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研究生:陳威廷
論文名稱:4',6-Diamidino-2-phenylindole(DAPI)與[d(CGCAAATTTGCG)]2複合物
指導教授:黃文彰
指導教授(外文):Wen-Chang Huang
學位類別:碩士
校院名稱:國立臺灣師範大學
系所名稱:化學研究所
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:115
中文關鍵詞:複合物動力結構
外文關鍵詞:DAPICGCAAATTTGCG
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DAPI全名為4’,6-diamidino-2-phenylindole,是一個重要的DNA鍵結藥物,這類的藥物能干擾DNA轉錄啟動子區域結構。過去研究指出,DAPI對DNA中富含AT鹼基對的序列位置,有相當高的鍵結親和力。而DAPI和DNA在AT鹼基對形成鍵結,阻擋了TBP與真核生物的TATA box位置結合。因而轉錄因子和無法和DNA於轉錄起始位置形成複合物,導致RNA 聚合酶 Ⅱ 無法辨識,致使轉錄作用無法起始。DAPI 因為其螢光特性,常常使用在生化或細胞化學的研究上當作染色劑,此高螢光的複合物,可用來當作DNA的探針。過去由分子足跡技術可知,DAPI對於兩個以上連續的AT鹼基對較容易鍵結,因此本實驗使用了雙股螺旋DNA序列d(CGCAAATTTGCG)2 ,藉由此一B-form 雙股螺旋DNA序列來觀察DAPI對DNA 之AT-rich區域鍵結,產生鍵結作用力的因子。本研究發現,DAPI鍵結在d(CGCAAATTTGCG)2的小凹槽,並以其indole(NH)官能基與DNA兩個對稱的T7分岔的氫鍵,而DAPI兩端amidino官能基,則會和DNA的A6產生氫鍵鍵結。此一複合物區域結構鍵結作用力的研究,可使吾人對TATA轉錄起始因子之鍵結位置的結構,有更進一步的瞭解。所得資訊也可應用在藥物設計上,藉以改善藥物結構與藥物對DNA鍵結位置的選擇性。

DAPI(4’,6-diamidino-2-phenylindole)is an important DNA-binding drugs that interferes with the activity of number of DNA transcription processing enzymes involved in regulatory and structural functions. Strong inhibitory activity of DAPI has been reported for RNA polymeraseⅡ and attributed to the high binding affinity of DAPI in the minor groove of AT-rich sequences. The strong DAPI-DNA interaction interferes with the binding of TBP (TATA-binding protein) to its consensus TATA box sequence, preventing formation of the transcription factors-DNA complex. This complex structure is required for RNA polymerase Ⅱ recognizes to initiate gene transcription. Traditionally, DAPI is used in biochemical and cytochemical studies as a specific fluorescent dye of DNA and chromosome. The special properties of DAPI—DNA can be used as a DNA probe of highly fluorescent complexes. It has been shown in footprinting experiments that DAPI will bind to two or more contiguous AT base pairs is covered when DAPI is bound. DAPI binds our d(CGCAAATTTGCG)2 in the minor groove of the dodecamer by forming a bifurcated hydrogen bond between its indole NH (imino proton) group and the two O2 of T7 in the central AAATTT region,where as two terminal amidinos form conventional hydrogen bonds with A6. Bifurcated or three-centered hydrogen bonds, in which the hydrogen atom is within bonding distance of two heteroatoms have been suggested to be present in selected small molecule structures with minor groove binding drugs. The detailed features of DAPI-d(CGCAAATTTGCG)2 complex revealed in this study can be used in designing a new drug of better selectivity.

