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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 温進德 | zh_TW |
| dc.contributor.advisor | Jin-Der Wen | en |
| dc.contributor.author | 王碩 | zh_TW |
| dc.contributor.author | Shuo Wang | en |
| dc.date.accessioned | 2025-08-19T16:15:54Z | - |
| dc.date.available | 2025-08-20 | - |
| dc.date.copyright | 2025-08-19 | - |
| dc.date.issued | 2025 | - |
| dc.date.submitted | 2025-08-05 | - |
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T., & Wen, J. D. (2021). Formation of frameshift-stimulating RNA pseudoknots is facilitated by remodeling of their folding intermediates. Nucleic Acids Res, 49(12), 6941–6957. https://doi.org/10.1093/nar/gkab512 Jones, C. P., & Ferré-D'Amaré, A. R. (2022). Crystal structure of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) frameshifting pseudoknot. Rna, 28(2), 239–249. https://doi.org/10.1261/rna.078825.121 Lan, T. C. T., Allan, M. F., Malsick, L. E., Woo, J. Z., Zhu, C., Zhang, F., Khandwala, S., Nyeo, S. S. Y., Sun, Y., Guo, J. U., Bathe, M., Näär, A., Griffiths, A., & Rouskin, S. (2022). Secondary structural ensembles of the SARS-CoV-2 RNA genome in infected cells. Nat Commun, 13(1), 1128. https://doi.org/10.1038/s41467-022-28603-2 Malone, B., Urakova, N., Snijder, E. J., & Campbell, E. A. (2022). Structures and functions of coronavirus replication-transcription complexes and their relevance for SARS-CoV-2 drug design. Nat Rev Mol Cell Biol, 23(1), 21–39. https://doi.org/10.1038/s41580-021-00432-z Moomau, C., Musalgaonkar, S., Khan, Y. A., Jones, J. E., & Dinman, J. D. (2016). Structural and Functional Characterization of Programmed Ribosomal Frameshift Signals in West Nile Virus Strains Reveals High Structural Plasticity Among cis-Acting RNA Elements. J Biol Chem, 291(30), 15788–15795. https://doi.org/10.1074/jbc.M116.735613 Mouzakis, K. D., Lang, A. L., Vander Meulen, K. A., Easterday, P. D., & Butcher, S. E. (2013). HIV-1 frameshift efficiency is primarily determined by the stability of base pairs positioned at the mRNA entrance channel of the ribosome. Nucleic Acids Res, 41(3), 1901–1913. https://doi.org/10.1093/nar/gks1254 Neuman, K. C., & Nagy, A. (2008). Single-molecule force spectroscopy: optical tweezers, magnetic tweezers and atomic force microscopy. Nat Methods, 5(6), 491–505. https://doi.org/10.1038/nmeth.1218 Neupane, K., Zhao, M., Lyons, A., Munshi, S., Ileperuma, S. M., Ritchie, D. B., Hoffer, N. Q., Narayan, A., & Woodside, M. T. (2021). Structural dynamics of single SARS-CoV-2 pseudoknot molecules reveal topologically distinct conformers. Nat Commun, 12(1), 4749. https://doi.org/10.1038/s41467-021- 25085-6 Omar, S. I., Zhao, M., Sekar, R. V., Moghadam, S. A., Tuszynski, J. A., & Woodside, M. T. (2021). Modeling the structure of the frameshift-stimulatory pseudoknot in SARS-CoV-2 reveals multiple possible conformers. PLoS Comput Biol, 17(1), e1008603. https://doi.org/10.1371/journal.pcbi.1008603 Pekarek, L., Zimmer, M. M., Gribling-Burrer, A. S., Buck, S., Smyth, R., & Caliskan, N. (2023). Cis-mediated interactions of the SARS-CoV-2 frameshift RNA alter its conformations and affect function. Nucleic Acids Res, 51(2), 728–743. https://doi.org/10.1093/nar/gkac1184 Ritchie, D. B., Foster, D. A., & Woodside, M. T. (2012). Programmed -1 frameshifting efficiency correlates with RNA pseudoknot conformational plasticity, not resistance to mechanical unfolding. Proc Natl Acad Sci U S A, 109(40), 16167–16172. https://doi.org/10.1073/pnas.1204114109 Tholstrup, J., Oddershede, L. B., & Sørensen, M. A. (2012). mRNA