Our laboratory is interested in developing methods that can be used for the control of gene expression. In this work, we are investigating the reaction of an intramolecular complex containing a triplex–duplex junction with partially complementary strands. We used a combination of isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), and spectroscopy techniques to determine standard thermodynamic profiles for these targeting reactions. Specifically, we have designed single strands to target one loop (CTTTC) or two loops (CTTTC and GCAA) of this complex. Both reactions yielded exothermic enthalpies of −66.3 and −82.8 kcal/mol by ITC, in excellent agreement with the reaction enthalpies of −72.7 and −88.7 kcal/mol, respectively, obtained from DSC Hess cycles. The favorable heat contributions result from the formation of base-pair stacks involving mainly the unpaired bases of the loops. This shows that each complementary strand is able to invade and disrupt the secondary structure. The simultaneous targeting of two loops yielded a more favorable reaction free energy, by approximately −8 kcal/mol, which corresponds to the formation of roughly four base-pair stacks involving the unpaired bases of the 5′-GCAA loop. The main conclusion is that the targeting of loops with a large number of unpaired bases results in a more favorable reaction free energy.

中文翻译：

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结合三链体-双链体域的DNA分子内复合物的展开和靶向热力学

我们的实验室对开发可用于控制基因表达的方法感兴趣。在这项工作中，我们正在研究分子内复合物的反应，该分子内复合物包含具有部分互补链的三链体-双链体连接。我们使用等温滴定热法（ITC），差示扫描量热法（DSC）和光谱技术的组合来确定这些靶向反应的标准热力学曲线。具体来说，我们设计了单链以靶向该复合物的一个环（CTTTC）或两个环（CTTTC和GCAA）。两种反应均通过ITC产生了-66.3和-82.8 kcal / mol的放热焓，分别与从DSC Hess循环获得的-72.7和-88.7 kcal / mol的反应焓非常吻合。有利的热贡献是由于形成了主要涉及环的未配对碱基的碱基对叠层。这表明每条互补链都能够侵入并破坏二级结构。同时靶向两个环产生约-8 kcal / mol的更有利的反应自由能，这对应于涉及5'-GCAA环未配对碱基的大约四个碱基对堆叠的形成。主要结论是，靶向具有大量不成对碱基的环可产生更有利的反应自由能。大约为-8 kcal / mol，这对应于涉及5'-GCAA环的未配对碱基的大约四个碱基对堆叠的形成。主要结论是，靶向具有大量不成对碱基的环导致了更有利的反应自由能。大约为-8 kcal / mol，这对应于涉及5'-GCAA环的未配对碱基的大约四个碱基对堆叠的形成。主要结论是，靶向具有大量不成对碱基的环导致了更有利的反应自由能。