本篇研究以探討 CGG 三重複序列的構型為主。依照先前的文獻，單股的 CGG 序列在不同的環境下會有不一樣的二級結構。為了解出最真實的構型，我們使用 了單分子螢光共振能量轉移光譜，我們的實驗結果顯示，CGG 三核苷酸重複序 列會傾向於折疊成髮夾型結構。這些髮夾型結構，會依照不同的重複次數會在髮 夾型的尾端有些許的不同。在較長的序列中，這樣的行為類似於髮夾型構型的滑 動。其中 d(CTG)n序列中相同的現象[27]。
另外，在我們也發現 CGG 序列在非生理條件下，可以摺疊成鳥嘌呤四聚體 的構型。其中最短的 d(CGG)4可以在高鉀離子的條件下折疊成鳥嘌呤四聚體。但 在較長的序列中，CGG 序列會傾向於折疊成髮夾型結構。即使加入了鳥嘌呤四 聚體的誘導劑，也僅能夠影響重複次數小於 12 的 CGG 序列，較長的序列在相同 的條件下，還是會以髮夾型結構為主。對於這樣的結果並不意外，因為較高重複 次數時，髮夾型結構能夠提供更多鹼基配對，能夠使構型更加穩定。
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4-2 AGG 對 CGG 序列之影響
在本研究的實驗結果，AGG 會讓 CGG 序列的構型發生變化。如果只插入一 組 AGG 時，會出現兩種不一樣的髮夾型結構，如圖 52 所示。同時也可以觀察兩 者之間會互相轉換。對於此結果，我們認為這與較長序列的滑動行為相同。
圖 52、單組 AGG 插入 CGG 之序列的情況。兩種髮夾行結構會 發生互換的現象。
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如圖 53 所示，當 CGG 序列中插入兩組 AGG 時，構型上會傾向形成突出一 組 CGG 之髮夾型的結構[24]，少部分的分子會維持在對齊型的髮夾型結構；從 動態觀察中，EFRET 並無明顯的變化。同樣地，這些序列也更不容易形成鳥嘌呤 四聚體。對於這樣的結果，進一步顯示出形成髮夾型結構更能夠形成鹼基間的配 對，以達到更穩定的二級結構。從實驗結果可以發現 AGG 的插入會使得原先的 髮夾型結構發生變化。若是插入 AGG 的數目變多，會使得構型更加穩定在露出 一組 CGG 之髮夾型結構。
圖 53、插入兩組 AGG 之 CGG 序列的構型示意圖，主要會以構型更加穩定在露出一 組 CGG 之髮夾型結構的構型為主。
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4-3 DNA 在人體中的擴張模型
綜合本研究的實驗結果，我們可以發現 CGG 三核苷酸重複序列在人體中是 以對齊型髮夾或是露出一個核苷酸髮夾結構為主。隨著重複次數的增加，髮夾型 結構會產生出滑動的現象。以本實驗的結果為依據，我們可以預測在人體中真實 的情況，髮夾型結構將會滑動。根據先前我們研究團隊所提出的 TGGAA 序列擴 張模型[26]，如圖 54 所示。DNA 可能會在複製、修復或是重組的過程中新增一 組重複序列單元；而形成突出型的髮夾型結構，此一構型較不利於繼續進行錯誤 的擴張。但因 DNA 的滑動現象將使少部分序列形成對齊型髮夾型結構，而此一 結構利於繼續擴張(圖 54 中紅色區域)。經由多次循環將擴張成更長序列[26]。
圖 54、TGGAA 序列擴張機制示意圖。
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根據前述的擴張模型以及我們的實驗結果，一般的 CGG 序列會傾向於折疊 成對齊型或是接近對齊的髮夾型結構。此構型較利於序列在複製、修復或重組時 擴張。然而若是插入 AGG 的 CGG 序列，將會使對齊型的髮夾型結構減少，平 衡會往突出一組 CGG 之髮夾型結構為主，而不利於 DNA 序列的擴張，並且滑 動現象並不復見，如圖 55 所示。因此我們認為這樣的結構差異是阻止序列擴張 的主要原因。
圖 55、CGG 序列可能的擴張模型示意圖。
對於先前研究所提出的 DNA 序列擴張模型是否確實可以應用在 CGG 序列 中。未來可以模擬出一個與 DNA 擴張(複製、修復、重組)相同的環境，觀察是 否也具有滑動的現象，以驗證此模型在 CGG 序列上的可行性。若經驗證即可病 患體內的 CGG 重複序列以基因療法或進行點突變 (point mutation) 以抑制疾病 並防止遺傳至子代在先前的研究中也有指出，可以透過調控甲基化的機制來減緩 過甲基化的現象，能夠使 FMR1 基因再次具有活性，得到其基因產物[48]。若能 夠加入藥物使序列擴張的情況減緩，就能夠抑制過甲基化的現象，藉此達到治療 疾病的效果。
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參考文獻
[1] Di Prospero, Nicholas A., and Kenneth H. Fischbeck. "Therapeutics development for triplet repeat expansion diseases." Nature Reviews Genetics,
2005, 6, 756-767.
