博碩士論文 103324018 詳細資訊




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姓名 陳奕儒(Yi-Ju 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 探討中性DNA與一般DNA雜交反應熱力學與結合機制之研究
(Studies of thermodynamic and mechanism for neutralized DNA (nDNA)/DNA and DNA/DNA duplex formation)
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摘要(中) 在過去的幾十年來,由人體基因表現以作疾病的預防與臨床醫療的研究逐漸蓬勃發展,並朝向精準醫學方向邁進。而為了提升基因晶片檢測目標核酸分子雜交的效率,本研究利用一種核酸類似物,骨幹上磷酸根所帶的負電由甲基化所遮蔽,使核酸分子呈現電中性,與互補股序列DNA雜交時因為靜電排斥力的下降而有更好的穩定性,期待改質過的探針對於序列的辨識性、靈敏性、專一性得到改善。
在生物檢測平台上,感測探針性質會影響檢測之效率,因此在一股DNA當中,如何設計中性DNA核酸的最適化序列與其互補股雜交,就變成一個很重要的課題。所以此研究利用恆溫滴定微卡計(ITC)、圓二色光譜儀(CD)、熔解溫度(Tm)的量測,以熱力學和分子結構的角度探討一般DNA與中性DNA雜交之交互作用機制,再結合分子模擬來做分析,並改變不同實驗條件,包含不同鹽濃度、nDNA改質數目、序列上的修飾位置、鹼基種類等。
從CD量測的結果可以證明nDNA/DNA雜交之二級結構的形成,且與DNA/DNA形成之雙股都是B-form構型。熱力學的分析上可以發現nDNA/DNA的雜交反應是以焓為主的驅動力伴隨著大量的熵補償,並且觀察到焓熵補償效應(EEC)之現象,因此推測穩定度的貢獻主要來自於氫鍵鍵結能、鹼基堆疊和水合作用力為主,這些作用力均屬放熱反應;系統亂度包含核酸分子結構熵和水分子熵變化的總和則是下降;而不論在高、低鹽濃度下,其結合常數也大於一般DNA/DNA之雜交,說明了靜電排斥力的降低致使雜交親和力上升,從熱穩定性(Tm)也可以得到相似結果。
另外,從不同實驗條件結果可以得到一些nDNA修飾於序列的原則。nDNA改質鳥嘌呤發現Tm值上升最多,推測與鹼基體積大小,因此有不同疏水作用力有關;改質位置於序列終端會來的比序列中間穩定度提高較多,且改質nDNA的數目必須視序列長度而定,修飾越多不一定穩定度就提升,這部分可能與中性DNA甲基化產生立體障礙效應有關。初步的中性核酸分子雜交熱力學機制探討,期望提供一個最佳化序列設計,以利探針於生物檢測平台之效率提升。
摘要(英) In the past few decades, detection and recognition of nucleic acid hybridization has been booming. In order to enhance the hybridization efficiency, we try to make a good use of neutralized DNA (nDNA) which is a DNA analogue with the backbone phosphate groups replaced by phosphate methylated groups. To reduce the electrostatic repulsion between the complementary DNA, we modified nDNA into DNA single strand and hope to improve the sensitivity and specificity of the nDNA probe. With the aim to know how the incorporation of nDNA is the optimized design, in this study we discuss a lot of different experimental conditions including nDNA numbers of modification, bases and position effect. We also proved that the nDNA sequences exhibit a stronger hybridization affinity than their corresponding DNA sequences no matter in general or the lower ionic strength buffer condition. In order to understand the hybridization thermodynamics and mechanism information from the combination of the calorimetric techniques, spectroscopic and molecular simulation were used. CD measurements highlight the modified duplexes adopt a B-form conformation that is similar to the unmodified duplexes. An entirely thermodynamic profile for nDNA/DNA hybridization suggested that nDNA-induced stability emanates from a favorable enthalpy change but a less favorable entropic term. Isothermal titration calorimetry (ITC) also showed that an increase in the binding constant between the two strands as we add nDNA while we also need to consider the DNA length. By ITC and melting studies, the modifications of nDNA in the terminal is more stable than that of in the central. And the guanine modification enhanced the thermal stability that was attributed to the stronger base stacking interaction. Our results indicated that the hybridization process of nDNA is more exothermic than the natural DNA resulting from greater hydrogen bonding especially in the lower salt concentration. We also evaluated the hybridization energies from the molecular simulation. The computed energies for the duplex involving nDNA are often larger than those for DNA/DNA due to the lower electrostatic interaction. By the experiment and simulation, we conclude the basic hybridization thermodynamic and mechanism information in order to provide a proper sequence design/modification guideline.
關鍵字(中) ★ 恆溫滴定微卡計
★ 圓二色光譜儀
★ 結合機制
★ 焓熵補償效應
★ 核酸分子
關鍵字(英) ★ isothermal titration calorimetry
★ circular dichroism
★ binding mechanism
★ enthalpy-entropy compensation
★ nucleic acid
論文目次 摘要 i
Abstract iii
誌謝 v
目錄 vii
圖目錄 x
表目錄 xiv
第一章 緒論 1
第二章 文獻回顧 3
2.1 核酸分子 3
2.1.1 核酸分子介紹 3
2.1.2 核酸結構 4
2.2 核酸類似物 5
2.2.1 肽核酸 6
2.2.2 鎖核酸 7
2.2.3 磷酸根甲基化去氧核醣核酸 9
2.3 不同系統於核酸分子雜交機制之研究 13
2.3.1 圓二色光譜儀 14
2.3.2 紫外光/可見光光譜儀 15
2.3.3 SYBR Green 18
2.3.4 分子模擬 20
2.4 單一核苷酸多形性 23
2.4.1 SNP簡介 23
2.4.2 錯誤配對辨識能力之研究 24
2.5 恆溫滴定微卡計 26
2.5.1 恆溫滴定微卡計介紹 26
2.5.2 吸附熱之計算 29
2.5.3 雜交行為之應用 30
第三章 實驗藥品、儀器設備與方法 32
3.1 實驗藥品 32
3.2 儀器設備 34
3.3 實驗方法 35
3.3.1 圓二色光譜儀實驗 35
3.3.2 UV-vis分光光譜儀實驗 37
3.3.3 恆溫滴定微卡計實驗 38
3.3.4 SYBR Green螢光熔點測量儀實驗 41
3.3.5 分子模擬 43
第四章 結果與討論 45
4.1 nDNA 修飾不同鹼基之探討 45
4.2 nDNA修飾不同序列位置之探討 49
4.2.1 焓熵補償效應 53
4.3 nDNA修飾數目之探討 55
4.3.1 改變鹽濃度 56
4.3.2 nDNA與LNA特性之比較 62
4.4 nDNA應用於生物檢測平台之探討 66
4.4.1 雜交熱力學之分析 66
4.4.2 FET之應用 69
4.5 nDNA辨識mismatch效果之探討 72
4.5.1 熱穩定性之分析 73
第五章 結論 83
第六章 參考文獻 86
附錄Appendixes 95
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指導教授 陳文逸(Wen-Yih Chen) 審核日期 2016-7-22
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