利用表面電漿共振儀和圓二色光譜儀來探討不同凝血酶核適體對凝血酶的作用機制

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姓名

陳人豪(Ren-hao chen) 查詢紙本館藏

畢業系所

化學工程與材料工程學系

論文名稱

利用表面電漿共振儀和圓二色光譜儀來探討不同凝血酶核適體對凝血酶的作用機制
(Studies of the Binding Mechanism between Aptamers and Thrombin by Surface Plasmon Resonance and Circular Dichroism)

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摘要(中)

本研究利用圓二色光譜儀(CD)和表面電漿共振儀(SPR)來探討凝血酶(thrombin)和其核酸核適體(aptamer)之間的作用行為。凝血酶是一種在人體的凝血機制中伴演重要角色的蛋白質，而為了抑制其活性來達到抗凝血效果。科學家於九零年代已利用SELEX技術篩選出兩條抗凝血酶核適體(15-mer aptamer和29-mer aptamer)，分別可結合於凝血酶上的纖維蛋白結合區和肝素結合區。近年來雖然有大量抗凝血酶核適體的相關研究，但大部份集中於15-mer aptamer上，因此29-mer aptamer在文獻的研究並不完整，且針對其和凝血酶的結合機制的文獻探討仍相當缺乏。因此本研究期望利用圓二色光譜儀獲得核適體構形資訊，並以表面電漿共振儀獲得凝血酶和其核適體間的動力學資訊，以探討不同抗凝血酶適合體和凝血酶之間的作用機制。
在本研究中我們設計不同實驗條件，包含不同鹽濃度、金屬離子種類、不同凝血酶濃度，並利用圓二色光譜儀(CD)觀測核適體的構形變化，以探討29-mer aptmer的折疊行為，並和20-mer aptamer(含有15-mer aptamer序列)作一系統的比較。由實驗結果發現29-mer aptmer以hairpin結構與凝血酶結合，且誘導hairpin結構中的duplex部份由B-form轉換為A-form，這和20-mer aptamer以G-guadruplex結構與凝血酶結合行為有很大的差異。此外我們利用了表面電漿共振儀(SPR)來觀測在不同操作環境下(鹽濃度、pH)，此兩條核適體對於凝血酶的結合的行為，並搭配反應速率式來計算分析凝血酶吸附和脫附曲線，以獲得動力學常數常數(Ka)。實驗結果發現，隨著溶液中鈉鹽濃度的上升導致20-mer aptamer與Ka下降，推測20-mer aptamer與凝血酶進行結合為靜電作用主導的過程；相反地隨著溶液中鈉鹽濃度的上升，導致29-mer aptamer與凝血酶的Ka上升，推測29-mer aptamer與凝血酶結合為疏水作用主導的過程。最終利用恆溫滴定卡計(ITC)觀測此兩條核適體對於凝血酶之反應焓 (△H)，也發現20-mer aptamer與凝血酶反應之放熱量大於29-mer aptamer，推測其為不同作用力主導的結合過程所造成的結果。總結上述，我們認為此兩條核適體對凝血酶擁有相當不同的結合機制，且核適體的結構為影響其辨認凝血酶的重要因素。

摘要(英)

This study characterizes the interaction mechanism between aptamers and human thrombin by surface plasmon resonance (SPR) and ciucular dichroism (CD). Thrombin, a multifunctional serine protease, has both procoagulant and anticoagulant functions in human blood. Thrombin involves two electropositive exosites；one is fibrinogen-binding site and the other is heparin-binding site. Two thrombin-binding aptamers have been selected by SELEX technique over the past decade, respectively. One is 15-mer aptamer binds at fibrinogen-binding site of thrombin, while 29-mer aptamer binds at the heparin binding site of thrombin. In the past years many papers have reported the interaction between 15-mer aptamer and thrombin, however the difference of the two aptamers bind to thrombin is still lacking and worth of investigation. In this study, we combined kinetics and conformational information to compare the binding mechanism between these two aptamers with thrombin.
Two experiments were mainly performed in this investigation. CD assay demonstrated the comformational feature of different aptamers binding to thrombin, while SPR provided kinetic constant (Ka) in different binding parameters of aqueous solution (salt concentration and pH). From the results, we found that 20-mer aptamer binding to thrombin by G-guadruplex structure and dominated by electrostatic interactions. The 29-mer aptamer binds to thrombin by hairpin structure and is driven by hydrophobic effects. Furthermore, we comfirmed this argument by Isothermal Titration Calometry measurements . By experimental results, we suggested that the structure of these two aptamers is an important factor to cause the different binding mechanism between these two aptamers with thrombin.

