臺灣博碩士論文加值系統

本論文的第一部分主要是探討有關DNA-胜肽網路異位交互作用的能量變化之研究。根據先前研究設計，十種以XPRK及XHypRK為根據並結合N-甲基吡咯（Py）的新型態胜肽，觀察與DNA股間的交互作用。
足跡法實驗發現，經過兩種不同類型的網路異位協同模式，胜肽通過交互作用並互補到DNA鏈上的多個結合位置。在溫度變化實驗中使用十二肽RY-12結果顯示，在較低溫度的環境下（25℃）展現出網路協同模式（circuit type），而在較高的溫度（31℃及37℃）則表現出局部協同模式（partial-circuit type）。在圓二色光譜的研究方面，胜肽明顯的誘導DNA結構變化並以二聚體的方式結合於DNA的小凹槽內。
等溫滴定微卡計使用了十二個胜肽來做能量上的相互比較，實驗結果發現與短片段DNA雙股相互結合後產生強烈的放熱反應，焓的範圍由-14.7至-74.4千卡莫耳，熵能變化範圍則為-6.5至-65.9千卡莫耳。熵-焓補償作用（EEC）關係可以經由12個胜肽以焓及熵能變化做圖，斜率趨近於1，而y截距則對應平均的自由能（ΔG）為-8.5千卡莫耳，而所觀察到的自由能範圍則為-8.2至-9.1千卡莫耳。另外RY-12變溫的能量變化，同樣符合熵-焓補償作用，使自由能能夠維持在恆定的範圍內，由三維圖可以更明顯的見到相互間的關係。
此篇論文的第二部分，聚丙烯胺凝膠電泳和MALDI-TOF兩項實驗表明，苯丁酸氮芥-胜肽綴合物:CLB-HyM-10和CLB-HyQ-10可以容易且有效率的在沒有加熱、化學輔助劑及UV輻射的幫忙下進行選擇性的切割DNA雙股序列。
圓二色光譜研究發現，CLB-HyM-10和CLB-HyQ-10可能以二聚體的形式結合至DNA小凹槽並產生結構上的變化。DNA-胜肽的結合通過等溫滴定微卡計測得各項能量參數，顯示結合模式是屬於焓驅動的。且在DNA-胜肽的相互作用中，維持恆定的自由能並符合熵-焓補償現象。最終推論出苯丁酸氮芥-胜肽以強而有力的焓驅動結合至DNA 並產生類核酸酶序列選擇性切割。

The first part of this thesis reports the study of the energetic basis of complex DNA–peptide interactions relating to allosteric interactions. In common with other designed peptides, ten new conjugates incorporating the XPRK or XHypRK motif (Hyp = hydroxyproline) attached to a N-methylpyrrole (Py) tract with a basic tail have been found to display cooperative binding to DNA involving multiple monodentate as well as interstrand bidentate interactions. Quantitative DNase I footprinting show that allosteric communication via cooperative binding to multiple sites on complementary DNA strands corresponds to two different types of DNA–peptide interaction network. Temperature variation experiments using a dodecapeptide RY-12 show that lower temperature (25 °C) favors a circuit type of allosteric interaction network, whereas higher temperatures (31 and 37 °C) afford only a partial-circuit type of network. Circular dichroism studies show that the peptides induce significant local conformational changes in DNA via the minor groove, with apparently dimeric binding stoichiometry.
Isothermal titration calorimetry reveals that the peptides are strongly exothermic upon binding to a model 13-mer DNA duplex, as characterized by ΔH ranging from −14.7 to −74.4 kcal mol−1, and also high TΔS ranging from −6.5 to −65.9 kcal mol−1. Distinctive enthalpy–entropy compensation (EEC) relationships are demonstrated for the interaction of all twelve designed peptides with DNA, affording a straight line of slope close to unity when ΔH is plotted versus TΔS, with a y-axis intercept (average ΔG) corresponding to −8.5 kcal mol−1, while the observed ΔG ranges from −8.2 to −9.1 kcal mol−1 for the peptides. The EEC seen with peptide RY-12 binding to the model duplex persists throughout various incubation temperatures. The net compensation of energy between the favorable negative ΔH and unfavorable negative ΔS components thus constrains the value of net binding free energy ΔG within a remarkably constant range, as is clearly visible in a 3-dimensional energetic plot.
For the second part of this thesis, both polyacrylamide gel electrophoresis and MALDI-TOF experiments showed that chlorambucil-peptides conjugates CLB-HyM-10 and CLB-HyQ-10 can readily cleave DNA duplexes very effectively and sequence-selectively in the absence of heat, chemical additives and UV irradiation.
Circular dichroism studies show that the conjugates CLB–HyM-10 and CLB–HyQ-10 induce significant local conformational changes in DNA via the minor groove, possibly with dimeric binding stoichiometry. The energetic basis of DNA binding by these conjugates has been investigated by isothermal titration calorimetry, revealing that the binding of both the peptides and their CLB conjugates is overwhelmingly enthalpy-driven.
The maintenance of a conserved negative binding free energy in DNA–conjugate interactions is a crucial feature of the universal enthalpy–entropy compensation phenomenon. It is concluded that the strongly enthalpy-driven binding of CLB–peptide conjugates to preferred loci in DNA furnishes the required proximity effect to generate the observed nuclease-like sequence-selective cleavage.

