臺灣博碩士論文加值系統

第三股寡核酸鏈必須利用鹼基配對的原則與核酸雙螺旋鍵結，才能夠形成核酸三螺旋。然而鹼基配對的原則卻限制了第三股寡核酸鏈必須與嘌呤股鍵結才能夠形成核酸三螺旋。我們已經証明，人工修飾的核酸：N4-(6-aminopyridin-2-yl)-2'-deoxycytidine (P)能夠利用氫鍵與CG鹼基對作用，形成穩定的P‧CG鹼基三聚體，進而解除鹼基序列辨識的限制。
於本論文中，我們設計了寡核酸P1 (5'-d-TTTTTTTPTTTTTTT) 及 P3 (5'-d-TTTTTPTPTPTTTTT)，且P1及P3能夠與它們本身互補的核酸雙螺旋R1或R3鍵結，形成核酸三螺旋。本實驗利用紫外線光譜及圓二色光譜等物理化學的方法，來探討含P之寡核酸鏈形成核酸三螺旋結構之穩定度；其中包括pH值的效應、鹽的種類及改變P在寡核酸鏈上的數目。從研究結果發現：(i)在相同的條件下，含三個P‧CG的核酸三螺旋體P3：R3之第三股寡核酸鏈熔解溫度值較含一個P之P1：R1的第三股寡核酸鏈熔解溫度值為低；(ii) 在酸鹼值的影響方面，含有P之寡核酸鏈P1:R1及P3:R3在中性及酸性的條件下的熔點變化並不明顯，所以pH值對含P之寡核酸鏈的影響是很小的；(iii)陽離子的添加能夠增加核酸三螺旋結構的穩定度，其增加穩定度大小的排列如下：spermine > Mg2+ > Na+。此外，在圓二色光譜的變溫實驗中發現，利用P1:R1在320nm的吸收值對不同溫度作圖或P3:R3在335nm的吸收值對不同溫度作圖，可各得一曲線，此曲線與紫外線熔點實驗之核酸三螺旋體的熔點溫度曲線相似，兩者都有兩個反曲點。以上研究証實P1可與含CG之雙螺旋鍵結形成三螺旋，可以打破中間鏈都是嫖呤之限制，且將可進一步地運用在基因治療上。

Oligonucleotide-directed triple helix formation is one of the most versatile methods for the sequence-specific recognition of double helical DNA. However, both sequence-specific recognition of dsDNA appears to be limited to mostly purine tracts. Design leads to expand the recognition code to pyrimidine-purine base pair (CG and AT). We have previously shown that N4-(6-aminopyridin-2-yl)-2'-deoxycytidine (P) is able to interact with the CG base pair through hydrogen bonds to form a stable P‧CG base triad.
In this thesis, the oligonucleotide P1 (5'-d-TTTTTTTPTTTTTTT) or P3 (5'-d-TTTTTPTPTPTTTTT) was designed to form a triplex with its complementary duplex R1 and R3, respectively. The physical properties of complexes P1:R1 and P3:R3 were investigated as a function of pH and cations, such as NaCl, MgCl2 and spermine, by UV thermal melting experiments and circular dichroism spectroscopy (CD). The physical studies showed that (i) the complex P3:R3 containing three P‧CG triads was less stable than P1:R1 with a single one under the same conditions; (ii) the triplex stability of P1:R1 or P3:R3 was not very sensitive to the solution pH; (iii) cations did enhance the triplex stability with an order: spermine ? Mg2+ ? Na+. The temperature-dependent studies of CD showed that the curve of CD absorbance monitored at 320 nm for P1:R1 or at 335 nm for P3:R3 was biphasic, which was in agreement with the Tm data of UV thermal melting experiments

第一章 緒論……………………………………………………1
1.1 前言…………………………………………………………1
1.2 核酸的基本結構……………………………………………4
1.2.1 核酸雙螺旋………………………………………………4
1.2.2 核酸三螺旋………………………………………………11
1.3 基因治療的原理與應用…………………………………15
1-4 研究動機…………………………………………………18
第二章 核酸螺旋結構之分析原理…………………………21
2.1 前言………………………………………………………21
2.2 紫外線吸收光譜在核酸分析之原理及應用……………21
2.2.1 紫外線光譜分析原理…………………………………21
2.2.2 核酸濃度測定…………………………………………24
2.2.3 熔點溫度的測定………………………………………25
2.3 圓二色光譜在核酸結構分析之應用……………………29
第三章 實驗方法與步驟……………………………………36
3.1 核酸及寡核酸鏈樣品的製備…………………………36
3.2 實驗條件…………………………………………………37
3.3 樣品之製備………………………………………………38
3.4 寡核酸鏈濃度測定………………………………………39
3.5 定波長變溫掃描實驗……………………………………40
3.6 紫外線熔點測定法數據之分析…………………………42
3.7 圓二色光譜實驗…………………………………………46
第四章 儀器與藥品…………………………………………48
4.1 儀器………………………………………………………49
第五章 結果與討論…………………………………………50
5.1 紫外線光譜之分析………………………………………50
5.2 紫外線光譜及紫外線熔點測定之分析…………………52
5.3 圓二色光譜測定之分析…………………………………61
結論…………………………………………………………69
參考文獻……………………………………………………70

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