部分磷酸根甲基化之反義去氧核醣核酸探針與微小核糖核酸雜交靈敏度與專一性之研究

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研究生：	張晴雯 Ching-Wen Chang
論文名稱：	部分磷酸根甲基化之反義去氧核醣核酸探針與微小核糖核酸雜交靈敏度與專一性之研究 Sensitive and specific microRNA hybridization using partially methylated phosphotriester antisense DNA probes
指導教授：	陳文逸 Wen-Yih Chen
口試委員:
學位類別：	碩士 Master
系所名稱：	工學院 - 化學工程與材料工程學系 Department of Chemical & Materials Engineering
論文出版年：	2022
畢業學年度：	110
語文別：	中文
論文頁數：	123
中文關鍵詞：	合成寡核苷酸、核酸類似物、定量即時聚合酶連鎖反應、酵素結合免疫吸附分析法、微核糖核酸、直腸結腸癌
外文關鍵詞：	Synthesized oligonucleotides, Nucleic acid analogues, qPCR, ELISA, microRNA, colorectal cancer
相關次數：	點閱：7 下載：0
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MicroRNAs (miRNAs)為一種非編碼的小片段RNA，在調節基因表達中發揮著重要的作用。然而，目前使用反義寡核苷酸與目標miRNA雜交作為檢測或抑制miRNA的方法，而這些方法包含原位雜交技術(ISH)、RT-qPCR和miRNA微陣列分析技術，但上述這些方法皆缺乏雜交的專一性及親和力。因此為了改善這些問題，我們在核酸序列探針上修飾中性去氧核醣核酸 (nDNA)，nDNA為一種人工DNA類似物，其磷酸骨幹經過特定位置的甲基化改質後，使帶負電的磷酸根轉變成磷酸三酯鍵而形成電中性，其可減少核酸雙股同時帶負電造成的靜電排斥效應，因此具有較佳的雜交穩定性。
本研究表明，藉由RT-qPCR檢測結果所示，部分甲基化的反義nDNA探針(N4 nDNA，具有四個nDNA修飾)可比DNA 探針更有效地抑制在人體血漿培養基中的miR-21進行逆轉錄反應(N4 nDNA探針具有較高的Ct值)。接著，我們使用具有miR-21表達的人類結腸癌細胞(HCT116)進行原位雜交技術(ISH)的檢測，使用N4 nDNA探針檢測HCT116細胞中的miR-21，可顯示出比使用一般DNA探針檢測到更高的染色訊號強度且具有檢測專一性。同樣地，藉由酵素結合免疫吸附分析法(ELISA)表明N4 nDNA探針與miRNA雜交的效率高於DNA探針。此外，N4 nDNA探針在細胞中不會引起免疫反應(與國家衛生研究院紀雅惠博士合作)，並且N4 nDNA探針在體外無細胞蛋白質合成(CFPS)系統中可抑制GFP mRNA進行轉譯(與日本東京工業大學地球生命研究所的Kosuke Fujishima教授合作)。
由於N4 nDNA探針對於DNase I具有抵抗能力並且不會在細胞中引發強烈的免疫反應，因此未來在開發nDNA探針作為調節體內基因表達的治療性核酸藥物是具有可能性的。

MicroRNAs (miRNAs), a class of short non-coding RNA, play important roles in regulating gene expression. However, current miRNA hybridization methods for miRNA detection/inhibition using antisense oligonucleotides, including in situ hybridization (ISH), RT-qPCR, and miRNA microarray profiling technology, are lacking hybridization specificity and affinity. As such, to ameliorate these problems, the neutralized DNA, an emerging class of DNA oligonucleotides chemically synthesized with site-specific internucleoside methyl phosphotriester linkages was used as a hybridization probe instead of DNA. The reduction of inter-strand charge repulsion of nucleotide duplexes results in stronger binding between nDNA and other nucleic acids.
This study suggested that partially methylated antisense nDNA (N4 nDNA, with four modifications) probes inhibited reverse transcription of miR-21 more efficiently (higher Ct value) than DNA probes in the human plasma-like medium, as demonstrated by the RT-qPCR assays. Subsequently, we performed in situ hybridization analysis using a miR-21-expressing colorectal cancer cell line (HCT116). HCT116 cells stained with N4 nDNA antisense probes revealed a greater detection intensity and specificity than cells stained with DNA probes. Consistently, enzyme-linked immunosorbent assays (ELISA) suggested that the miRNA hybridization efficiency of N4 nDNA antisense probes was greater than that of DNA probes. In addition, the cell-based immune response of the N4 nDNA antisense probe was immune-negative (cooperated with Dr. Ya-Hui Chi at National Health Research Institutes) and the N4 nDNA antisense prob was also inhibited in vitro GFP mRNA translation (cooperated with Prof. Kosuke Fujishima at Earth-Life Science Institute).
As N4 nDNA probes are resistant to DNase I and do not elicit a strong immune response in cells, future development of nDNA probes as therapeutic nucleic acid drugs for in vivo gene expression modulation is possible.

