以熱力學的觀點探討鈉離子與錯誤鹼基配對在一般核酸和中性DNA雜交形成雙 股螺旋之機制

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吳孟威(Meng-Wei Wu) 查詢紙本館藏

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化學工程與材料工程學系

論文名稱

以熱力學的觀點探討鈉離子與錯誤鹼基配對在一般核酸和中性DNA雜交形成雙股螺旋之機制
(The thermodynamic aspects of the Na+ and the mismatch discrimination on the formation of double stranded DNA containing site-specific methyl phosphotriester linkages)

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摘要(中)

近年來，隨著生物技術的進步，核酸類似物例如：Locked nucleic acid(LNA)、Peptide nucleic acid(PNA)等成為新型生物技術應用於分子檢測的理想選擇。為了增加檢測的專一性與辨識性，我們實驗室發展一種新的電中性核酸類似物，phosphate-methylated DNA，並將其簡稱為nDNA。nDNA磷酸骨幹上不同核苷酸間的氧可以選擇性地經由甲基化(-CH3)，使帶負電的磷酸二酯鍵(O=P-O-，phosphodiester linkages)轉變成磷酸三酯鍵(O=P-O-CH3，methyl phosphotriester linkages)而成為電中性。此甲基化結果會使nDNA和其互補股核酸之間的靜電排斥力下降，而增加雙股的穩定性，但同時nDNA磷酸骨幹上的甲基也會在雙股螺旋形成時導致立體障礙的影響產生，並使結構不穩定。依此，就有機會藉由改變特定甲基化位置及不同數目，我們就能選擇與控制nDNA和互補股DNA雜交之性質，以為特定功能使用。
實驗結果方面，根據電泳我們可以分辨出不同數目nDNA的帶電量，並且證實nDNA能夠與一般DNA形成雜交，且circular dichroism spectroscopy(CD)的量測結果發現在波長275nm時出現一正峰，在245nm時出現負峰，指出nDNA和一般DNA雜交仍會形成B-form結構，值得注意的是我們藉由恆溫滴定微卡計(ITC)和melting temperature(Tm)的量測發現nDNA與一般DNA雜交，有可能會使雙股螺旋不穩定。而從∆G和Na+的斜率可以看到當雙股螺旋形成時，一般需要大量鹽離子以穩定結構，但若將其中一股換成nDNA能夠使Na+的吸收減少，同時我們也從熱力學參數探討無論是鈉離子或者是修飾甲基對於遮蔽磷酸骨幹上負電荷的影響，這一方面也使我們更加了解nDNA與其互補股之間的雜交機制。
最後，文獻指出辨識SNP特別是在GC-rich的序列是十分困難的，因為perfect match和mismatch之間的Tm值差異很小，所以我們選用了C型肝炎的其中一種分型(HCV-3b)當作目標物，此一序列為GC含量75%的序列，藉由改變nDNA修飾的位置與間隔不同鹼基數目修飾nDNA，發現間隔兩個鹼基設計nDNA或者是將nDNA設計在mismatch周圍，都能提高∆Tm，使得辨識能力上升，提供最佳化之設計的參考。

摘要(英)

For the upsurging needs of disease detection and genetic analysis, the uses of synthetic DNA containing modified nucleotides such as LNA or PNA are becoming desired for novel biotech applications. To enhance the specificity of detections, we used newly developed DNA oligonucleotide (nDNA), which contains site-specific neutral internucleoside methyl phosphotriester (MPTE) linkages. The electrostatic repulsion between nDNA and its complementary strand may decrease and enhance the duplex formation. However, the methyl group of MPTEs may sterically hinder duplex formation and destabilize the duplexes. Therefore, the uses of nDNA offer additional elements of maneuvering the stability of nucleic acid duplexes. Here we used gel electrophoresis to examine the charges of the obtained nDNA, CD measurements showed that nDNA and complementary native DNA exhibited a positive peak at 275nm and a negative peak at 245nm which mainly in B-form duplexes. Surprisingly we found that nDNA destabilized the formation of DNA duplexes by using a calorimeter and Tm measurements. Slope of ∆G vs [Na+] showed that methylation of backbone phosphates leads to a lower uptake of Na+ in hybridization. We also revealed the contrary thermodynamic effects of reducing inter-stand negative-charge repulsion by shielding with sodium cations and by neutralizing the backbone negative charge with methyl groups. These later discoveries provide understanding the driving forces of formation of nucleic acid duplexes with modified nucleotides and its applications. Lastly, nDNA were applied to discriminate oligonucleotides of single-nucleotide polymorphism (SNP), Studies showed that discriminating SNP especially in GC-rich regions is often difficult because of the small difference between the melting temperature of the perfect match and the mismatch. So we chose HCV-3b which is one of the HCV genotypes as the target, by introducing different position and different density of the nDNA modification, we tried to find optimized nDNA design that can enhance the discrimination ability.

關鍵字(中)

★ 恆溫滴定微卡計
★ 結合機制
★ 單一核苷酸多型性
★ 核酸分子
★ GC 含量

關鍵字(英)

★ isothermal titration calorimetry
★ binding mechanism
★ single nucleotide polymorphism
★ nucleic acid
★ GC content

論文目次

摘要 i
Abstract iii
誌謝 v
目錄 vi
圖目錄 xiv
表目錄 xvii
第一章緒論 1
第二章文獻回顧 3
2.1 核酸分子 3
2.1.1 去氧核醣核酸結構 5
2.1.2核醣核酸結構 7
2.1.3 鹼基含量之研究 8
2.2 核酸類似物 9
2.2.1 肽核酸 9
2.2.2 鎖核酸 11
2.2.3嗎啉基寡核苷酸 13
2.2.4 中性去氧核醣核酸 14
2.3 單一核苷酸多型性 18
2.3.1 SNP簡介 18
2.3.2 錯誤配對辨識能力之研究 19
2-4核酸分子雜交熱力學機制之研究 20
2.4.1 恆溫滴定微卡計 21
2.4.2 圓二色光譜儀 24
2.4.3 SYBR green 25
第三章實驗藥品、儀器設備與方法 27
3.1 實驗藥品 27
3.2 儀器設備 29
3.3 實驗方法 30
3.3.1 nDNA之質譜分析與保存方法 30
3.3.1 PAGE電泳 32
3.3.2 離子交換層析法 33
3.3.3 圓二色光譜儀實驗 34
3.3.4 恆溫滴定微卡計實驗 35
3.3.5 SYBR Green 螢光熔點測量儀實驗 38
第四章結果與討論 40
4.1修飾不同數目nDNA之電荷量探討 40
4.1.1 以凝膠電泳判別電荷量 40
4.1.2 以離子交換層析法判別電荷量 43
4-2 nDNA與鹽離子(Na+)關係之探討 44
4-2-1鹽濃度及nDNA修飾數目對雙股螺旋二級結構之影響 45
4-2-2 以熱力學的觀點探討nDNA與鹽離子(Na+)之關係 47
4-2-3 焓熵補償效應 53
4-3 不同nDNA修飾辨識錯誤鹼基配對之能力 54
4-3-1 RNA/DNA與RNA/nDNA雜交之二級結構影響 55
4-3-2 nDNA鄰近效應之探討 57
4-3-3 nDNA修飾不同位置之探討 62
4-3-4 改變鹽濃度 68
第五章結論與未來展望 72
5.1 結論 72
5.2 未來展望 74
第六章參考文獻 75
附錄 Appendixes 83

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指導教授

陳文逸(Wen-Yih Chen)

審核日期

2018-7-12

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