Lee Hui-Ting, Carr Caroline, Siebler Hollie, Waters Lela, Khutsishvili Irine, Iseka Fany, Domack Brian, Olsen Chris M, Marky Luis A
Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, Nebraska, USA.
Methods Enzymol. 2011;492:1-26. doi: 10.1016/B978-0-12-381268-1.00013-6.
The main focus of our investigations is to further our understanding of the physicochemical properties of nucleic acid structures. We report on a thermodynamic approach to study the reaction of a variety of intramolecular nucleic acid structures with their respective complementary strands. Specifically, we have used a combination of isothermal titration (ITC) and differential scanning calorimetry (DSC) and spectroscopy techniques to determine standard thermodynamic profiles for the reaction of a triplex, G-quadruplex, hairpin loops, pseudoknot, and three-arm junctions with their complementary strands. Reaction enthalpies are measured directly in ITC titrations, and compared with those obtained indirectly from Hess cycles using DSC unfolding data. All reactions investigated yielded favorable free energy contributions, indicating that each single strand is able to invade and disrupt the corresponding intramolecular DNA structure. These favorable free energy terms are enthalpy-driven, resulting from a favorable compensation of exothermic contributions due to the formation of additional base-pair stacks in the duplex product, and endothermic contributions, from the disruption of base stacking contributions of the reactant single strands. The overall results provide a thermodynamic approach that can be used in the targeting of nucleic acids, especially the secondary structures formed by mRNA, with oligonucleotides for the control of gene expression.
我们研究的主要重点是进一步加深对核酸结构物理化学性质的理解。我们报告了一种热力学方法，用于研究各种分子内核酸结构与其各自互补链的反应。具体而言，我们结合了等温滴定量热法（ITC）、差示扫描量热法（DSC）和光谱技术，以确定三链体、G-四链体、发夹环、假结和三臂连接体与其互补链反应的标准热力学曲线。反应焓在ITC滴定中直接测量，并与使用DSC解链数据从赫斯循环间接获得的结果进行比较。所有研究的反应都产生了有利的自由能贡献，表明每条单链都能够侵入并破坏相应的分子内DNA结构。这些有利的自由能项是由焓驱动的，这是由于双链产物中形成额外碱基对堆积所产生的放热贡献与反应物单链碱基堆积贡献的破坏所产生的吸热贡献之间的有利补偿。总体结果提供了一种热力学方法，可用于利用寡核苷酸靶向核酸，特别是由mRNA形成的二级结构，以控制基因表达。