[2] Nicholas A. Di Prospero and Kenneth H. Fischbeck. "Clinical features of spinal and bulbar muscular atrophy. " Brain, 2009, 132, 3242-3251.
[3] Sergei M. Mirkin. "Expandable DNA repeats and human disease " Nature, 2007,
447, 932-940.
[4] Soochul Shin, Kwangbeom Hyun, Jaehoon Kim, and Sungchul Hohng "ATP Binding to Rad initiates Replication Fork Reversal by Inducing the Unwinding of the Leading arm and the Formation of the Lolliday Junction" Cell Reports,
2018, 23, Issue6, 1831-1939.
[5] Catherine B Volle, Sarah Delaney. "AGG/CCT interruptions affect nucleosome formation and positioning of heathy-length CGG/CCG triplet repeats" BMC
Biochemistry, 2013, 14-33,
[6] 侯家瑋 王作仁 "Ｘ染色體脆折症之臨床及分子生物學研究" 台灣醫界 [7] Lubs HA. "A marker X-chromosome." Am J Hum Genet, 1969, 21, 231-244 [8] K Sugimoto, T Okazaki, and R Okazaki. ." Mechanism of DNA chain growth,
II. Accumulation of newly synthesized short chains in E. coli infected with ligase-defective T4 phages. " PNAS, 1968, 60(4), 1356-1362.
[9] De Vries BBA, Halley DJJ, Oostra BA, et al "The fragile X syndrome." J Med
Genet, 1998, 35, 579-589.
[10] Turner G, Eastman C, Casey J, et al "X-linked mental retardation associated with macro-orchidism. " J Med Genet, 1975, 12, 367-371.
64
[11] Sutherland GR "Fragile sites on human chromosome: demonstration of their dependence on the type of tissue culture medium." Science, 1977, 197, 265-266.
[12] Verkerk AJMH, Pteretti M, Sutcliff JS, et al "Identification of a gene (FMR-1) containing a CGG repeat coincident with a breakpoint cluster region exhibiting length variation in fragile X syndrome. " Cell, 1991, 65, 905-914.
[13] Pembrey ME, Winter RM, Davies KE, "A premutation that generates a defect at crossing over explains the inheritance of fragile X retardation. " Am J Med
Genet, 1985, 21, 709-717.
[14] Manel Esteller "CpG island hypermethylation and tumor suppressor genes:a booming present, a brighter future. " Oncogene, 2002, 21, 5427-5440
[15]
Greger V, Passarge E, Höpping W, Messmer E, Horsthemke B. "Epigenetic
changes may contribute to the formation and spontaneous regression of retinoblastoma." Human Genetics, 1989, 83 (2), 155–8.[16] Gacy, A. M., Goellner, G., Juranic, N., Macura, S. & McMurray, C. T.
"Trinucleotide repeats that expand in human disease form hairpin structures in vitro." Cell, 1995, 81, 533–540
[17] Nancy H. Campbell, Stephen Neidle, "G-Quadruplex and metal ions." Metal
Ions in Life Science, 2012, 10, 119-133
[18] K. Usdin, K.J.Woodford, "CGG repeats associated with DNA instability and chromosome fragility form structures Mithat block DNA synthesis in vitro. "
Nucleic Acid Research, 1995, 23(20), 4202-4209.
[19] Hagar Mor-Shaked, Rachel Eiges, "Revaluation of FMR1 hypermethylation Timing in Fragile X Syndrome." Frontiers in Molecular Neuroscience, 2018,
Vol.11, Article 31.
65
[20] Matthew J. Bottomley, et al "Structural and Functional Analysis of the Human HDAC4 Catalytic Domain Reveals a Regulatory Structural Zinc-binding Domain" JBC, 2008, 283(39), 26694-26704.
[21] Renciuk D et.al "Quadruplex-forming properties of FRAXA (CGG) repeats interrupted by (AGG) triplet. " Biochime, 2009, 91, 416-422.
[22] Vorlícková M, Bednárová K, Kypr J. "Ethanol is a better inducer of DNA guanine tetrahedral than potassium. " Biopolymers, 2006, 82, 253-260.
[23] Daniel A Jarem, Lauren V.Huckaby, Sarah Delaney "AGG interruptions in (CGG)n DNA repeat tract modulate the structure and thermodynamics of non-B conformations in vitro" Biochemistry, 2010, 49, 6826-6837
[24] S. Amrane, J.-L. Mergny "Length and pH-dependent energetics of (CCG)n and (CGG)n trinucleotide repeats." Biochime, 2006, 88, 1125-1134.
[25] Petr Fojtík, et al."The guanine-rich fragile X chromosome repeats are reluctant to form tetraplexes." Nucleic Acids Research, 2004, Vol.32, 298-306.
[26] Tze-Yun Huang, et al, "Parity-dependent hairpin configurations of repetitive DNA sequence promote slippage associated with DNA expansion. " PNAS,
2017, 114, 9535-9540.
