Khutsishvili Irine, Johnson Sarah, Lee Hui-Ting, Marky Luis A
Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center, Omaha, Nebraska, USA.
Methods Enzymol. 2009;466:477-502. doi: 10.1016/S0076-6879(09)66020-1. Epub 2009 Nov 13.
Our laboratory is interested in predicting the thermal stability and melting behavior of nucleic acids from knowledge of their sequence. One focus is to understand how sequence, duplex and triplex stabilities, and solution conditions affect the melting behavior of complex DNA structures, such as intramolecular DNA complexes containing triplex and duplex motifs. For these reasons, in this chapter, we used a combination of UV and circular dichroism (CD) spectroscopies and differential scanning calorimetry (DSC) techniques to obtain a full thermodynamic description of the melting behavior of six intramolecular DNA complexes with joined triplex and duplex motifs. The CD spectra at low temperatures indicated that these complexes maintained the "B" conformation. UV and DSC melting curves of each complex show biphasic or triphasic transitions. However, their corresponding transition temperatures (T(m)s) remained constant with increasing strand concentration, confirming their intramolecular formation. Deconvolution of the DSC thermograms allowed us to determine standard thermodynamic profiles for the transitions of each complex. For each transition, the favorable free energy terms result from the characteristic compensation of a favorable enthalpy and unfavorable entropy contributions. The magnitude of these thermodynamic parameters (and associated T(m)s) indicate that the overall folding of each complex depends on several factors: (a) the extent of the favorable heat contributions (formation of base-pair and base-triplet stacks) that are compensated with both the ordering of the oligonucleotide and the putative uptake of protons and ions; (b) inclusion of the more stable C(+)GC base triplets; (c) stabilizing the duplex stem of the complex; and (d) solution conditions, such as pH and salt concentration. Overall, the temperature-induced unfolding of each complex corresponds to the initial disruption of the triplex motif (removal of the third strand) followed by the partial or full unfolding of the duplex stem.
我们实验室致力于从核酸序列信息预测其热稳定性和熔解行为。其中一个重点是了解序列、双链和三链稳定性以及溶液条件如何影响复杂DNA结构（如包含三链和双链基序的分子内DNA复合物）的熔解行为。基于这些原因，在本章中中中我们结合使用紫外光谱、圆二色光谱（CD）和差示扫描量热法（DSC）技术，以全面热力学描述六个具有相连三链和双链基序的分子内DNA复合物的熔解行为。低温下的CD光谱表明这些复合物保持“B”构象。每个复合物的紫外和DSC熔解曲线显示出双相或三相转变。然而，随着链浓度增加，其相应的转变温度（Tm）保持恒定，证实了它们的分子内形成。对DSC热谱图进行去卷积处理使我们能够确定每个复合物转变的标准热力学概况。对于每个转变，有利的自由能项源于有利的焓贡献和不利的熵贡献之间的特征性补偿。这些热力学参数（以及相关的Tm）的大小表明每个复合物的整体折叠取决于几个因素：（a）有利的热贡献程度（碱基对和碱基三联体堆积的形成），这与寡核苷酸的有序排列以及质子和离子的假定摄取相互补偿；（b）包含更稳定的C(+)GC碱基三联体；（c）稳定复合物的双链茎；（d）溶液条件，如pH和盐浓度。总体而言，每个复合物的温度诱导解折叠对应于三链基序的初始破坏（第三条链的去除），随后是双链茎的部分或完全解折叠。
