Thermodynamics of an intramolecular DNA triple helix: a calorimetric and spectroscopic study of the pH and salt dependence of thermally induced structural transitions.
Thermodynamics of an intramolecular DNA triple helix: a calorimetric and spectroscopic study of the pH and salt dependence of thermally induced structural transitions.
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DOI：
10.1006/jmbi.1995.0251
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发表时间：
1995-05
影响因子：
5.6
通讯作者：
Plum Ge;K. Breslauer
中科院分区：
文献类型：
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作者：
Plum Ge;K. Breslauer
我们对DNA 31-mer寡核苷酸（5'- gaagaggtttttcctcttctttttcttcccc -3'）的熔融转变进行了热力学表征，该寡核苷酸被设计成分子内三螺旋结构。前19个残基在自身上折叠，形成具有T5环的反平行沃森-克里克发夹双工。3'端7个残基通过第二个T5环连接到沃森-克里克发夹双链，在沃森-克里克发夹的主槽中形成Hoogsteen相互作用。从紫外（UV）熔融研究中，我们发现31-mer根据溶液条件表现出一个或两个转变。我们使用依赖于pH和温度的圆二色性（CD）来分配与每个转变相关的初始和最终状态。我们发现Hoogsteen发夹的断裂伴随着质子的释放和钠离子的摄取，而Watson-Crick发夹的断裂伴随着钠离子的释放，但质子化状态没有改变。根据这些数据，我们构建了分子内DNA三螺旋结构随pH、钠离子浓度和温度变化的相图。我们用范霍夫分析和差示扫描量热法（DSC）表征了每个转变的能量学。值得注意的是，DSC数据提供了与模型无关的热诱导转变的热力学表征。通过结合光谱和量热数据，我们开发了一个半经验模型，该模型描述了31-mer的状态作为pH、钠离子浓度和温度的函数。利用该模型，我们成功地预测了31-mer的特性，这些特性超出了建立模型所使用的数据（例如，Hoogsteen链表观pKa的盐依赖性）。这种半经验模型可以作为开发预测分子内三螺旋体系相图方法的原型。
We have characterized thermodynamically the melting transitions of a DNA 31-mer oligonucleotide (5'-GAAGAGGTTTTTCCTCTTCTTTTTCTTCTCC-3') which is designed to fold into an intramolecular triple helix. The first 19 residues fold back on themselves to form an antiparallel Watson-Crick hairpin duplex with a T5 loop. The 3'-terminal seven residues, which are connected to the Watson-Crick hairpin duplex by a second T5 loop, form Hoogsteen interactions in the major groove of the Watson-Crick hairpin. From ultraviolet (UV) melting studies we find that the 31-mer exhibits either one or two transitions, depending on solution conditions. We use pH- and temperature-dependent circular dichroism (CD) to assign the initial and final states associated with each transition. We find that the disruption of the Hoogsteen hairpin is accompanied by a release of protons and an uptake of sodium ions while the disruption of the Watson-Crick hairpin is accompanied by a release of sodium ions with no change in protonation state. From these data, we construct a phase diagram for this intramolecular DNA triple helix as a function of pH, sodium ion concentration, and temperature. We characterize the energetics of each transition using a van't Hoff analysis and differential scanning calorimetry (DSC). Significantly, the DSC data provide a model-independent thermodynamic characterization of the thermally induced transitions of this triplex. By combining the spectroscopic and calorimetric data, we develop a semi-empirical model which describes the state of the 31-mer as a function of pH, sodium ion concentration, and temperature. With this model we successfully predict characteristics of the 31-mer, which are beyond the data which are used in establishing the model (for example, the salt dependence of the apparent pKa of the Hoogsteen strand). This semi-empirical model may serve as a prototype for developing a method to predict the phase diagrams of intramolecular triple helix systems.