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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 溫進德(Jin-Der Wen) | |
dc.contributor.advisor | 溫進德(Jin-Der Wen | jdwen@ntu.edu.tw | ), | |
dc.contributor.author | Ping-Chang Wu | en |
dc.contributor.author | 吳秉強 | zh_TW |
dc.date.accessioned | 2023-03-19T23:45:10Z | - |
dc.date.copyright | 2022-09-14 | |
dc.date.issued | 2022 | |
dc.date.submitted | 2022-08-30 | |
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Detection of caspase-3 activation in single cells by fluorescence resonance energy transfer during photodynamic therapy induced apoptosis. Cancer Letters, 235(2), 239–247. doi:10.1016/j.canlet.2005.04.036 李翊廷學姊論文Study of interaction between frameshift-stimulating mRNA pseudoknots and ribosomes by single-molecule FRET (2020) 杜睿芸學姊論文Exploring how the ribosome affects frameshifting-stimulating pseudoknots by using optical tweezers (2020) | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/86256 | - |
dc.description.abstract | 許多種類的病毒或細菌等擁有較短信使核醣核酸(mRNA)的物種會藉由核醣體重複讀取一個核苷酸,使下游核苷酸密碼子重新排列進而合成出不同的蛋白質,這種機制稱為-1框架位移,而-1框架位移發生的條件需要有滑動序列以及距離下游5-7個核苷酸的二級結構(例如偽結)。本次實驗我們使用SARS-CoV-2偽結RNA作為實驗材料,SARS-CoV-2為一RNA病毒並且為COVID-19的感染源,值得注意的是SARS-CoV-2偽結RNA具典型冠狀病毒RNA的三莖三環結構,並有著15-30%的-1框架位移效率。 我們透過與互補DNA的黏合解開SARS-CoV-2偽結的二級結構,再將互補DNA由3’端到5’端的逐步與RNA分離以模擬核醣體在mRNA轉譯的過程,並透過單分子螢光共振能量轉移 (smFRET)技術測量SARS-CoV-2偽結於再摺疊過程中特定兩點的距離變化,以推斷其構型的改變及探討中間態形成與否與其對-1框架位移效率的影響。我們分別使用DNA polymerase I, Klenow Fragment、Exonuclease V (RecBCD)、phi29 DNA polymerase進行RNA/DNA的分離,發現Klenow fragment無法成功將互補的DNA去除。而Exonuclease V能有效去除DNA並使SARS-CoV-2偽結重新摺疊,然而,由於Exonuclease V的作用是雙向並可能會造成RNA結構不穩定。而SARS-CoV-2 RNA 在phi29 DNA polymerase作用下能明顯觀察出RNA逐步形成偽結的過程,並能觀察到部份RNA再摺疊的過程中有中間態短暫的形成,這些中間態是否會影響-1框架位移效率目前尚不清楚,仍需要後續不同具框架位移機制的RNA互相比較。 | zh_TW |
dc.description.abstract | Minus-one programmed ribosomal frameshifting (-1 PRF) is a translational mechanism in several types of viruses and bacteria. It occurs when the ribosome rereads a nucleotide and therefore changes the reading frame in order to express multiple proteins from a single mRNA. In order to stimulate -1 PRF, a slippery sequence and a well-spaced downstream RNA secondary structure, such as a pseudoknot, should be contained in mRNA. In this experiment, we used the frameshift-stimulating pseudoknot RNA derived from SARS-CoV-2, which is an RNA virus that causes COVID-19, as our model. It is noteworthy that the SARS-CoV-2 pseudoknot RNA is a traditional three stem-three loop pseudoknot structure and its –1 frameshift efficiency is about 15-30%. We used single-molecule Förster resonance energy transfer (smFRET) to observe the folding of SARS-CoV-2 pseudoknot RNA. Native and intermediate conformations may be formed when