很多神經退化性疾病都是由重複序列異常擴張所引起，包含脊髓小腦萎縮症 36 型 (Spinocerebellar ataxia 36, SCA36)。他是由位於 Nop56 基因中的 GGCCTG 六核苷酸重複序列擴張所造成的，會造成一些神經功能障礙，像是肢體不協調及小腦萎縮等症狀。此類重複序列通常會折疊成二級結構，而這些二級結構被認為是在複製、修復或重組過程中造成不正常序列擴張的原因。
我們使用單分子螢光共振能量轉移 (single-molecule fluorescence resonance energy transfer, smFRET) 研究重複次數為 3 ~ 8、11 及位於預突變 (premutation) 範圍的 17 的 (GGCCTG)n 重複序列的結構動態學，以及在精胺 (為與 GGCCTG 具有特異性結合的多電荷胺類) 影響下的動力學。我們發現偶數重複的 (GGCCTG)n 會穩定形成 2 個核酸 (2-nt) 突出髮夾結構 (2-nt overhang hairpin)；而奇數重複的 (GGCCTG)n 會在 2-nt 突出髮夾結構與 8-nt 突出髮夾結構 (8-nt overhang hairpin) 之間做轉換，且平衡利於 2-nt 突出髮夾結構。精胺的添加使平衡傾向於 2-nt 突出髮夾結構，使其可能具有更高的重複序列擴張的趨勢。

Abnormal repeat expansion is a major cause for many neurodegenerative diseases, including Spinocerebellar Ataxia 36 (SCA36). GGCCTG hexanucleotide repeat expansion in the Nop56 gene is responsible for nervous system dysfunctions, including incoordination and cerebellar atrophy. These tandem repeats DNA usually fold into a secondary structure, which is believed to be crucial in abnormal expansion during replication, repair or recombination process.
We used single-molecule fluorescence resonance energy transfer (smFRET) microscopy to study conformational dynamics of (GGCCTG)n sequences with n = 3 ~ 8, 11 and 17, as well as the dynamics under the influence of spermine, a polycharged amine that binds to GGCCTG tandem repeat specifically. We found that even-numbered repeats of (GGCCTG)n form stable 2-nt overhang hairpin structure while odd-numbered repeats form hairpin interconverting between a 2-nt and 8-nt overhang structures, with the equilibrium favors a 2-nt overhang hairpin structure. The addition of spermine makes the equilibrium futher lean toward the 2-nt overhang hairpin structure, which may have higher tendency to undergo repeat expansions.

[1] Ashizawa, T.;Oz, G.;Paulson, H. L., Spinocerebellar Ataxias: Prospects and Challenges for Therapy Development. Nat, Rev. Neurol. 2018, 14 (10), 590-605.
[2] Sullivan, R.;Yau, W. Y.;O'Connor, E.;Houlden, H., Spinocerebellar Ataxia: an Update. J. Neurol. 2019, 266 (2), 533-544.
[3] Kobayashi, H.;Abe, K.;Matsuura, T.;Ikeda, Y.;Hitomi, T.;Akechi, Y.;Habu, T.;Liu, W.;Okuda, H.;Koizumi, A., Expansion of Intronic GGCCTG Hexanucleotide Repeat in NOP56 Causes SCA36, a Type of Spinocerebellar Ataxia Accompanied by Motor Neuron Involvement. Am. J. Hum. Genet. 2011, 89 (1), 121-30.
[4] Lopez, S.;He, F., Spinocerebellar Ataxia 36: From Mutations Toward Therapies. Front. Genet. 2022, 13.
[5] Yi, J.;Wan, L.;Liu, Y.;Lam, S. L.;Chan, H. Y. E.;Han, D.;Guo, P., NMR Solution Structures of d(GGCCTG)n Repeats Associated with Spinocerebellar Ataxia Type 36. Int. J. Biol. Macromol. 2022, 201, 607-615.
[6] Furuta, N.;Tsukagoshi, S.;Hirayanagi, K.;Ikeda, Y., Suppression of the Yeast Elongation Factor Spt4 Ortholog Reduces Expanded SCA36 GGCCUG Repeat Aggregation and Cytotoxicity, Brain Res. 2019, 1711, 29-40.
[7] Hirayanagi, K.;Ozaki, H.;Tsukagoshi, S.;Furuta, N.;Ikeda, Y., Porphyrins Ameliorate Spinocerebellar Ataxia Type 36 GGCCTG Repeat Expansion-Mediated Cytotoxicity. Neurosci. Res. 2021, 171, 92-102.
[8] Maizels, N., G4-associated Human Diseases. EMBO Rep. 2015, 16 (8), 910-22.
[9] Varshney, D.;Spiegel, J.;Zyner, K.;Tannahill, D.;Balasubramanian, S., The Regulation and Functions of DNA and RNA G-quadruplexes. Nat. Rev. Mol. Cell Biol. 2020, 21 (8), 459-474.
