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研究生： 蔡佩璇
Pei-Hsun Tsai
論文名稱： 利用恆溫吸附曲線及恆溫滴定微卡計探討單股及雙股DNA與Hydroxyapatite間交互作用之機制與熱力學
Isotherm and Isothermal Titration Microcalorimetric Studies of Interaction Mechanism and Thermodynamics between ssDNA and dsDNA with Hydroxyapatite
指導教授： 陳文逸
Wen-Yih Chen
口試委員:
學位類別： 碩士
Master
系所名稱： 工學院 - 化學工程與材料工程學系
Department of Chemical & Materials Engineering
畢業學年度： 93
語文別： 中文
論文頁數： 101
中文關鍵詞： 恆溫滴定微卡計 、恆溫吸附曲線 、Hydroxyapatite(HA) 、雙股去氧核醣核酸 、單股去氧核醣核酸
外文關鍵詞： binding isotherm, ssDNA, Hydroxyapatite(HA), dsDNA, ITC
相關次數： 點閱：7  下載：0 
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    • Hydroxyapatite(HA) chromatography 已經廣泛的被利用來分離單股DNA和雙股DNA，此技術是利用單股DNA對HA之吸附親和力較其雙股DNA小的原理來做分離，但是之間的交互作用與分離機制目前並無完整的研究討論。因此本研究主要是利用等溫吸附線與恆溫滴定微卡計(ITC)來探討HA與單股DNA和雙股DNA的吸附機制與熱力學分析。在實驗之部分，我們設計不同的條件，包括不同的環境因子(溫度、鹽濃度和pH)以及DNA組成(GC content、length、GC stacking rich & CG stacking rich)等來探討各種不同的效應對吸附行為的影響。
    由實驗結果顯示，在等溫吸附線的分析部分，發現對於雙股DNA而言，對HA的交互作用主要是以靜電作用力為主，因此疏水作用力或結構之改變並不會對其造成太大的影響；而對於單股DNA，因為其鹼基是暴露在外，所以與HA之交互作用會受較多因素之影響，例如靜電作用力、疏水作用力與結構之穩定性等，因此在不同的環境因子或是DNA組成的不同，都會造成單股DNA與HA之親和力改變。而且我們也發現單股DNA有多層吸附（multi-layer adsorption）之現象，尤其是在高鹽的環境中，這也說明了單股DNA在吸附過程中受到多重作用力的影響。
    而在熱力學分析的部分，發現在不同的鹽濃度下，雙股DNA及單股DNA皆為吸熱的反應，所以此吸附過程為entropy driven，表示去水合在此貢獻中相當重要。並且發現單股DNA之吸附焓皆大於雙股DNA，表示對於單股DNA，疏水作用力之貢獻較大，然而對於雙股DNA，則是靜電作用力貢獻較大。
    藉由等溫吸附線與熱力學分析，我們可以清楚了解不同環境因子對於雙股DNA和單股DNA與HA之鍵結行為和機制的影響，此研究結果可提供生物辨識作用方面的基礎資訊。

    Hydroxyapatite(HA) chromatography has been used extensively for the separation of single-strand DNA and double-strand DNA. This technique is based on the fact that ssDNA have less affinity for hydroxylapatite than their dsDNA. But the details of the mechanism of the separation of ssDNA and dsDNA by hydroxyapatite are still not clear. In this study, we discussed the effects of environmental factors (i.e. salt concentration, temperature and pH) and different kinds of ssDNA and dsDNA (i.e. GC content, length, secondary structure effects and GC or CG stacking rich…) on the binding behavior. By equilibrium batch analysis, we measure the adsorption isotherm to evaluate the affinity ssDNA and dsDNA with HA, while isothermal titration microcalorimetric was used to measure the adsorption enthalpy. By the kinetics and thermodynamics analysis, we established an interaction mechanism to explain the ssDNA and dsDNA with HA interaction behavior.
    By isotherm analysis, we realized that dsDNA mainly use electrostatic force to bind with HA. So in high salt concentration, the phosphate of dsDNA interaction was shielded Na+ and the affinity would be decreased. Because the surface of ssDNA molecule contains hydrophobic bases and negative charge phosphate backbone, so both of the hydrophobic and electrostatic interactions affect the affinity of ssDNA for HA. In addition, stability of structure is also important for ssDNA, so increasing in the structural order of ssDNA would increase the binding affinity of ssDNA with HA. However, for oligo ssDNAs (15mer and 60mer) in this study, the hydrophobic interaction is more revealed and multi- layers adsorption were observed.