[27] 倪丞緯。2017。以單分子光譜觀測 CTG 重複序列的滑動現象。碩士學位 論文。台北:國立臺灣師範大學化學所
[28] F. Ritort. "Single-molecule experiments in biological physics: methods and applications. " J. Phys. Condens. Matter, 2006, 18, 531-583.
[29] A. Ashkin. "Acceleration and Trapping of Particles by Radiation Pressure. "
Phys. Rev. Lett, 1970, 24, 156–159.
[30] Hellen C. Ishikawa-Ankerhold, Richard Ankerhold and Gregor P. C. Drummen.
"Advanced Fluorescence Microscopy Techniques" FRAP, FLIP, FLAP, FRET
and FLIM. Molecules, 2012, 17, 4047-4132.
66
[31] Brtce T. Bajor, Emily S. Wang, Shu Zhang, Micheal Z. Lin, Jun Chu. "A Guide to Lluorescent Protein FRET Pairs." Sensors, 2006, 16,1488.
[32] 黃子芸。2016。利用單分子技術研究小腦失調症第 31 型特殊連續 TGGAA 重複序列結構動態學。碩士學位論文。台中：國立中興大學基因體暨生物 資訊學研究所。
[33] Sulfo-Cyanine3 NHS ester , Lumiprobe Corporation
<http://www.lumiprobe.com/p/sulfo-cy3-nhs-ester.>
[34] 5' Amino Modifier C6, Integrated DNA Technologies, Inc
<https://sg.idtdna.com/site/Catalog/Modifications/Product/1082>.
[35] Hellen C. Ishikawa-Ankerhold, Richard Ankerhold and Gregor P. C. Drummen.
"Advanced Fluorescence Microscopy Techniques." FRAP, FLIP, FLAP, FRET
and FLIM. Molecules, 2012, 17, 4047-4132.
[36] Thorben Cordes, Jan Vogelsang and Philip Tinnefeld. "On the Mechanism of Trolox as Antiblinking and Antibleaching Reagent. " J. Am. Chem. Soc., 2009,
131, 5018–5019.
[37] Theodorus H. de Koker, Michael D. Mozuch, Daniel Cullen, Jill Gaskell and Philip J. Kersten. "Isolation and Purification of Pyranose 2-Oxidase from Phanerochaete chrysosporium and Characterization of Gene Structure and Regulation." Applied and Environmental Microbiology, 2004, 70(10), 5794–
5800
[38] Andrey Ahirak, Uri Seroussi, Elisha Gootwine and Eyal Seroussi. "Sequence motifs capable of forming DNA stem-loop structures act as a replication diode."
FEBS Open Bio, 2017, 7, 944-952.
[39] Rahul, R., Hohng, S. & Ha, T. "A Practical Guide to Single Molecule FRET. "
67
Nat. Methods, 2008, 5, 507–516.
[40] Mckinney, S. A., Joo, C. & Ha, T."Analysis of Single-Molecule FRET Trajectories Using Hidden Markov Modeling." Biophys J, 2006, 91,1941–1951.
[41] 許顥頤。2016。以單分子螢光共振能量轉移光譜研究人類端粒序列形成的 鳥嘌呤四股結構之構型變化與動力學數據分析在不同實驗因素下的影響。
碩士學位論文。台北：國立臺灣師範大學。
[42] Amarjit Saini, Sarabjit Mastana,Fiona Myers, Mark Peter Lewis "‘From Death, Lead Me to Immortality’ – Mantra of Ageing Skeletal Muscle" Curr Genomics,
2013, 14, 256-267.
[43] Saccà B, Lacroix L, Mergny JL "The effect of chemical modifications on the thermal stability of different G-quadruplex-forming oligonucleotides." Nucleic
Acid Research, 2005, 33, 1182-1192.
[44] Watson JD, Baker TA, Bell SP, Gann A, Levine M, Losick R. Molecular Biology of the Gene. 5th ed. Pearson Benjamin Cummings: CSHL Press.
[45] Huppert J. L., Balasubramanian S. "Prevalence of quadruplexes in the human genome. " Nucleic Acids Research, 2005, 33, 2908–2916.
[46] AuroreDe Rache, Jean-LouisMergny, "Assessment of selectivity of G-quadruplex ligands via an optimised FRET melting assay. " Biochime, 2015,
115, 194-202.
[47] Koirala D, Dhakal S, Ashbridge B, Sannohe Y, Rodriguez R, Sugiyama H, Balasubramanian S, Mao H, "A single-molecule platform for investigation of interactions between G-quadruplexes and small-molecule ligand. " Nat Chem,
2011, 3(10), 782-787.
[48] X. ShawnLiu, HaoWu,, et al. "Rescue of Fragile X Syndrome Neurons by DNA Methylation Editing of the FMR1 Gene" Cell, 2018, 172, 979-992.
68
[49] DZ Torrone, JS Kuriakose, K Moors, H Jiang, MM Niedzwiecki, FF Perera and RL MillerEmail author. "Reproducibility and intraindividual variation over days in buccal cell DNA methylation of two asthma genes, interferon γ (IFNγ) and inducible nitric oxide synthase (iNOS)" Clinical Epigenetics, 2010, 4:3