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研究生： 顏誠威
Yen, Chan-Wei
論文名稱： 利用SPR與ITC探討APMED與DNA之親和力
Investigating the affinity of APMED to DNA using SPR and ITC
指導教授： 吳見明
Wu, Chien-Ming
口試委員:
學位類別： 碩士
Master
系所名稱： 原子科學院 - 生醫工程與環境科學系
Department of Biomedical Engineering and Environmental Sciences
論文出版年： 2010
畢業學年度： 98
語文別： 中文
論文頁數： 88
中文關鍵詞： 表面電漿共振 、恆溫卡熱計 、藥物親和力
外文關鍵詞： Surface Plasmon Resonance, Isothermal Titration Calorimetry, drug affinity, λ-DNA
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    • APMED是一前導化合物，其結構類似DNA鹼基對，可嵌入DNA的雙股結構達到結合效果，期許發展為DNA的標靶藥物。DNA在不同細胞內，或位於不同的細胞週期，DNA可能具有不同的立體結構。本文選定表面電漿共振儀(SPR)與恆溫卡熱計(ITC)進行實驗，共同對APMED與不同DNA形式之間的親和力進行探討。SPR藉由偵測晶片上DNA與APMED反應的質量變化，推算兩者之間的親和力；ITC利用APMED對DNA進行滴定，偵測滴定過程中微弱的熱焓變化。
    本研究首先利用SPR比較APMED在不同DNA長度上的結果差異。利用18bp與30bp進行實驗，在短序列18bp對APMED的結合得到較高的親和力。接著探討晶片的重複固化對於APMED與DNA結合的影響，發現再生後晶片的DNA固化，反應後使得APMED結合的訊號上升，推測再生反應破壞晶片表面的齊整度，降低了不同實驗結果之間的 和 差異，導致最終的親和力趨向一致性。最後希望採用λ-DNA應證上列長度結果，卻發現λ-DNA表面電漿共振晶片上的固化不易，於是採用ITC對λ-DNA進行APMED的相關探討。
    將λ-DNA經65℃加熱後形成鏈狀λ-DNA。利用ITC得到18bp、30bp和鏈狀λ-DNA對於APMED的親和力較為接近，環狀λ-DNA所得到的親和力結果比鏈狀λ-DNA小，推測DNA長度對於親和力的影響不如立體結構。於是分析環狀、鏈狀λ-DNA對APMED結合情況的不同，發現環狀λ-DNA在三維空間中的摺疊，可能提供了更多APMED的結合位，但降低了環狀λ-DNA對於APMED的親和力，說明了18bp、30bp和鏈狀λ-DNA，在對於APMED親和力大於環狀λ-DNA的結果。
    整理SPR與ITC的結果，藉由本研究中探討多種DNA型式與APMED的親和力結果及相關分析，希望能更幫助了解APMED在DNA上的結合情形，以及未來應用在DNA相關藥物的發展方向。

    第一章 緒論 1 1.1　前言 1 1.2　文獻回顧 3 1.2.1　DNA藥物的親和力 3 1.2.2　APMED (N-(2-Aminoethy)-N’-pyrene-1-ylmethylethane-1,2-diamine) 8 1.2.3　酵素結合免疫吸附法 (Enzyme-linked immunosobent assay; ELISA) 12 1.2.4　表面電漿共振 (Surface Plasmon Resonance; SPR) 16 1.2.5　恆溫卡熱計 (Isothermal Titration Calorimetry; ITC) 21 1.3　研究目的 25 第二章　材料與方法 26 2.1　表面電漿共振 26 2.1.1　實驗材料 26 2.1.2　實驗儀器 27 2.1.3　微流道清洗 28 2.1.4　Biotin-λ-DNA的製備 29 2.1.5　晶片固化 32 2.1.6　藥物的配製與注射 34 2.1.7　數據擬合 36 2.1.8　再生重複性 37 2.2　恆溫卡熱計 38 2.2.1　實驗材料 38 2.2.2　儀器清洗 39 2.2.3　實驗溶液的配製 40 2.2.4　反應物的置放 41 2.2.5　ITC參數設定 43 2.2.6　數據擬合 44 第三章　結果與討論 45 3.1　表面電漿共振 45 3.1.1　晶片測試 45 3.1.2　表面固化條件 46 3.1.3　以表面電漿共振儀進行觀測分析 48 3.1.4　非專一性測試 50 3.1.5　流速對訊號的影響 52 3.1.6　APMED在18bp、30bp DNA的反應曲線 53 3.1.7　0.05% SDS進行再生反應(regeneration) 58 3.1.8　再生後，APMED在18bp、30bp DNA的反應曲線。 60 3.1.9　biotin-λ-DNA的固化，APMED的結合。 67 3.2　恆溫卡熱計 69 3.2.1　30bp-DNA在一般室溫溶液狀態的互補情形 69 3.2.2　以30bp-DNA與APMED反應，尋找最好的反應比例 70 3.2.3　18bp-DNA、30bp-DNA與APMED的滴定結果 75 3.2.4　λ-DNA與APMED的滴定結果 76 第四章　結論 82 參考文獻 85

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