    By thermodynamics analysis, we found that the adsorption enthalpies of ssDNA and dsDNA are all endothermic in this study and the dehydration step in the binding process plays a key role. This investigation offers useful knowledge of ssDNA and dsDNA with HA binding behavior and fundamental thermodynamics information in biorecognition system.

    第一章 緒論 1 第二章 文獻回顧與應用 3 2.1 核酸介紹 3 2.1.1 核酸研究歷史 3 2.1.2 去氧核糖核酸結構 6 2.1.3影響DNA結構穩定性之因子 14 2.1 Hydroxyapatite的介紹 18 2.3核酸在HA管柱的層析行為 20 2.3.1 HA管柱層析之發展 20 2.3.2 影響核酸分子與HA交互作用之因子 21 2.3.2.1 核酸長度之影響 21 2.3.2.2 核酸鹼基組成之影響 22 2.3.2.3 溫度效應以及對SNP分離之影響 24 2.3.2.4 環境pH值之影響 30 2.3.2.5 不同的HA種類對DNA分離效果之探討 33 2.4 鍵結模式的討論 35 2.5 恆溫滴定微卡計 37 2.5.1 卡計之基本介紹 37 2.5.2 恆溫滴定微卡計之基本介紹 38 2.5.3 利用恆溫滴定微卡計研究吸附行為 40 第三章 實驗藥品、儀器及方法 43 3.1 實驗藥品 43 3.2 實驗儀器設備 45 3.3實驗方法 46 3.3.1 等溫吸附線的量測 46 3.3.2 恆溫微卡計量測鍵結焓 47 3.3.2.1恆溫滴定微卡計之操作步驟 48 第四章 實驗結果與討論 50 4.1 Mfold模擬核酸序列之結果 50 4.2 單股和雙股DNA與HA間之交互作用研究 55 4.2.1 鹽類效應 55 4.2.2 溫度效應 61 4.2.3 pH效應 64 4.2.4 DNA之不同GC content之影響 66 4.2.5 具明顯與不明顯二級結構之影響 71 4.2.6 不同長度DNA之影響 73 4.2.7 DNA之GC stacking rich與CG stacking rich 之影響 75 4.3 DNA與HA吸附行為的動力學探討 78 4.4 利用恆溫微卡計探討DNA與HA之間的熱力學 82 4.4.1 鹽類效應 83 4.4.2 不同GC content之DNA的影響 87 第五章 結論 90 第六章 參考文獻 94 圖 目 錄 圖2.1 核苷酸化學結構 7 圖2.2胸腺嘧啶(thymine)、胞嘧啶(cytosine)、腺嘌呤(adenine)與 7 鳥糞嘌呤(guanine)之化學結構 7 圖2.3 去氧核醣核苷酸結構 7 圖2.4 聚核苷酸鏈(polynucleotide)的鹼基序列 8 圖2.5 DNA雙股螺旋結構 9 圖2.6 DNA的鹼基排列配對 10 圖2.7 A-T鹼基對與G-C鹼基對間之距離 10 圖2.9 雙股DNA之三級結構 13 圖2.10 鹼基芳香環的π-π stacking 14 圖2.11 單股DNA之二級結構 15 圖2.11 DNA分子變性及復性之示意圖 17 圖2.12 DNA的溶解曲線（melting curve） 17 圖2.13 DNA與HA結合之示意圖 20 圖2.14 AMP與ATP之溶析曲線 21 圖2.15 雙股DNA之GC content與elution molarity (ME)之關係 22 圖2.16 不同GC content之單股DNA在HA管柱中之行為 23 圖2.17 雙股DNA及單股DNA之溶析曲線與溫度之關係 25 圖2.18 SNP之示意圖 26 圖2.19 HA管柱分析Homoduplexes以及Heteroduplexes之方法 26 圖2.20 Homoduplexes與Hetroduplexes的溶析曲線 27 圖2.21 吸附點數目（number of binding site）的概念 28 圖2.22 溫度對Homoduplexes和Hetroduplexes之吸附點數目影響 29 圖2.23 溫度對Homoduplexes和Hetroduplexes之溶析鹽濃度影響 29 圖2.24 溫度對Homoduplexes和Hetroduplexes之溶析位置VR之影響 30 圖2.25 在不同的pH值之下，雙股DNA與HA結合的能力不同 31 圖2.26 吸附點數目（number of binding site）與pH之關係 32 圖2.27 溶析鹽濃度IR與pH之關係 32 圖2.28比較HA typeI 和HA typeII的層析實驗中，DNA的滯留(retention)行為。 