摘要.......................................................i
ABSTRACT.................................................iii
致謝.......................................................v
目錄.....................................................vii
圖目錄....................................................xi
表目錄...................................................xiv
一、緒論...................................................1
二、文獻回顧................................................3
1核酸分子介紹.............................................3
1.1核酸分子..............................................3
1.2去氧核醣核酸(Deoxyribonucleic acid, DNA)...............4
1.3核醣核酸(Ribonucleic acid, RNA).......................5
1.4微小核醣核酸(microRNA).................................7
2核酸類似物..............................................9
2.1鎖核酸(Lock nucleic acid, LNA)........................9
2.2肽核酸(Peptide nucleic acid, PNA)....................11
2.3中性去氧核醣核酸(Neutralized DNA, nDNA)...............12
3反義寡核苷酸對基因的調控.................................18
4基因分子檢測............................................20
4.1聚合酶鏈鎖反應(Polymerase chain reaction, PCR)........20
4.2即時定量聚合酶鏈鎖反應(Quantitative real-time polymerase chain reaction, qPCR).....................................23
5原位雜交技術(in situ hybridization, ISH)................26
5.1原位雜交技術的基本原理與發展歷程.......................26
5.2原位雜交技術之訊號放大.................................29
6酵素結合免疫吸附分析法(Enzyme-linked immunosorbent assay, ELISA)....................................................32
7無細胞蛋白質合成(Cell-free protein synthesis system, CFPS)系統......................................................34
三、實驗方法與儀器設備......................................36
1實驗藥品...............................................36
1.1細胞培養.............................................36
1.2序列設計.............................................36
1.3核酸探針3′端DIG合成...................................37
1.4原位雜交技術(in situ hybridization, ISH)..............37
1.5模擬生物體內miRNA雜交.................................38
1.6細胞轉染.............................................38
1.7核酸萃取.............................................38
1.8即時聚合酶鏈式反應....................................38
1.9洋菜凝膠電泳(Agarose gel).............................39
2儀器設備...............................................40
3實驗方法...............................................41
3.1細胞解凍.............................................41
3.2細胞冷凍保存..........................................42
3.3細胞培養.............................................43
3.4核酸探針3′端DIG合成...................................44
3.5原位雜交技術 (in situ hybridization, ISH).............45
3.6模擬生物體內miRNA雜交.................................47
3.7細胞轉染.............................................48
3.8微小核醣核酸萃取......................................49
3.9逆轉錄及時聚合酶鍊式反應(qRT-PCR)......................51
3.10酵素結合免疫吸附分析法(Enzyme-linked immunosorbent assay, ELISA).............................................55
3.11洋菜凝膠電泳(Agarose gel)............................57
四、實驗結果與討論.........................................58
1建立反義寡核苷酸探針在Human plasma-like medium中抑制miRNA的逆轉錄....................................................58
1.1不同修飾數目nDNA探針對miR-21逆轉錄的抑制能力............65
1.2nDNA探針對DNase I的抵抗能力...........................68
1.3nDNA探針在不同逆轉錄時間的抑制能力.....................70
1.4不同nDNA探針濃度對miRNA逆轉錄的抑制能力................72
2通過N4 nDNA探針在癌細胞HCT116中定量miR-21................74
2.1使用原位雜交技術通過N4 nDNA探針定量miR-21..............75
2.2使用ELISA通過N4 nDNA探針定量miR-21....................81
3利用nDNA探針在人類結腸癌細胞HCT116中抑制miR-21...........85
五、結論..................................................87
六、未來展望...............................................89
七、參考文獻...............................................90
八、附錄..................................................96
1使用利用高效液相層析法(High performance liquid chromatography, HPLC)分析DNA及N4 nDNA產物.................96
2不同探針在加熱/未加熱DNase I之p-value....................97
3通過N4 nDNA探針在無細胞蛋白質合成系統中抑制GFP mRNA.......97
4不同去除保護基試劑對於合成nDNA序列分子量的影響............99
                                

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