椅型核酸四股結構在不同陰離子影響下結構穩定度之研究

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Title:	椅型核酸四股結構在不同陰離子影響下結構穩定度之研究
Authors:	劉正鈞
Contributors:	應用化學研究所
Keywords:	四股結構結構穩定度凝血酶適體
Date:	2010
Issue Date:	2011-11-02 14:16:35 (UTC+8)
Abstract:	本論文以凝血酶適體(thrombin aptamer)為研究標的，其寡核苷酸序列為5’-d-GGTTGGTGTGGTTGG，在適當溶液環境中可自我摺疊生成椅型核酸四股結構，實驗中利用紫外光可見光分光光譜儀與圓二色光譜儀進行定量與定性分析，探討其自我摺疊結構的形成和結構的穩定度。本論文探討的實驗條件，包含不同濃度的鹽類、陽離子種類(鉀離子與鈉離子)、陰離子種類(氯離子與硫酸根離子)及溫度等。由實驗結果顯示，凝血酶適體之椅型核酸四股結構，其結構穩定度和陰離子種類與濃度有密切的關係，隨著硫酸根離子(具有二價負電荷)濃度增加，其與核酸骨架磷酸酯鍵間的靜電排斥力，使得椅型核酸四股結構解離；此現象反映在紫外光吸收值變化量，隨著硫酸根濃度增加而降低(ΔAbs從0.063降至0.037)。另一方面，氯離子(具有一價負電荷)與磷酸酯骨架之靜電排斥力較小，所以當氯離子濃度提升時，紫外光吸收值變化量相對較少。陽離子(鈉離子和鉀離子)可使本實驗核酸樣品之紫外熔點溫度升高，幫助椅型核酸四股結構更加穩定。當樣品中加入140 mM氯化鉀，鉀離子嵌入所生成的椅型核酸四股結構，其紫外熔點溫度約51 ℃。當樣品僅加入氯化鈉，鈉離子嵌入椅型核酸四股結構中，其紫外熔點溫度最高約21 ℃(鈉離子濃度高於1.2 mM以上)。由此可知當樣品中添加鉀離子比鈉離子更有助於穩定生成椅型核酸四股結構。 The thrombin aptamer (5'-d-GGTTGGTGTGGTTGG) can fold itself into a chair-type G-quadruplex in a suitable solution. The formation and the structural stability of G-quadruplex was studied by UV-VIS melting experiments and circular dichroism spectra to conduct quantative and qualitaive analyses. According to the experimental result, the structural stability of chair-type G-quadruplex of thrombin aptamer was closely related to the nature and concentration of anions. The electrostatic repulsion between the divalent anion SO42－and phosphate backbone would destabilize the structure of the G-quadruplex. This phenomenon also reflected on the UV absorption. The hypochromic effect (ΔAbs:0.063→0.037) at 295 nm of G-quadruplex was decreasing as the concentration of SO42－was elevated. On the other hand, the electrostatic replusion between Na+ (a monovalent anion) and phosphate backbone was smaller than that between SO42－ and phosphate backbone. The hypochromic values did not change a lot when the concentrations of chloride were elevated from 0.1 mM to 145 mM. Cations (such as K+, Na+, etc.) stabilize chair-type G-quadruplex. When 140 mM potassium chloride was added into sample solution, the melting temperature of chair-type G-quadruplex with K+ was about 51 ℃. In the case of sodium chloride, the melting temperature of chair-type G-quadruplex with Na+ was at most 21 ℃ as the concentration of Na+ above 1.2 mM. This result indicates that K+ enhance chair-type G-quadruplex formation better than Na+ does.
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