34 圖2.29 在不同溫度下燒結而成的HA，其分離效果的比較 34 圖2.30 恆溫滴定微卡計 37 圖2.31 恆溫滴定微卡計之恆溫部分 39 圖2.32 恆溫滴定微卡計之注射與反應部份 39 圖2.33 ITC典型放熱圖譜 42 圖2.34 ITC典型吸熱圖譜 42 圖3.1 測量等溫吸附線之步驟圖示 47 圖3.2 恆溫滴定微卡計實驗步驟之圖示 49 圖4.1 以Mfold預測核酸序列s60_2nd於25℃及20℃，不同濃度下之hairpin構造及計算之△G (a) s60_2nd於0.03M SPB buffer、25℃(b) s60_2nd於0.12M SPB buffer、25℃(c)s60_2nd於0.03M SPB buffer、20℃(d) s60_2nd於0.12M SPB buffer、20℃ 52 圖4.2 以Mfold預測核酸序列s60_non於25℃及20℃，不同濃度下之hairpin構造及計算之△G。(a) s60_non於0.03M SPB buffer、25℃ (b) s60_non於0.12M SPB buffer、25℃（c）s60_non於0.03M SPB buffer、20℃ (d) s60_non於0.12M SPB buffer、20℃ 52 圖4.3 以Mfold預測核酸序列編號s15_GC33於25℃，不同濃度下之hairpin構造及計算之△G。(a) s15_GC33於0.03M SPB buffer (b) s15_GC33於0.12M SPB buffer 53 圖4.4 以Mfold預測核酸序列編號s15_GC53於25℃，不同濃度下之hairpin構造及計算之△G。(a) s15_GC53於0.03M SPB buffer (b) s15_GC53於0.12M SPB buffer 53 圖4.5 以Mfold預測核酸序列編號s15_GC67於25℃，不同濃度下之hairpin構造及計算之△G。(a) s15_GC67於0.03M SPB buffer (b) s15_GC67於0.12M SPB buffer 53 圖4.6 以Mfold預測核酸序列編號s15_CG33於25℃，不同濃度下之hairpin構造及計算之△G。 (a) s15_CG33於0.03M SPB buffer (b) s15_CG33於0.12M SPB buffer 54 圖4.7 以Mfold預測核酸序列編號s15_CG53於25℃，不同濃度下之hairpin構造及計算之△G。 (a) s15_CG53於0.03M SPB buffer (b) s15_CG53於0.12M SPB buffer 54 圖4.8 以Mfold預測核酸序列編號s15_CG67於25℃，不同濃度下之hairpin構造及計算之△G。 (a) s15_CG67於0.03M SPB buffer (b) s15_CG67於0.12M SPB buffer 54 圖 4.9 33% GC且長度為15mer的單股DNA及雙股DNA在不同鹽濃度(0.03M,0.12M)，pH6.8的SPB buffer，溫度25℃之等溫吸附線 57 圖 4.10 53% GC且長度為15mer的單股DNA及雙股DNA在不同鹽濃度(0.03M,0.12M)，pH6.8的SPB buffer，溫度25℃之等溫吸附線 57 圖 4.11 67% GC且長度為15mer的單股DNA及雙股DNA在不同鹽濃度(0.03M,0.12M)，pH6.8的SPB buffer，溫度25℃之等溫吸附線 57 圖 4.12 33% CG且長度為15mer的單股DNA及雙股DNA在不同鹽濃度(0.03M,0.12M)，pH6.8的SPB buffer，溫度25℃之等溫吸附線 58 圖 4.13 53% CG且長度為15mer的單股DNA及雙股DNA在不同鹽濃度(0.03M,0.12M)，pH6.8的SPB buffer，溫度25℃之等溫吸附線 58 圖 4.14 67% CG且長度為15mer的單股DNA及雙股DNA在不同鹽濃度(0.03M,0.12M)，pH6.8的SPB