pseudoknot structures can act as ribosomal roadblocks. Nucleic Acids Res, 40(1), 303–313. https://doi.org/10.1093/nar/gkr686 V'Kovski, P., Kratzel, A., Steiner, S., Stalder, H., & Thiel, V. (2021). Coronavirus biology and replication: implications for SARS-CoV-2. Nat Rev Microbiol, 19(3), 155–170. https://doi.org/10.1038/s41579-020-00468-6 Wu, B., Zhang, H., Sun, R., Peng, S., Cooperman, B. S., Goldman, Y. E., & Chen, C. (2018). Translocation kinetics and structural dynamics of ribosomes are modulated by the conformational plasticity of downstream pseudoknots. Nucleic Acids Res, 46(18), 9736–9748. https://doi.org/10.1093/nar/gky636 Zhang, K., Zheludev, I. N., Hagey, R. J., Haslecker, R., Hou, Y. J., Kretsch, R., Pintilie, G. D., Rangan, R., Kladwang, W., Li, S., Wu, M. T., Pham, E. A., Bernardin-Souibgui, C., Baric, R. S., Sheahan, T. P., D'Souza, V., Glenn, J. S., Chiu, W., & Das, R. (2021). Cryo-EM and antisense targeting of the 28-kDa frameshift stimulation element from the SARS-CoV-2 RNA genome. Nat Struct Mol Biol, 28(9), 747–754. https://doi.org/10.1038/s41594-021-00653-y 吳郁涵. (2024). 利用光鉗探討小分子藥物對於框架位移刺激子RNA 偽結的效應. 國立台灣大學生命科學院分子與細胞生物學研究所碩士論文. 杜睿芸. (2020). 利用光鉗技術探討核醣體如何影響誘導框架位移的偽結. 國立台灣大學生命科學院分子與細胞生物學研究所碩士論文. 汪健州. (2023). 影響轉譯框架位移的核醣核酸結構和此結構與聚核醣體相互作用之研究. 國立台灣大學生命科學院分子與細胞生物學研究所博士論文. 黃瑀彤. (2023). 利用光鉗技術探討RNA 偽結結構對核醣體框架位移之影響. 國立台灣大學生命科學院分子與細胞生物學研究所碩士論文. | - |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/98803 | - |
| dc.description.abstract | 由 SARS-CoV-2 病毒引起的嚴重急性呼吸道症候群 COVID-19 疫情自 2019年末開始傳播，造成數億人感染和數百萬人死亡，對人類健康與生活造成重大影響和威脅。計畫性核醣體框架位移(-1 PRF)作為 SARS-CoV-2 複製過程中必要的機制，成為潛在的對抗病毒的研究目標。引發-1 PRF 的 RNA 結構偽結作為核醣體轉譯過程中的路障，其特性會影響-1 PRF 發生的效率，是研究-1 PRF 的關鍵。
本實驗透過單分子技術光鉗研究 SARS-CoV-2 偽結的序列可能摺疊的各種結構及中間產物，分析組成偽結的二級結構 ST1、ST3 各自的特性及兩者摺疊時的競爭關係，並透過覆蓋 ST2 序列的方式找到偽結在動力學上偏好的摺疊路徑為ST1 接著 ST3 和 ST2，且 ST1 上游序列露出的鹼基數量可能影響最終的偽結構型。 已有相關研究發現在細胞中 SARS-CoV-2 gRNA 並不偏好形成偽結，而是形成包含部分偽結序列的上游競爭結構 AS1，並可能與核醣體共同調控偽結的摺疊。本實驗透過簡化的 AS1 配合光鉗技術模擬核醣體轉譯時對偽結摺疊路徑的調控過程，發現受到上游競爭結構影響的偽結其摺疊路徑發生改變、形成的機率提升、結構塑性降低，且透過突變穩定上游競爭結構可以進一步增強這些效果。根據相關研究表示，偽結的結構塑性降低可能使其引發-1 PRF 的效率降低。 我們認為核醣體和上游競爭結構 AS1 可能透過調控偽結的摺疊順序，使特定構型的偽結形成，降低其結構塑性，最終使-1 PRF 效率下降。 | zh_TW |
| dc.description.abstract | The COVID-19 pandemic, caused by the SARS-CoV-2 virus, broke in late 2019 and has resulted in hundreds of millions of infections and millions of deaths, posing a major threat to human health and daily life. Programmed -1 ribosomal frameshifting (-1 PRF), a crucial step in the replication of SARS-CoV-2, has emerged as a potential target for antiviral research. The RNA pseudoknot that triggers -1 PRF serves as a roadblock to ribosome progression during translation. Its structural properties significantly influence the efficiency of -1 PRF, making it a key subject in the study of frameshifting mechanism.
In this study, we used single-molecule optical tweezers to analyze various folding conformations and intermediates of the pseudoknot sequence. We examined the characteristics of individual stem-loops ST1 and ST3, as well as their competitive interactions during folding. By covering the ST2 sequence with DNA handle, we identified a kinetically preferred folding pathway of the pseudoknot: ST1 folds first, followed by ST3 and ST2. Additionally, the number of available nucleotides upstream of ST1 may affect the final pseudoknot structure. Previous studies have found that SARS-CoV-2 RNA does not preferentially form the pseudoknot structure in cells. Instead, it tends to form upstream competing structures that include parts of the pseudoknot sequence and may co-regulate pseudoknot folding with