Counterion and polythymidine loop-length-dependent folding and thermodynamic stability of DNA hairpins reveal the unusual counterion-dependent stability of tetraloop hairpins.
Counterion and polythymidine loop-length-dependent folding and thermodynamic stability of DNA hairpins reveal the unusual counterion-dependent stability of tetraloop hairpins.
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DOI：
10.1021/jp404832d
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发表时间：
2013-11
期刊：
影响因子：
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通讯作者：
R. Nayak;A. Van Orden
中科院分区：
文献类型：
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作者：
R. Nayak;A. Van Orden
利用热力学解链分析和停流动力学研究了含有5个碱基对（bp）茎和单链多聚胸苷环的茎环DNA发夹。这些研究揭示了热力学稳定性和折叠动力学作为环长度和抗衡离子浓度的函数。我们的研究结果表明，与较长的环长度发夹相比，四环或4聚（dT）环发夹具有异常高的热力学稳定性。此外，这种特殊的稳定性是高度依赖于反离子的。例如，在50 mM NaCl和更高的较高浓度浓度方案中，与较长的环长度发夹相比，四环发夹显示出增强的稳定性。然而，在25 mM NaCl和更低的较低抗衡离子浓度下，四环发夹的热稳定性与较长的环发夹一致。四环发夹在较高浓度下的稳定性增强可以解释为基于环闭合以及环区域中的碱基堆积的组合熵效应。在所有浓度下的较长环长度发夹以及在较低浓度下的四环发夹的稳定性可以单独基于环闭合的熵效应来解释。在较低和较高的浓度的热力学参数进行了测定，以量化增强的稳定性的基础上堆积效应发生在较高的浓度。例如，对于100 mM NaCl，由于四环内的碱基堆积而导致的过量吉布斯能和焓分别测量为-1.2 ± 0.14和-3.28 ± 0.32 kcal/mol，而对于0、5、10和25 mM NaCl没有观察到过量的吉布斯能和焓。这些研究结果表明，显着的碱基堆积相互作用发生在环区的四环发夹在较高的浓度和较低的浓度制度，在较低的浓度制度不太显着的碱基堆积的相互作用。我们建议，在较高的反离子浓度，疏水性的崩溃的核苷酸在环中可能会增强由于极性的溶剂，从而增强碱基堆积的相互作用，有助于异常高的稳定性。
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.