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研究生(外文):Tzu-Yun Huang
論文名稱(外文):Single-molecule Study on the Structural Dynamics of TGGAA Tandem Repeats Associated with Spinocerebellar Ataxia Type 31 Disease
指導教授(外文):Min-Hon HouI-Ren Lee
口試委員(外文):Ying-Chieh Sun
外文關鍵詞:tandem repeatsTGGAASpinocerebellar Ataxiasingle-molecule spectroscopy
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TGGAA五核苷酸重複序列為造成神經退行性疾病脊髓小腦失調症第31型 (Spinocerebellar ataxia type 31, SCA31)的主要致病原因,患者16號染色體q22.1位置上的BEAN和TK2兩基因間會插入一段長度2.5~3.8kb、且包含至少110次重複 TGGAA序列的插入片段,並會於小腦內的浦肯亞神經細胞 (purkinje neurons)形成具細胞毒性的RNA凝聚體,在前人的研究中曾發現發病子代的插入片段比發病親代要來得更長,而隨著該片段長度越長、發病年齡亦越早,其中造成序列增長的可能原因為複製、修復或重組過程中,重複序列因非典型摺疊而滑動。本篇以單分子螢光共振能量轉移光譜觀察TGGAA二到八次重複序列如何摺疊,結果顯示三次以上重複者會形成類髮夾結構,而重複次數為奇數時,會形成頭對尾構型,重複次數為偶數時,會以少一個重複單位參與鍵結的方式形成類髮夾結構;最特別的是在6與8次重複序列中,有觀察到前述兩種構型間轉換的動態行為,分析所得結果後,本篇為TGGAA重複序列於合成時,因滑動而增長提出一可能機制。

Tandem pentanucleotide repeats of TGGAA is a specific sequence that has been shown to be responsible to the neurodegenerative disease Spinocerebellar Ataxia type 31. An insertion of TGGAA repeats with length of 2.5-3.8 kb is found between BEAN (brain expressed, associated with Nedd4) and TK2 (thymidine kinase 2 gene) on chromosome 16q22.1 exclusively in every patient. This insertion contains at least 110 TGGAA repeats and ultimately lead to the production of the toxic RNA foci in Purkinje neuron. The onset age is highly correlated to the length of the insertion and the expansion of this insertion was found in the inherited younger generation patients. This expansion is possibly due to the DNA slippage induced by a non-canonical folded structure during replication, repair, or recombination process. We use single-molecule spectroscopy to study the folding of 2 to 8 repeats of TGGAA sequences. Our result shows that the tandem repeats of TGGAA fold into a hairpin-like structure when the repeat number is greater than 2. Moreover, an end-to-end aligned hairpin-like structure was found in the molecules with odd repeats, while a single-repeat overhang that does not participate in the folding in addition to the hairpin-like structure is found in the case of even repeats. Interestingly, we also observed a dynamic behavior of interconversion of the abovementioned conformations in the molecules with 6 and 8 repeats. We propose a slippage mechanism that may lead to the expansion of the TGGAA repeats during DNA synthesis.

摘要 i
Abstract ii
目錄 iii
圖目錄 v
表目錄 vii
第一章、緒論 1
一、 文獻探討 1
(一) 核苷酸重複序列 1
(二) 脊髓小腦失調症第31型 2
(三) 已解出之TGGAA結構 10
二、 研究動機與目的 13
第二章、實驗儀器與方法 14
一、 實驗技術與儀器 14
(一) 單分子實驗技術 14
(二) 螢光共振能量轉移 15
(三) 全內反射螢光顯微鏡 17
二、 實驗樣品製備與流程 18
(一) 實驗玻片處理 18
(二) 實驗玻片組裝 20
(三) DNA序列 20
(四) 螢光染料Cy3標記 21
(五) DNA黏合反應 (DNA annealing) 23
(六) 令DNA附於微通道樣品槽 23
(七) 顯像緩衝溶液 (imaging buffer) 24
(八) 數據分析與處理 26
第三章、實驗結果 29
一、 實驗設計 29
二、 TGGAA重複序列頭對尾結構模擬 30
三、 TGGAA 2~8次重複序列摺疊情形 31
四、 利用模擬構型確認偶數組重複序列構型 34
五、 (TGGAA)8動態追蹤 35
六、 (TGGAA)6動態追蹤 36
七、 其餘(TGGAA)重複序列軌跡追蹤 39
第四章、討論 41
第五章、未來展望 44
第六章、參考資料 45

[1]Guy Franck Richard, ‘Shortening Trinucleotide Repeats Using Highly Specific Endonucleases: A Possible Approach to Gene Therapy?’, Trends in Genetics, 31.4 (2015), 177–86. doi.org/10.1016/j.tig.2015.02.003.