buffer，溫度25℃之等溫吸附線 58 圖 4.15 具明顯二級結構且長度為60mer的單股DNA及雙股DNA在不同鹽濃度(0.03M,0.12M)，pH6.8的SPB buffer，溫度25℃之等溫吸附線 59 圖 4.16 不明顯二級結構且長度為60mer的單股DNA及雙股DNA在不同鹽濃度(0.03M,0.12M)，pH6.8的SPB buffer，溫度25℃之等溫吸附線 59 圖4.17 單股DNA吸附於HA上之雙層吸附示意圖 60 圖4.18 33% CG且長度為15mer的單股DNA在不同溫度（25℃和20℃）在鹽濃度為0.03M和0.12M，pH6.8的SPB buffer下之等溫吸附線 62 圖4.19 53% CG且長度為15mer的單股DNA在不同溫度（25℃和20℃）在鹽濃度為0.03M和0.12M，pH6.8的SPB buffer下之等溫吸附線 62 圖4.20 67% CG且長度為15mer的單股DNA在不同溫度（25℃和20℃）在鹽濃度為0.03M和0.12M，pH6.8的SPB buffer下之等溫吸附線 62 圖 4.21 具明顯二級結構且長度為60mer的單股DNA在不同溫度（25℃和20℃）在鹽濃度為0.03M和0.12M，pH6.8的SPB buffer下之等溫吸附線 63 圖 4.22 不明顯二級結構且長度為60mer的單股DNA在不同溫度（25℃和20℃）在鹽濃度為0.03M和0.12M，pH6.8的SPB buffer下之等溫吸附線 63 圖 4.23 具明顯二級結構且長度為60mer的單股DNA在不同pH值（pH=6.8和pH=8.0）在鹽濃度為0.03M的SPB buffer及溫度為20℃下之等溫吸附線 65 圖 4.24不明顯二級結構且長度為60mer的單股DNA在不同pH值（pH=6.8和pH=8.0）在鹽濃度為0.03M的SPB buffer及溫度為20℃下之等溫吸附線 65 圖4.25 不同GC content（33%、53%、67%）的單股DNA在溫度為25℃時之等溫吸附線。（a）GC stacking rich之單股DNA， pH 6.8的0.03M SPB buffer（b）CG stacking rich之單股DNA， pH 6.8的0.03M SPB buffer（c）GC stacking rich之單股DNA， pH 6.8的0.12M SPB buffer（d）CG stacking rich之單股DNA， pH 6.8的0.12M SPB buffer 68 圖4.26 不同CG content（33%、53%、67%）的單股DNA在溫度為25℃時之等溫吸附線。（a）CG stacking rich之單股DNA， pH 6.8的0.03M SPB buffer（b）CG stacking rich之單股DNA， pH 6.8的0.12M SPB buffer 69 圖4.27 不同GC content（33%、53%、67%）的雙股DNA在溫度為25℃時之等溫吸附線。（a）GC stacking rich之雙股DNA， pH 6.8的0.03M SPB buffer（b）CG stacking rich之雙股DNA， pH 6.8的0.03M SPB buffer（c）GC stacking rich之雙股DNA， pH 6.8的0.12M SPB buffer（d）CG stacking rich之雙股DNA， pH 6.8的0.12M SPB buffer 70 圖 4.28 具明顯二級結構及不明顯二級結構的單股DNA在不同鹽濃度（0.03M和0.12M），pH6.8的SPB buffer，溫度為25℃下之等溫吸附線 72 圖 4.29 具明顯二級結構及不明顯二級結構的單股DNA在不同鹽濃度（0.03M和0.12M），pH6.8的SPB buffer，溫度為20℃下之等溫吸附線 72 圖 4.30 不同長度之單股DNA（15mer與60mer）的比較在不同鹽濃度（0.03M和0.12M），pH6.8的SPB buffer，溫度為25℃下之等溫吸附線 74 圖 4.31 不同長度之單股DNA（15mer與60mer）的比較在不同鹽濃度（0.03M和0.12M），pH6.8的SPB buffer，溫度為20℃下之等溫吸附線 74 圖 4.32 不同長度之雙股DNA（15mer與60mer）的比較在不同鹽濃度（0.03M和0.12M），pH6.8的SPB buffer，溫度為25℃下之等溫吸附線 75 圖 4.33 不同的序列的排列（GC