 将是所采用的实验技术。 实验对象 主要是哑铃状DNA双链DNA的两端都是交联的 通过单链环。 实验将根据以下方面进行分析 最近邻序列相关的、数值精确的、统计的 DNA熔解的热力学理论 有七个具体目标， 该项目(1)发夹环自由能的计算 由Xn（X-A，T，G，C; n - 2-14）核苷酸组成的环的形成 残基(2)评估异质循环序列对 DNA发夹的稳定性，来自DNA发夹的解链研究， 四个基本环序列X-Y-Y-X（X，Y-A，T，G，C，X不等于Y）。.（三） 评估第一个碱基对的序列同一性的影响， 从DNA的解链研究中，双链体茎成核为发夹环 具有通过Y 4末端环连接的双链体序列5 'GGATAX 3'的发夹（X，Y - A、T、G、C）。(4)评估最近邻碱基对堆叠 DNA中的自由能是序列结构域长度的函数。(5)评价 形成小的（2-14个碱基对）和大的（大于 80个碱基对）在DNA中的两个完整的 螺旋区(6)确定最近邻序列的影响， 序列错配对DNA三链体稳定性的影响 哑铃和单股。(7)确定烷基（十二烷基和 十六烷基）链共价连接到DNA双链体的末端， 双链体区域的稳定性和限制酶切割。

Before analytical theories can be developed, tested and implemented to construct physical maps of genes and accurately predict the formation of secondary structure from primary sequence alone, the data base surrounding the sequence dependent stability of DNA, the energetics of hairpin and internal loop formation and the sequence dependence of triplex formation must be considerably expanded. The goal of this proposal is to expand the basis of thermodynamic knowledge surrounding the sequence dependence of these DNA intramolecular structures. Thermal denaturation and differential scanning calorimetry will be the experimental techniques employed. The experimental subjects will primarily be DNA dumbbells; duplex DNA's cross-linked on both ends by single strand loops. Experiments will be analyzed in terms of the nearest-neighbor sequence dependent, numerically exact, statistical thermodynamic theory of DNA melting. There are seven specific aims of the project. (1) Evaluation of the free-energies of hairpin loop formation for loops comprised of Xn (X - A,T,G,C; n - 2-14) nucleotide residues. (2) Evaluate the effects of heterogeneous loop sequence on the stability of DNA hairpins from melting studies of DNA hairpins with the four base loop sequences X-Y-Y-X (X,Y - A,T,G,C, X not equal to Y). . (3) Evaluate the influence of the sequence identity of the first base-pair of the duplex stem nucleating a hairpin loop from melting studies of DNA hairpins with the duplex sequence 5'GGATAX3'linked by Y4 end-loops (X,Y - A,T,G,C). (4) Evaluate the nearest-neighbor base-pair stacking free-energies in DNA as function of sequence domain length. (5) Evaluate the energetics of forming small (2-14 basepairs) and larger (greater than 80 base - pairs) internally melted loops in DNA between two intact helical regions. (6) Determine effects of nearest-neighbor sequence and sequence mis-matches on the stability of DNA triplexes formed from DNA dumbbells and single strands. (7) Determine effects of alkyl (dodecyl and hexadecyl) chains covalently attached to the ends of a DNA duplex on the stability and restriction enzyme cleavage of the duplex region.