the ribosome. In this study, we mimicked ribosomal regulation of pseudoknot folding pathways by simplifying these upstream competing structures. We observed that the presence of such structures alters the folding pathway of the pseudoknot, increases its formation probability, and reduces its structural plasticity. Furthermore, mutations that stabilize the upstream competing structure enhance these effects. According to related research, reduced structural plasticity of the pseudoknot may lead to decreased -1 PRF efficiency. We propose that the ribosome and upstream competing structure may regulate the folding sequence of the pseudoknot, promoting the formation of a specific conformation with reduced structural plasticity, ultimately decreasing -1 PRF efficiency. | en |
| dc.description.provenance | Submitted by admin ntu (admin@lib.ntu.edu.tw) on 2025-08-19T16:15:54Z No. of bitstreams: 0 | en |
| dc.description.provenance | Made available in DSpace on 2025-08-19T16:15:54Z (GMT). No. of bitstreams: 0 | en |
| dc.description.tableofcontents | 口試委員審定書............................................................................................................. i
致謝.............................................................................................................................. iii 摘要................................................................................................................................ v Abstract ........................................................................................................................ vii 目次............................................................................................................................... ix 圖次............................................................................................................................... xi 表次............................................................................................................................ xiii 一、 緒論 ....................................................................................................................... 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optical tweezers ............................................................................... 3 二、 研究動機與目的 ................................................................................................... 5 三、 材料與方法 ........................................................................................................... 6 3.1 Construct 建構 ..................................................................................................... 6 3.1.1 Vector 製備 ................................................................................................... 6 3.1.2 Insert 製備 .................................................................................................... 6 3.1.3 Ligation、transformation 和plasmid 純化 .................................................. 8 3.1.4 in vitro transcription ...................................................................................... 9 3.1.5 5’ handle 製備和DIG 修飾 ......................................................................... 9 3.1.6 3’ handle 製備 ............................................................................................. 11 3.1.7 RNA 與DNA handle 黏合 ......................................................................... 11 3.2 單分子實驗optical tweezers ............................................................................ 12 3.2.1 AD bead 製備 .............................................................................................. 