目錄 I
圖表目錄 VI
中文摘要 1
Abstract 2
第一章、 緒論 4
1.1.1 DAPI(4’,6-diamidino-2-phenylindole) 4
1.1.2 DNA(DeoxyriboNucleic Acid)的基本結構 5
1.2 DAPI對DNA構形探討 6
1.3 動機 8
第二章、 分子結構分析原理 19
2.0 前言 19
2.1.0 核磁共振發展歷史 19
2.1.1 核磁共振理論基礎 20
2.1.2 連續波(Continuous Wave, CW)與
脈衝式(Pulsed)NMR 21
2.1.3 向量模型 22
2.1.3.1古典向量模型 22
2.1.3.2半古典向量模型 23
2.1.3.3密度運算子方法 (density operator approach) 23
2.1.4 積運算子型式 (product operator) 25
2.1.5 二維光譜基本原理 27
2.1.6 Through space coupling二維光譜 29
2.1.6.1 Nuclear Overhauser Effect (NOE) 29
2.1.6.2 NOESY (Nuclear Overhauser Effect Spectroscopy)實驗 30
2.1.7 Through bond coupling二維光譜 31
2.1.7.1 DQF-COSY (Double Quantum Filter COSY) 實驗 31
2.1.7.2 TOCSY (Total Correlation Spectroscopy) 實驗 31
2.2.0 分子模擬基本理論 33
2.2.1 位能表面(Potential Energy Surface) 33
2.2.2 力場(Forcefield) 34
2.2.2.1 AMBER力場 34
2.2.3 分子動力學(Molecular Dynamics) 34
2.2.3.1 動態模擬粹熄(dynamic simulated annealing) 36
2.2.4 距離幾何理論(Distance Geometry theory) 37
第三章、 實驗的方法及步驟 42
3.1 樣品製備(Sample preparation) 42
3.1.1 合成(synthesis) 42
3.2 樣品準備 42
3.2.1 去除保護基(deprotection) 43
3.2.2 純化(purification)及去鹽(desalting) 43
3.3 樣品的濃度與吸收係數(extinction coefficients) 44
3.4 UV變溫實驗 44
3.5 NMR實驗樣品之製備 45
3.5.1 純DNA-d(CGCAAATTTGCG)2 45
3.5.2 退火(Annealing) 45
3.5.3 DNA與DAPI複合物的配製 46
3.6 NMR實驗數據 46
3.7.1 1D NMR 47
3.7.2 2D NMR 47
3.7.2.1 溶劑是D2O的二維光譜 47
3.7.2.2 溶劑是90% H2O/10% D2O的二維光譜 48
3.8 NMR實驗數據處理 48
3.9 光譜數據分析 49
3.9.1 循序判定法(Sequential assignment) 49
3.9.2 距離設限(distance constraints) 50
3.10 分子三度空間結構 51
3.10.1 建構迴文對稱序列 51
3.10.1 設限模擬粹熄 (restrained Simulated Annealing) 51
3.10.2設限分子動力模擬(Restrainted Molecular Dynamics) 52
第四章、 Free DNA的實驗結果與討論 57
4.1 d(CGCAAATTTGCG)2之free DNA部分 57
4.1.1 Free DNA一維光譜圖 58
4.1.2 Free DNA二維光譜圖 58
4.1.2.1 確認非交換的氫(Assignment of nonexchangeable proton)58
4.1.2.2 確認可交換性質子(Assignment of exchangeable proton) 59
4.2 d(CGCAAATTTGCG)2之free DNA結構分析 60
第五章、 DNA-DAPI complex的實驗結果與討論 72
5.1 d(CGCAAATTTGCG)2-DAPI之複合物部分 72
5.1.1 DNA-DAPI complex一維光譜圖 72
5.1.2 DNA-DAPI complex二維光譜圖 73
5.1.2.1確認DNA非交換的氫
(Assignment of nonexchangeable proton) 73
5.1.2.2確認DNA可交換性質子
(Assignment of exchangeable proton) 74
5.2.3 判斷DAPI質子 75
5.3 d(CGCAAATTTGCG)2-DAPI之複合物結構分析 76
5.3.1 DNA部分 76
5.3.2 藥物束縛部分 76
5.3.3 使複合物穩定的作用力 77
第六章、 結構分析與結論 93
6.1 Free DNA與DAPI-DNA complex 結構比較 93
6.1.1 兩股間鹼基對的配對方式 94
6.1.2 上下相鄰兩鹼基對間的堆疊方式 95
6.1.3 鹼基對相較於螺旋主軸的排列方式 96
6.2 糖苷鍵與χ扭角(torsion angle) 97
6.3 醣基的摺疊情況 97
6.4 凹槽寬度(groove width)的差異性 97
6.5 結論 98
參考文獻 113

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