RNA refolds during ribosomal translation. To simplify the experiment and mimic the ribosomal translation, we annealed SARS-CoV-2 pseudoknot RNA with its complementary DNA and then the RNA was released from the 5’ to 3’ end gradually by removing the DNA strand. DNA polymerase I Klenow Fragment, Exonuclease V (RecBCD), and phi29 DNA polymerase were used to release RNA. We found that DNA polymerase I Klenow Fragment isn’t able to displace the RNA strand. Exonuclease V can digest the complementary DNA to release the pseudoknot RNA, but it acts bidirectionally, which can also digest from the opposite end of DNA. When treating with phi29 DNA polymerase, which has stronger strand displacement ability than the Klenow Fragment, transient intermediates can be observed during RNA refolding to pseudoknots. However, whether these intermediates influence -1 PRF is still unknown and should be compared to other frameshift-stimulating RNA in the future. | en |
dc.description.provenance | Made available in DSpace on 2023-03-19T23:45:10Z (GMT). No. of bitstreams: 1 U0001-2908202219193000.pdf: 4547872 bytes, checksum: 1d2c4a7dd28a4502f6db6ae232dfdc97 (MD5) Previous issue date: 2022 | en |
dc.description.tableofcontents | 口試委員審定書 i 誌謝 ii 中文摘要 iii Abstract iv 目錄 vi 圖目錄 viii 表目錄 x 一、導論 1 1.1 計畫性核醣體框架位移 1 1.2 嚴重急性呼吸道症候群冠狀病毒2型(SARS-CoV-2) 1 1.3 單分子技術 2 1.3.1 簡介 2 1.3.2 單分子技術應用 3 1.3.3 螢光能量共振轉移 (Förster Resonance Energy Transfer, FRET) 3 1.4 酵素 4 1.4.1 Exonuclease V (RecBCD) 4 1.4.2 DNA polymerase I, Large(Klenow) Fragment 4 1.4.3 phi29 polymerase 4 1.5 研究動機 5 二、材料與方法 6 2.1 材料 6 2.1.1 溶液 6 2.1.2 藥品 8 2.1.3 Constructs 10 2.1.4 酶 11 2.1.5 Kits 11 2.1.6 序列 12 2.2 方法 15 三、結果 26 3.1 SARS-CoV-2偽結RNA製備 26 3.1.1 CoV2-UTR 26 3.1.2 Dye labeling 27 3.1.3 SARS-CoV-2 full RNA 27 3.1.4 互補ssDNA製備 28 3.2 SARS-CoV-2偽結的再摺疊 28 3.2.1 SARS-CoV-2偽結RNA 29 3.2.2 DNA polymerase I, Klenow Fragment 29 3.2.3 Exonuclease V (RecBCD) 30 3.2.4 phi29 DNA polymerase 33 四、討論 35 參考資料 38 圖1、核醣體轉譯過程中可能形成intermediate影響-1 PRF效率 43 圖2、SARS-CoV-2 full RNA construct 44 圖3、HDV ribozyme系統 45 圖4、HDV自切效率比較 46 圖5、RNA-dye FPLC純化 47 圖6、SARS-CoV-2接合產物 48 圖7、CoV2 ssDNA的製備 49 圖8、SARS-CoV-2偽結結構、FRET值分布與FRET隨時間變化圖 51 圖9、SARS-CoV-2/ DNA hybrid結構、FRET值分布與FRET隨時間變化圖 52 圖10、SARS-CoV-2再摺疊實驗設計 (聚合酶) 53 圖11、SARS-CoV-2再摺疊實驗設計 (外切酶) 54 圖12、經Klenow Fragment + 0.2 mM dNTPs treatment SARS-CoV-2 RNA FRET值分布與FRET隨時間變化圖 55 圖13、SARS-CoV-2 RNA在Exonuclease V無ATP作用之FRET分布與隨時間變化圖 56 圖14、經Exonuclease V + 1 mM ATP treatment SARS-CoV-2 RNA FRET隨時間變化圖 57 圖15、在Exonuclease V與不同ATP濃度作用當下SARS-CoV-2 RNA不同構型的比例 58 圖16、在Exonuclease V 與不同ATP濃度作用當下SARS-CoV-2 RNA動態轉換速率比較 59 圖17、在Exonuclease V與不同ATP濃度作用後SARS-CoV-2 RNA不同構型的比例 61 圖1、核醣體轉譯過程中可能形成intermediate影響-1 PRF效率 43 圖2、SARS-CoV-2 