Department of Pharmaceutical Sciences, University of Nebraska Medical Center, 986025 Nebraska Medical Center, Omaha, Nebraska 68198-6025, USA.
J Phys Chem B. 2010 Jan 14;114(1):541-8. doi: 10.1021/jp9084074.
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. Nucleic acid oligonucleotides (ODNs), as drugs, present an exquisite selectivity and affinity that can be used in antigene and antisense strategies for the control of gene expression. In this work, we try to answer the following question: How does the molecularity of a DNA complex affect its overall stability and melting behavior? We used a combination of temperature-dependent UV spectroscopy and calorimetric (DSC) techniques to investigate the melting behavior of DNA complexes with a similar helical stem sequence, TC(+)TC(+)TC(+)T/AGAGAGACGCG/CGCGTCTCTCT, but formed with different strand molecularity. We determined standard thermodynamic profiles, and the differential binding of protons and counterions accompanying their unfolding. The formation of a DNA complex is accompanied by a favorable free energy term resulting from the typical compensation of favorable enthalpy-unfavorable entropy contributions. As expected, acidic pH stabilized each complex by allowing protonation of the cytosines in the third strand; however, the percentage of protonation increases as the molecularity decreases. The results help in the design of oligonucleotide sequences as targeting reagents that could effectively react with DNA or RNA sequences involved in human diseases, thereby increasing the feasibility of using the antigene and antisense strategies, respectively, for therapeutic purposes.
我们实验室的研究兴趣在于从核酸序列知识中预测核酸的热稳定性和熔化行为。其中一个重点是研究序列、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）的熔化行为。作为药物的核酸寡核苷酸 (ODN) 具有极高的选择性和亲和力，可用于反基因和反义策略来控制基因表达。在这项工作中，我们试图回答以下问题：DNA 复合物的分子数如何影响其整体稳定性和熔化行为？我们使用温度依赖的紫外光谱和量热法（DSC）技术来研究具有相似螺旋茎序列的 DNA 复合物的熔化行为，TC(+)TC(+)TC(+)T/AGAGAGACGCG/CGCGTCTCTCT，但形成不同的链分子数。我们确定了标准热力学曲线，并测定了伴随其解折叠的质子和抗衡离子的差分结合。DNA 复合物的形成伴随着有利的自由能，这是由于典型的焓有利熵不利贡献的补偿。正如预期的那样，酸性 pH 通过允许第三链中的胞嘧啶质子化来稳定每个复合物；然而，随着分子数的减少，质子化的百分比增加。这些结果有助于设计作为靶向试剂的寡核苷酸序列，这些靶向试剂可以有效地与涉及人类疾病的 DNA 或 RNA 序列反应，从而分别增加使用反基因和反义策略进行治疗的可行性。