關鍵字(中)

★ 凝血酶
★ 核酸核適體
★ 圓二色光譜儀
★ 表面電漿共振儀

關鍵字(英)

★ aptamer
★ thrombin
★ surface plasmon resonance
★ ciucular dichroism

論文目次

中文摘要 I
Abstract II
誌謝 III
目錄 IV
圖目錄 VII
表目錄 XI
第一章緒論 1
第二章文獻回顧 3
2.1 核酸核適體 3
2.1.1 核酸核適體(Aptamers)介紹 3
2.1.2 Systematic Evolution of Ligands by Exponential Enrichment (SELEX) 6
2.1.3 核酸核適體之應用 8
2.1.3.1 Biosensors之應用 9
2.1.3.2毛細管電泳與管柱層析之應用 12
2.1.3.3 藥物發展、治療與臨床之應用 13
2.1.3.4 奈米科技之應用 15
2.1.4 抗凝血酶核酸核適體 17
2.1.4.1 15-mer aptamer 17
2.1.4.1 29-mer aptamer 20
2.2凝血酶 23
2.2.1 凝血機制 23
2.2.2 凝血酶介紹 25
2.1.2凝血酶結構 27
2.3 凝血酶和其核酸核適體作用機制之研究 30
2.3.1 表面電漿共振儀 31
2.3.2 圓二色光譜儀 32
2.3.3 恆溫滴定卡計(ITC) 34
第三章實驗藥品與儀器設備 36
3.1 實驗藥品 36
3.2 實驗儀器 38
3.3 實驗方法 39
3.3.1 緩衝溶液之配製 39
3.3.2 表面電漿共振儀實驗 40
3.3.2.1 製備蛋白質溶液 40
3.3.2.2 表面電漿共振感測儀晶片的改質 40
3.3.3圓二色光譜儀實驗 44
3.3.3.1製備核酸核適體和凝血酶溶液 44
3.3.3.2 圓二色光譜儀之操作 44
3.3.4 恆溫滴定卡計實驗 45
3.3.4.1製備核酸核適體和凝血酶溶液 45
3.3.4.2恆溫滴定卡計之操作 45
第四章結果與討論 47
4.1 抗凝血核酸核適體折疊行為之探討 47
4.1.1 抗凝血酶核酸核適體在不同鹽類實驗條件下之構形變化 47
4.1.1.1 氯化鈉對抗凝血酶核酸核適體構形之影響 50
4.1.1.2 氯化鉀對抗凝血酶核酸核適體構形之影響 52
4.1.1.3 氯化鎂對抗凝血酶核酸核適體構形之影響 54
4.1.2 抗凝血核酸核適體適體和凝血酶反應之構形變化 55
4.1.2.1 20-mer aptamer與凝血酶反應之構形變化 56
4.1.2.2 29-mer aptamer與凝血酶反應之構形變化 59
4.2凝血酶和其核酸核適體結合行為之動力學分析 63
4.2.1 SPR晶片改質與測試 63
4.2.1.1 晶片改質 63
4.2.1.2 凝血酶專一性測試 68
4.2.2 核酸核適體與凝血酶之動力學分析 71
4.2.2.1 在不同鈉鹽濃度下對凝血酶和其核酸核適體結合行為之影響 72
4.2.2.2 在不同pH環境下對凝血酶和其核酸核適體結合行為之影響 79
4.3凝血酶和其核酸核適體結合行為之熱力學分析 84
第五章結論 88
第六章參考文獻 90

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指導教授

陳文逸(Wen-yih chen)

審核日期

2010-7-29

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