Energetics of a stable intramolecular DNA triple helix formation.
Energetics of a stable intramolecular DNA triple helix formation.
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稳定的分子内 DNA 三螺旋形成的能量学。
DOI：
10.1006/jmbi.1993.1241
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发表时间：
1993
影响因子：
5.6
通讯作者：
H. Klump
中科院分区：
文献类型：
--
作者：
J. Völker;D. P. Botes;G. Lindsey;H. Klump
我们用常规化学方法设计并合成了一个由5 ′ d（Pu）10 d（C）4d（Py）10 d（T）4d（Py）10（3 ′）序列组成的38聚体寡核苷酸。该寡核苷酸在pH 12时呈现随机卷曲构象。在pH 8.0下，在其5'嘌呤十聚体序列和连续的嘧啶十聚体之间形成发夹螺旋，留下第二个嘧啶十聚体作为悬空无序的3'延伸。在将pH降低至4.5时，该第二嘧啶十聚体折叠回到发夹螺旋的大沟上，导致分子内三链茎环结构。我们已经使用了各种生物化学（凝胶迁移率，P1核酸酶消化）和生物物理（紫外光和圆二色光谱，荧光，微量热）技术来表征不同的构象，它们的稳定性和折叠成分子内三螺旋的途径。在100 mM-Na+中，分子内三链的热力学性质为：tm，71 ℃; Δ HvH，119.4（+/- 11.9）kcal mol-1; Δ Hcal，121.9（+/- 6.1）kcal mol-1，pH 4.5;发夹的那些是：tm，63 ℃; Δ HvH，71.7（+/- 4.0）kcal mol-1; Δ Hcal，69.9（+/- 3.5）kcal mol-1（pH 8.0）。在中等pH值下，三链体到卷曲转变分解成其组分三链体到发夹和发夹到卷曲转变，其热力学性质为：tm，41摄氏度; Δ HvH，58.7（+/- 4.2）kcal mol-1; Δ Hcal，39.8（+/- 2.0）kcal mol-1;和tm，63 ℃; Δ HvH，71.7（+/- 4.0）kcal mol-1; Δ Hcal，69.6（+/- 3.5）kcal mol-1，pH 6.7。
We have designed and synthesized by conventional chemical techniques a 38mer oligonucleotide consisting of a 5'd(Pu)10d(C)4d(Py)10d(T)4d(Py)10(3') sequence. This oligonucleotide assumes a randomly coiled conformation at pH 12. At pH 8.0 a hairpin helix forms between its 5' purine decamer sequence and the consecutive pyrimidine decamer leaving the second pyrimidine decamer as a dangling disordered 3' extension. On reducing the pH to 4.5 this second pyrimidine decamer folds back onto the major groove of the hairpin helix resulting in an intramolecular triple-stranded stem-loop structure. We have used a variety of biochemical (gel mobility, P1 nuclease digestion) and biophysical (ultraviolet light and circular dichroism spectroscopy, fluorimetry, microcalorimetry) techniques to characterize the different conformers, their stability and the folding pathway into an intramolecular triple helix. The thermodynamic properties of this intramolecular triple strand in 100 mM-Na+ are: tm, 71 degrees C; delta HvH, 119.4(+/- 11.9) kcal mol-1; delta Hcal, 121.9 (+/- 6.1) kcal mol-1 at pH 4.5; those of the hairpin are: tm, 63 degrees C; delta HvH, 71.7(+/- 4.0) kcal mol-1; delta Hcal, 69.9(+/- 3.5) kcal mol-1 at pH 8.0. At intermediate pH values, the triplex to coil transition breaks up into its component triplex to hairpin and hairpin to coil transitions with thermodynamic properties: tm, 41 degrees C; delta HvH, 58.7(+/- 4.2) kcal mol-1; delta Hcal, 39.8(+/- 2.0) kcal mol-1; and tm, 63 degrees C; delta HvH, 71.7(+/- 4.0) kcal mol-1; delta Hcal, 69.6(+/- 3.5) kcal mol-1 at pH 6.7.