12 3.2.2 SA bead 製備............................................................................................... 12 3.2.2 Optical tweezers 操作 ................................................................................. 13 3.2.3 實驗數據分析 ............................................................................................ 13 四、 結果 ..................................................................................................................... 14 4.1 SARS-CoV-2 偽結的特性 ................................................................................. 14 4.1.1 SARS-CoV-2 PK 中的Stem 1 與Stem 3 .................................................. 14 4.1.2 Stem 1 與stem 3 的競爭關係 .................................................................... 15 4.1.3 SARS-CoV-2 PK 序列形成的結構 ............................................................ 16 4.1.4 SARS-CoV-2 PK 的摺疊路徑 .................................................................... 17 4.1.5 5’ spacer 長度對SARS-CoV-2 PK 造成的影響 ....................................... 18 4.2 上游競爭結構對SARS-CoV-2 偽結的影響 ................................................... 19 4.2.1 SARS-CoV-2 PK 上游的競爭結構 ............................................................ 19 4.2.2 Stem 2 和stem 3 組成的SARS-CoV-2 PK 中間產物 .............................. 20 4.2.3 原生的上游競爭結構對SARS-CoV-2 PK 的影響 .................................. 21 4.2.4 上游競爭結構與核醣體調控SARS-CoV-2 PK 摺疊的假說 .................. 22 4.2.5 簡化的上游競爭結構 ................................................................................ 23 4.2.6 簡化的上游競爭結構對SARS-CoV-2 PK 的影響 .................................. 24 4.2.7 簡化的上游競爭結構對SARS-CoV-2 PK 摺疊順序的影響 .................. 25 4.2.8 突變的上游競爭結構 ................................................................................ 27 4.2.9 突變的上游競爭結構對SARS-CoV-2 PK 的影響 .................................. 28 4.2.10 突變的上游競爭結構對SARS-CoV-2 PK 摺疊順序的影響 ................ 29 五、 討論 ..................................................................................................................... 32 六、 參考文獻 ............................................................................................................. 37 圖表.............................................................................................................................. 41 | - |
| dc.language.iso | zh_TW | - |
| dc.subject | 計畫性核醣體框架位移 | zh_TW |
| dc.subject | SARS-CoV-2 | zh_TW |
| dc.subject | 光鉗 | zh_TW |
| dc.subject | 單分子 | zh_TW |
| dc.subject | 偽結 | zh_TW |
| dc.subject | Pseudoknot | en |
| dc.subject | Single-molecular | en |
| dc.subject | Optical tweezers | en |
| dc.subject | -1 PRF | en |
| dc.subject | SARS-CoV-2 | en |
| dc.title | 以光鉗技術研究SARS-CoV-2 病毒中引發計畫性核醣體框架位移的RNA 結構之摺疊 | zh_TW |
| dc.title | Investigating the folding of programmed -1 ribosomal frameshifting-stimulating RNA in SARS-CoV-2 virus using optical tweezers | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 113-2 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 張功耀;余建泓 | zh_TW |
| dc.contributor.oralexamcommittee | Kung-Yao Chang;Chien-Hung Yu | en |
| dc.subject.keyword | SARS-CoV-2,計畫性核醣體框架位移,偽結,單分子,光鉗, | zh_TW |
| dc.subject.keyword | SARS-CoV-2,-1 PRF,Pseudoknot,Single-molecular,Optical tweezers, | en |
| dc.relation.page | 104 | - |
| dc.identifier.doi | 10.6342/NTU202503842 | - |
| dc.rights.note | 同意授權(限校園內公開) | - |
| dc.date.accepted | 2025-08-11 | - |
| dc.contributor.author-college | 生命科學院 | - |
| dc.contributor.author-dept | 分子與細胞生物學研究所 | - |
| dc.date.embargo-lift | 2025-08-20 | - |
| 顯示於系所單位： | 分子與細胞生物學研究所 | |
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