[2]Marcy E. MacDonald and others, ‘A Novel Gene Containing a Trinucleotide Repeat That Is Expanded and Unstable on Huntington’s Disease Chromosomes’, Cell, 72.6 (1993), 971–83 . doi.org/10.1016/0092-8674(93)90585-E.
[3]H T Orr and others, ‘Expansion of an Unstable Trinucleotide CAG Repeat in Spinocerebellar Ataxia Type 1.’, Nature Genetics, 4.3 (1993), 221–26. doi.org/10.1038/ng0793-221.
[4]L. E. Almaguer-Mederos and others, ‘Estimation of the Age at Onset in Spinocerebellar Ataxia Type 2 Cuban Patients by Survival Analysis’, Clinical Genetics, 78.2 (2010), 169–74. dx.doi.org/10.1111/j.1399-0004.2009.01358.x.
[5]Yoshio Ikeda, Randy S. Daughters and Laura P W Ranum, ‘Bidirectional Expression of the SCA8 Expansion Mutation: One Mutation, Two Genes’, Cerebellum, 2008, 150–58. doi.org/10.1007/s12311-008-0010-7.
[6]Se Holmes and Ee O’Hearn, ‘Expansion of a Novel CAG Trinucleotide Repeat in the 5′ Region of PPP2R2B Is Associated with SCA12’, Nature …, 23.december (1999), 391–92 . doi.org/10.1038/70493.
[7]Mariely DeJesus-Hernandez and others, ‘Expanded GGGGCC Hexanucleotide Repeat in Noncoding Region of C9ORF72 Causes Chromosome 9p-Linked FTD and ALS’, Neuron, 72.2 (2011), 245–56. doi.org/10.1016/j.neuron.2011.09.011.
[8]T a Kunkel, ‘Nucleotide Repeats. Slippery DNA and Diseases.’, Nature, 365.6443 (1993), 207–8. doi.org/10.1038/365207a0.
[9]Sergei M. Mirkin, ‘Expandable DNA Repeats and Human Disease.’, Nature, 447.7147 (2007), 932–40. doi.org/10.1038/nature05977.
[10]Robert D. Wells and others, ‘Advances in Mechanisms of Genetic Instability Related to Hereditary Neurological Diseases’, Nucleic Acids Research, 33.12 (2005), 3785–98. doi.org/10.1093/nar/gki697.
[11]Sergei M. Mirkin, ‘DNA Structures, Repeat Expansions and Human Hereditary Disorders’, Current Opinion in Structural Biology, 2006, 351–58. doi.org/10.1016/j.sbi.2006.05.004.
[12]Christopher E. Pearson and Richard R. Sinden, ‘Trinucleotide Repeat DNA Structures: Dynamic Mutations from Dynamic DNA’, Current Opinion in Structural Biology, 1998, 321–30. doi.org/10.1016/S0959-440X(98)80065-1.
[13]K Usdin and K J Woodford, ‘CGG Repeats Associated with DNA Instability and Chromosome Fragility Form Structures That Block DNA Synthesis in Vitro.’, Nucleic Acids Research, 23.20 (1995), 4202–9 <http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=307363&tool=pmcentrez&rendertype=abstract>.
[14]Keiichi Ohshima and Robert D. Wells, ‘Hairpin Formation during DNA Synthesis Primer Realignment in Vitro in Triplet Repeat Sequences from Human Hereditary Disease Genes’, Journal of Biological Chemistry, 272.27 (1997), 16798–806. doi.org/10.1074/jbc.272.27.16798.
[15]U Nagaoka and others, ‘A Gene on SCA4 Locus Causes Dominantly Inherited Pure Cerebellar Ataxia.’, Neurology, 54.10 (2000), 1971–75.
[16]Kunihiro Yoshida and others, ‘Severity and Progression Rate of Cerebellar Ataxia in 16q-Linked Autosomal Dominant Cerebellar Ataxia (16q-ADCA) in the Endemic Nagano Area of Japan’, Cerebellum, 8.1 (2009), 46–51. doi.org/10.1007/s12311-008-0062-8.
[17]Kinya Ishikawa and others, ‘An Autosomal Dominant Cerebellar Ataxia Linked to Chromosome 16q22.1 Is Associated with a Single-Nucleotide Substitution in the 5’ Untranslated Region of the Gene Encoding a Protein with Spectrin Repeat and Rho Guanine-Nucleotide Exchange-Factor Domains.’, American Journal of Human Genetics, 77.2 (2005), 280–96. doi.org/10.1086/432518.
[18]Takako Ohata and others, ‘A -16C>T Substitution in the 5’ UTR of the Puratrophin-1 Gene Is Prevalent in Autosomal Dominant Cerebellar Ataxia in Nagano’, Journal of Human Genetics, 51.5 (2006), 461–66. doi.org/10.1007/s10038-006-0385-6.
