Department of Chemistry, Colorado State University , Fort Collins, Colorado 80523, United States.
J Phys Chem B. 2013 Nov 14;117(45):13956-66. doi: 10.1021/jp404832d. Epub 2013 Nov 5.
Stem-loop DNA hairpins containing a 5-base-pair (bp) stem and single-stranded polythymidine loop were investigated using thermodynamic melting analysis and stopped-flow kinetics. These studies revealed the thermodynamic stability and folding kinetics as a function of loop length and counterion concentration. Our results show the unusually high thermodynamic stability for tetraloop or 4 poly(dT) loop hairpin as compared with longer loop length hairpins. Furthermore, this exceptional stability is highly counterion-dependent. For example, in the higher counterion concentration regime of 50 mM NaCl and above, the tetraloop hairpin displays enhanced stability as compared with longer loop length hairpins. However, at lower counterion concentration of 25 mM NaCl and below, the thermal stability of tetraloop hairpin is consistent with the longer loop hairpins. The enhanced stability of tetraloop hairpins at higher counterion concentration can be explained on the basis of the combined entropic effect of loop closure as well as base stacking in the loop regions. The stability of longer loop length hairpins at all counterion concentrations as well as tetraloop hairpin at lower counterion concentration can be explained on the basis of entropic effect of loop closure alone. The thermodynamic parameters at lower and higher counterion concentrations were determined to quantify the enhanced stability of base-stacking effects occurring at higher counterion concentrations. For example, for 100 mM NaCl, excess Gibbs energy and enthalpy due to base stacking within the tetraloops were measured to be -1.2 ± 0.14 and -3.28 ± 0.32 kcal/mol, respectively, whereas, no excess of Gibbs energy and enthalpy was observed for 0, 5, 10, and 25 mM NaCl. These findings suggest significant base-stacking interactions occurring in the loop region of the tetraloop hairpins at higher counterion concentration and less significant base-stacking interactions in the lower counterion concentration regime. We suggest that at higher counterion concentrations, hydrophobic collapse of the nucleotides in the loop may be enhanced due to the increased polarity of the solvent, thereby enhancing base-stacking interactions that contribute to unusually high stability.
采用热力学熔融分析和停流动力学研究了含有 5 个碱基对（bp）茎和单链聚胸腺嘧啶环的茎环 DNA 发夹。这些研究揭示了环长度和抗衡离子浓度的功能的热力学稳定性和折叠动力学。我们的结果表明，与较长环长度发夹相比，四聚体或 4 聚（dT）环发夹具有异常高的热力学稳定性。此外，这种异常稳定性高度依赖于抗衡离子。例如，在更高的抗衡离子浓度 50 mM NaCl 及以上的范围内，四聚体发夹与较长环长度发夹相比显示出增强的稳定性。然而，在较低的 25 mM NaCl 及以下的抗衡离子浓度下，四聚体发夹的热稳定性与较长环发夹一致。在较高的抗衡离子浓度下，四聚体发夹的增强稳定性可以基于环闭合的熵效应以及环区碱基堆积的组合来解释。在所有抗衡离子浓度下的较长环长度发夹以及在较低抗衡离子浓度下的四聚体发夹的稳定性可以基于环闭合的熵效应来解释。在较低和较高抗衡离子浓度下确定热力学参数以量化在较高抗衡离子浓度下发生的碱基堆积效应的增强稳定性。例如，对于 100 mM NaCl，在四聚体环内碱基堆积的超额吉布斯自由能和焓分别测量为-1.2 ± 0.14 和-3.28 ± 0.32 kcal/mol，而对于 0、5、10 和 25 mM NaCl 则没有观察到超额吉布斯自由能和焓。这些发现表明在较高抗衡离子浓度下，四聚体发夹环区发生显著的碱基堆积相互作用，而在较低抗衡离子浓度下的碱基堆积相互作用则不那么显著。我们建议，在较高的抗衡离子浓度下，由于溶剂极性的增加，环中核苷酸的疏水塌陷可能会增强，从而增强有助于异常高稳定性的碱基堆积相互作用。