stacking rich與CG stacking rich）之33%GC與33%CG雙股DNA在不同鹽濃度（0.03M和0.12M），pH6.8的SPB buffer，溫度為25℃下之等溫吸附線 76 圖 4.34 不同的序列的排列（GC stacking rich與CG stacking rich）之53%GC與53%CG雙股DNA在不同鹽濃度（0.03M和0.12M），pH6.8的SPB buffer，溫度為25℃下之等溫吸附線 76 圖 4.35 不同的序列的排列（GC stacking rich與CG stacking rich）之67%GC與67%CG雙股DNA在不同鹽濃度（0.03M和0.12M），pH6.8的SPB buffer，溫度為25℃下之等溫吸附線 77 圖 4.36 33% CG的單股DNA及雙股DNA（15mer）在不同鹽濃度(0.03M,0.12M)，pH6.8的SPB buffer，溫度25℃之吸附焓之變化] 85 圖 4.37 53% CG的單股DNA及雙股DNA（15mer）在不同鹽濃度(0.03M,0.12M)，pH6.8的SPB buffer，溫度25℃之吸附焓之變化 86 圖 4.38 67% CG的單股DNA及雙股DNA（15mer）在不同鹽濃度(0.03M,0.12M)，pH6.8的SPB buffer，溫度25℃之吸附焓之變化 86 圖 4.39 不同的CG content（33%CG、53%CG、67%CG）的單股DNA（15mer）在鹽濃度為0.03M，pH6.8的SPB buffer，溫度25℃之吸附焓之變化 88 圖 4.40 不同的CG content（33%CG、53%CG、67%CG）的單股DNA（15mer）在鹽濃度為0.12M，pH6.8的SPB buffer，溫度25℃之吸附焓之變化 88 圖 4.41 不同的CG content（33%CG、53%CG、67%CG）的雙股DNA（15mer）在鹽濃度為0.03M，pH6.8的SPB buffer，溫度25℃之吸附焓之變化 89 圖 4.42 不同的CG content（33%CG、53%CG、67%CG）的雙股DNA（15mer）在鹽濃度為0.12M，pH6.8的SPB buffer，溫度25℃之吸附焓之變化 89 表 目 錄 表2.1基因及核酸之研究發展 4 表2.2 一般常見的鹼基、核苷以及核苷酸的名稱 8 表2.3 B-form、A-form和Z-form DNA雙螺旋結構比較 13 表2.3 相鄰的兩核苷酸對的Tm值 15 表3.1 實驗所使用之單股去氧核醣核酸分子序列表及其代號 44 表3.2 實驗所使用之雙股去氧核醣核酸分子序列表及其代號 44 表4.1：預測於不同濃度及溫度下計算核酸序列之自由能 51 表4.2：預測於不同濃度下計算核酸序列之自由能（25℃） 51 表4.3 利用Langmuir isotherm model 所計算出不同的單股DNA和雙股DNA（GC stacking rich）與HA在溫度為25℃且鹽濃度為0.03M和0.12M，pH6.8之SPB buffer中的平衡常數Kd 79 表4.4 利用Langmuir isotherm model 所計算出不同的單股DNA和雙股DNA（CG stacking rich）與HA在溫度為25℃且鹽濃度為0.03M和0.12M，pH6.8之SPB buffer中的平衡常數Kd 80 表4.5 利用Langmuir isotherm model 所計算出不同的單股DNA（CG stacking rich）與HA在溫度為20℃且鹽濃度為0.03M和0.12M，pH6.8之SPB buffer中的平衡常數Kd 80 表4.6 利用Langmuir isotherm model 所計算出具有明顯二級結構及非明顯二級結構之的單股DNA和雙股DNA與HA在溫度為25℃且鹽濃度為0.03M和0.12M，pH6.8之SPB buffer中的平衡常數Kd 81 表4.7 利用Langmuir isotherm model 所計算出具有明顯二級結構及非明顯二級結構之的單股DNA和雙股DNA與HA在溫度為20℃且鹽濃度為0.03M和0.12M，pH6.8及pH8.0之SPB buffer中的平衡常數Kd 81 表4.8 15mer的ssDNA和dsDNA分別在0.03M和0.12M的SPB buffer以及溫度為25℃下與HA鍵結的相關熱力學參數 87

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