[19]Takeshi Amino and others, ‘Redefining the Disease Locus of 16q22.1-Linked Autosomal Dominant Cerebellar Ataxia’, Journal of Human Genetics, 52.8 (2007), 643–49. doi.org/10.1007/s10038-007-0154-1.
[20]Nozomu Sato and others, ‘Spinocerebellar Ataxia Type 31 Is Associated with “Inserted” Penta-Nucleotide Repeats Containing (TGGAA)n’, American Journal of Human Genetics, 85.5 (2009), 544–57. doi.org/10.1016/j.ajhg.2009.09.019.
[21]Yusuke Niimi and others, ‘Abnormal RNA Structures (RNA Foci) Containing a Penta-Nucleotide Repeat (UGGAA)n in the Purkinje Cell Nucleus Is Associated with Spinocerebellar Ataxia Type 31 Pathogenesis’, Neuropathology, 33.6 (2013), 600–611. doi.org/10.1111/neup.12032.
[22]Maria Assunta Biscotti and others, ‘Transcription of Tandemly Repetitive DNA: Functional Roles’, Chromosome Research, 23.3 (2015), 463–77. doi.org/10.1007/s10577-015-9494-4.
[23]J. Prosser and others, ‘Sequence Relationships of Three Human Satellite DNAs’, Journal of Molecular Biology, 187.2 (1986), 145–55. doi.org/10.1016/0022-2836(86)90224-X.
[24]S H Chou, L Zhu and B R Reid, ‘The Unusual Structure of the Human Centromere (GGA)2 Motif. Unpaired Guanosine Residues Stacked between Sheared G.A Pairs.’, Journal of Molecular Biology, 244.3 (1994), 259–68. doi.org/10.1006/jmbi.1994.1727.
[26]L Zhu, S H Chou and B R Reid, ‘A Single G-to-C Change Causes Human Centromere TGGAA Repeats to Fold Back into Hairpins.’, Proceedings of the National Academy of Sciences of the United States of America, 93.22 (1996), 12159–64. doi.org/10.1073/pnas.93.22.12159.
[27]Y S Lo, Y F Kao, and M H Hou. (2014). ‘Crystal structure of d(GTGGAATGGAAC)’. Retrieved 16 June 2016, from Protein Data Bank <http://www.rcsb.org/pdb/explore/explore.do?structureId=4RZN>.
[28]David Dulin and others, ‘Studying Genomic Processes at the Single-Molecule Level: Introducing the Tools and Applications.’, Nature Reviews. Genetics, 14.1 (2013), 9–22. doi.org/10.1038/nrg3316.
[30]Sulfo-Cyanine3 NHS ester , Lumiprobe Corporation <http://www.lumiprobe.com/p/sulfo-cy3-nhs-ester.
[31]5'' Amino Modifier C6, Integrated DNA Technologies, Inc <https://sg.idtdna.com/site/Catalog/Modifications/Product/1082>.
[32]Thorben Cordes, Jan Vogelsang and Philip Tinnefeld, ‘On the Mechanism of Trolox as Antiblinking and Antibleaching Reagent’, Journal of the American Chemical Society, 131.14 (2009), 5018–19. doi.org/10.1021/ja809117z.
[33]Theodorus H. De Koker and others, ‘Isolation and Purification of Pyranose 2-Oxidase from Phanerochaete Chrysosporium and Characterization of Gene Structure and Regulation’, Applied and Environmental Microbiology, 70.10 (2004), 5794–5800. doi.org/10.1128/AEM.70.10.5794-5800.2004.
[34]Rahul Roy, Sungchul Hohng and Taekjip Ha, ‘A Practical Guide to Single-Molecule FRET.’, Nat. Methods, 5.6 (2008), 507–16. doi.org/10.1038/nmeth.1208.
[35]Chandran R. Sabanayagam, John S. Eid and Amit Meller, ‘Using Fluorescence Resonance Energy Transfer to Measure Distances along Individual DNA Molecules: Corrections due to Nonideal Transfer’, Journal of Chemical Physics, 122.6 (2005). doi.org/10.1063/1.1854120.
[36]Paul Held (2005). ‘An Introduction to Fluorescence Resonance Energy Transfer (FRET) Technology and its Application in Bioscience’. Retrieved 16 June 2016, from BioTek Instruments, Inc. <http://www.biotek.com/resources/articles/fluorescence-resonance-energy-transfer.html>.
[37]Sean A McKinney, Chirlmin Joo and Taekjip Ha, ‘Analysis of Single-Molecule FRET Trajectories Using Hidden Markov Modeling.’, Biophysical Journal, 91.5 (2006), 1941–51. doi.org/10.1529/biophysj.106.082487.

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