目 錄
誌謝
中文摘要 Ⅰ
英文摘要 Ⅲ
目錄 Ⅴ
圖目錄 Ⅵ
表目錄 Ⅷ
一、緒論 1
二、材料與方法 13
三、結果與討論 29
四、結論與未來展望 82
五、文獻參考 84
六、附錄 89
圖 目 錄
圖1.1. 核酸結合分子的運用性 2
圖1.2. SPRK序列β-turn結構圖 4
圖1.3. Netropsin與Distamycin之結構圖 6
圖1.4. Netropsin、Distamycin與DNA結合示意圖 7
圖1.5. pBR322 以及U4A-L4T和XU4A-XL4T 的核酸序列 9
圖1.6. 苯芥丁酸（Chlorambucil, CLB）之結構 11
圖1.7. 鹼基被烷化位置 11
圖 1.8. Guanine 鹼基被烷化之水解產物結構圖 11
圖 1.9. Guanine 鹼基被烷化 Cross-linking 12
圖2.1. 固相胜肽合成法示意圖 14
圖2.2. DNase I footprinting原理 16
圖2.3. 線偏極光示意圖 18
圖2.4. 右圓偏極光示意圖 18
圖2.5. 橢圓偏極光示意圖 19
圖2.6. 基質輔助雷射脫附游離飛行時間質譜儀（MALDI - TOF/ MS）儀器構造圖 21
圖2.7. 恆溫滴定微卡計偵測熱變化概要圖 22
圖3.1. HyM-10、HyQ-10對pBR322 DNA 片段進行去氧核醣核酸足跡試驗之放射自顯圖 31
圖3.2. HyS-10、HyE-10對pBR322 DNA 片段進行去氧核醣核酸足跡試驗之放射自顯圖 32
圖3.3. PyMK-10、PyQK-10對pBR322 DNA 片段進行去氧核醣核酸足跡試驗之放射自顯圖 33
圖3.4. CLB-HyM-10、CLB-HyQ-10 對pBR322 DNA 片段進行去氧核醣核酸足跡試驗之放射自顯圖 34
圖3.5. 各胜肽對去氧核醣核酸酶切割pBR322 158 mer Upper 片段及135 mer Lower片段核酸序列之切割差異度 36
圖3.6. 各胜肽對於去氧核醣核酸酶切割pBR322 158 mer Upper 片段及135 mer Lower 片段核酸序列之網路合作性假設模型 38
圖3.7. HyS-10、HyE-10對於U4A-L4T結合之CD光譜 42
圖3.8. [ peptide ] / [ DNA ] ratio對Δθ 的關係圖 44
圖3.9. 不同胜肽之ITC熱量變化 46
圖3.10. 以各胜肽的△H及T△S做圖（25℃） 50
圖3.11. 以各RY-12 不同溫度的△H及T△S做圖 53
圖3.12. 以各胜肽的△H及T△S做圖（包含RY-12變溫數據） 55
圖3.13. 各胜肽EEC三維圖 55
圖3.14. CLB-HyM-10對U4A-L4T 700-1000 m/z 57
圖3.15. CLB-HyM-10對U4A-L4T 1000-2000 m/z 58
圖3.16. CLB-HyM-10對U4A-L4T 2000-3000 m/z 60
圖3.17. CLB-HyM-10對XU4A-XL4T 700-1000 m/z 62
圖3.18. CLB-HyM-10對XU4A-XL4T 1000-1800 m/z 64
圖3.19. CLB-HyM-10對XU4A-XL4T 1800-2000 m/z 65
圖3.20. CLB-HyM-10對XU4A-XL4T 2000-3000 m/z 66
圖3.21. CLB-HyQ-10對U4A-L4T 700-1000 m/z 67
圖3.22. CLB-HyQ-10對U4A-L4T 1000-2000 m/z 69
圖3.23. CLB-HyQ-10對U4A-L4T 2000-3000 m/z 70
圖3.24. CLB-HyQ-10對XU4A-XL4T 700-1000 m/z 72
圖3.25. CLB-HyQ-10對XU4A-XL4T 1000-1800 m/z 73
圖3.26. CLB-HyQ-10對XU4A-XL4T 1800-2000 m/z 74
圖3.27. CLB-HyQ-10對XU4A-XL4T 2000-3000 m/z 75
圖3.28. 胜肽對DNA片段切割圖 76
圖3.29. 胜肽對DNA 片段切割強度圖 77
圖 3.30. CLB-HyM-10與CLB-HyQ-10的切割差異直條圖。 79
圖3.31. CLB-HyM-10、CLB-HyQ-10最常切割的片段曲線圖 81
表 目 錄
表 1. 胜肽序列表 12
表2. 各胜肽對於去氧核醣核酸酶切割pBR322 158 mer Upper
片段及135 mer Lower 片段核酸序列之結合常數（Ka）、
合作性（nH : Hill coefficient）與結合位置 40
表3. 各溫度之RY-12 對於去氧核醣核酸酶切割pBR322 158
mer Upper 片段及135 mer Lower片段核酸序列之結
合常數（Ka）、合作性（nH : Hill coefficient）與結合位置 40
表4. 各胜肽的ITC相關數據 47
表 5. CLB-HyM-10與CLB-HyQ-10最常切割的片段 78
附 錄
附錄 1. CLB-HyM-10對U4A-L4T mass片段整理 89
附錄2. CLB-HyM-10 對XU4A-XL4T mass片段整理 90
附錄3. CLB-HyQ-10 對U4A-L4T mass片段整理 91
附錄4. CLB-HyQ-10 對XU4A-XL4T mass片段整理 92
附錄5. CLB-HyM-10與CLB-HyQ-10的CLB切割片段。 93
附錄6. CLB-HyM-10與CLB-HyQ-10的熱力學各項數據。 94
附錄7. 各胜肽之結構圖 95

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