full RNA construct 44 圖3、HDV ribozyme系統 45 圖4、HDV自切效率比較 46 圖5、RNA-dye FPLC純化 47 圖6、SARS-CoV-2接合產物 48 圖7、CoV2 ssDNA的製備 49 圖8、SARS-CoV-2偽結結構、FRET值分布與FRET隨時間變化圖 51 圖9、SARS-CoV-2/ DNA hybrid結構、FRET值分布與FRET隨時間變化圖 52 圖10、SARS-CoV-2再摺疊實驗設計 (聚合酶) 53 圖11、SARS-CoV-2再摺疊實驗設計 (外切酶) 54 圖12、經Klenow Fragment + 0.2 mM dNTPs treatment SARS-CoV-2 RNA FRET值分布與FRET隨時間變化圖 55 圖13、SARS-CoV-2 RNA在Exonuclease V無ATP作用之FRET分布與隨時間變化圖 56 圖14、經Exonuclease V + 1 mM ATP treatment SARS-CoV-2 RNA FRET隨時間變化圖 57 圖15、在Exonuclease V與不同ATP濃度作用當下SARS-CoV-2 RNA不同構型的比例 58 圖16、在Exonuclease V 與不同ATP濃度作用當下SARS-CoV-2 RNA動態轉換速率比較 59 圖17、在Exonuclease V與不同ATP濃度作用後SARS-CoV-2 RNA不同構型的比例 61 圖18、在Exonuclease V 與不同ATP濃度作用後SARS-CoV-2 RNA動態轉換速率比較(無enzyme及ATP) 62 圖19、SARS-CoV-2 RNA經Exonuclease V 與不同ATP濃度作用並穩定後FRET分布圖 64 圖20、SARS-CoV-2 RNA在phi29 DNA polymerase無dNTPs作用之FRET分布與隨時間變化圖 65 圖21、經phi29 DNA polymerase + 0.2 mM dNTPs treatment SARS-CoV-2 RNA FRET隨時間變化圖 66 圖22、在phi29 DNA polymerase與0.2 mM、0.5 mM dNTPs濃度作用當下SARS-CoV-2 RNA不同構型的比例 68 圖23、在phi29 DNA polymerase與0.2 mM、0.5 mM dNTPs濃度作用後SARS-CoV-2 RNA不同構型的比例 69 圖24、SARS-CoV-2 RNA經phi29 DNA polymerase 與不同dNTPs濃度作用並穩定後FRET分布圖 70 圖25、Exonuclease V作用後動態變化結構的推測 71 表1、CoV2u-Cy3與CoV2d-Cy5 label效率 72 表2、SARS-CoV-2 RNA在Exonuclease V與不同濃度ATP作用下各分子於各型態的比例 73 表3a、SARS-CoV-2 RNA在Exonuclease V與不同濃度ATP作用下各state的FRET值 73 表3b、SARS-CoV-2 RNA在Exonuclease V與不同濃度ATP作用下各state間的轉換速率 (1/s) 73 表4、SARS-CoV-2 RNA在Exonuclease V與不同濃度ATP作用15分鐘並洗掉後各分子於各型態的比例 74 表5a、SARS-CoV-2 RNA在Exonuclease V與不同濃度ATP作用15分鐘並洗掉後各state的FRET值 74 表5b、SARS-CoV-2 RNA在Exonuclease V與不同濃度ATP作用15分鐘並洗掉後各state間的轉換速率 (1/s) 74 表6、SARS-CoV-2 RNA在phi29 DNA polymerase與不同濃度dNTPs作用下各分子於各型態的比例 75 表7、SARS-CoV-2 RNA在phi29 DNA polymerase與不同濃度dNTPs作用15分鐘並洗掉後各分子於各型態的比例 75 | |
dc.language.iso | zh-TW | |
dc.title | 利用單分子螢光共振能量轉移技術探討SARS-CoV-2偽結的摺疊 | zh_TW |
dc.title | Study of the folding of frameshift-stimulating SARS-CoV-2 pseudoknot using single-molecule FRET | en |
dc.type | Thesis | |
dc.date.schoolyear | 110-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 張功耀(Kung-Yao Chang),余建泓(Chien-Hung Yu) | |
dc.subject.keyword | -1框架位移,SARS-CoV-2偽結,單分子螢光共振能量轉移,DNA polymerase I,Klenow Fragment,Exonuclease V (RecBCD),phi29 DNA polymerase, | zh_TW |
dc.subject.keyword | Minus-one programmed ribosomal frameshifting (-1 PRF),SARS-CoV-2 pseudoknot,smFRET,DNA polymerase I,Klenow Fragment,Exonuclease V (RecBCD),phi29 DNA polymerase, | en |
dc.relation.page | 75 | |
dc.identifier.doi | 10.6342/NTU202202949 | |
dc.rights.note | 同意授權(全球公開) | |
dc.date.accepted | 2022-08-30 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 分子與細胞生物學研究所 | zh_TW |
dc.date.embargo-lift | 2022-09-14 | - |
顯示於系所單位: | 分子與細胞生物學研究所 |
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