THERMODYNAMIC STUDIES OF DNA INTRAMOLECULAR STRUCTURES

在开发、测试和实施分析理论之前 构建基因的物理图谱并准确预测 二级结构仅由一级序列、数据库组成 围绕着DNA的序列依赖稳定性， 发夹和内环的形成及其序列依赖性 必须大幅扩大三联体的形成。 这项提议的目标是扩大热力学的基础。 关于这些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 将主要是DNA哑铃；两端的双链DNA交联 通过单股环路。实验将从以下方面进行分析 最近邻序列依赖、数值精确、统计 DNA融化的热力学理论。有七个具体目标 这个项目。(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 残留物。(2)评估异构环序列对系统性能的影响。 DNA发夹熔融研究中DNA发夹的稳定性 四个基环序列X-Y-Y-X(X，Y-A，T，G，C，X不等于Y)。。(3) 评估第一个碱基对的序列同一性的影响 DNA融化研究中形成发夹环的双链茎 具有由Y4末端环(X，Y-)连接的双链序列5‘GGATAX3’的发夹 A、T、G、C)。(4)评估最近邻碱基对堆积 DNA中的自由能是序列结构域长度的函数。(5)评估 形成较小(2-14个基准点)和较大(大于 80个碱基对)在两个完整的DNA之间内部融化的环 螺旋区域。(6)确定最近邻序列的影响，并 序列错配对DNA三联体稳定性的影响 哑铃和单股。(7)测定烷基(十二烷基和十二烷基)的影响 十六)链共价连接到DNA双链的末端 双链区的稳定性和限制性内切酶的切割。

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.