[10] Khristich, A. N.;Mirkin, S. M., On the Wrong DNA Track: Molecular Mechanisms of Repeat-Mediated Genome Instability. J. Biol. Chem. 2020, 295 (13), 4134-4170.
[11] Mirkin, S. M., DNA Structures, Repeat Expansions and Human Hereditary Disorders. Curr. Opin. Struct. Biol. 2006, 16 (3), 351-8.
[12] Huang, T. Y.;Chang, C. K.;Kao, Y. F.;Chin, C. H.;Ni, C. W.;Hsu, H. Y.;Hu, N. J.;Hsieh, L. C.;Chou, S. H.;Lee, I. R.;Hou, M. H., Parity-dependent Hairpin Configurations of Repetitive DNA Sequence Promote Slippage Associated with DNA Expansion. Proc. Natl. Acad. Sci. U.S.A. 2017, 114 (36), 9535-9540.
[13] Ni, C. W.;Wei, Y. J.;Shen, Y. I.;Lee, I. R., Long-Range Hairpin Slippage Reconfiguration Dynamics in Trinucleotide Repeat Sequences. J. Phys. Chem. Lett. 2019, 10 (14), 3985-3990.
[14] Mounce Bryan, C.;Olsen Michelle, E.;Vignuzzi, M.;Connor John, H., Polyamines and Their Role in Virus Infection. Microbiol. Mol. Biol. Rev. 2017, 81 (4), e00029-17.
[15] Handa, A. K.;Fatima, T.;Mattoo, A. K., Polyamines: Bio-Molecules with Diverse Functions in Plant and Human Health and Disease. Front. Chem. 2018, 6, 10.
[16] Kanemura, A.;Yoshikawa, Y.;Fukuda, W.;Tsumoto, K.;Kenmotsu, T.;Yoshikawa, K., Opposite Effect of Polyamines on In Vitro Gene Expression: Enhancement at Low Concentrations but Inhibition at High Concentrations. PLoS One 2018, 13 (3), e0193595.
[17] Brooks, W. H., Increased Polyamines Alter Chromatin and Stabilize Autoantigens in Autoimmune Diseases. Front. Immunol. 2013, 4, 91.
[18] Madeo, F.;Eisenberg, T.;Pietrocola, F.;Kroemer, G., Spermidine in Health and Disease. Science 2018, 359 (6374), eaan2788.
[19] Minois, N.;Carmona-Gutierrez, D.;Madeo, F., Polyamines in Aging and Disease. Aging 2011, 3 (8), 716-732.
[20] Lewandowski Nicole, M.;Ju, S.;Verbitsky, M.;Ross, B.;Geddie Melissa, L.;Rockenstein, E.;Adame, A.;Muhammad, A.;Vonsattel Jean, P.;Ringe, D.;Cote, L.;Lindquist, S.;Masliah, E.;Petsko Gregory, A.;Marder, K.;Clark Lorraine, N.;Small Scott, A., Polyamine Pathway Contributes to the Pathogenesis of Parkinson Disease. Proc. Natl. Acad. Sci. U.S.A. 2010, 107 (39), 16970-16975.
[21] Ritort, F., Single-molecule Experiments in Biological Physics: Methods and Applications. J. Phys. Condens. Matter 2006, 18 (32), R531-83.
[22] Kankanamge, D.;Ratnayake, K.;Senarath, K.;Tennakoon, M.;Harmon, E.;Karunarathne, A., Optical Approaches for Single-Cell and Subcellular Analysis of GPCR–G Protein Signaling. Anal. Bioanal. Chem. 2019, 411 (19), 4481-4508.
[23] Sasmal, D. K.;Pulido, L. E.;Kasal, S.;Huang, J., Single-molecule Fluorescence Resonance Energy Transfer in Molecular Biology. Nanoscale 2016, 8 (48), 19928-19944.
[24] Ishikawa-Ankerhold, H. C.;Ankerhold, R.;Drummen, G. P. C., Advanced Fluorescence Microscopy Techniques—FRAP, FLIP, FLAP, FRET and FLIM. Molecules 2012, 17 (4).
[25] Martin-fernandez, M.;Tynan, C.;Webb, S., A ‘Pocket Guide’ to Total Internal Reflection Fluorescence. J. Microsc. 2013, 252.
[26] 李以仁，許顥頤，秦志皞，吳佳諭, 單分子螢光共振能量轉移光譜簡介. 化學 2015, 73 (4), 303-312.
[27] Wylie, D., Evidence for DNA Oxidation in Single Molecule Fluorescence Studies. 2006.
[28] Silva, I.;Rausch, V.;Seitz, H. K.;Mueller, S., Does Hypoxia Cause Carcinogenic Iron Accumulation in Alcoholic Liver Disease (ALD)? Cancers 2017, 9 (11).
[29] Roy, R.;Hohng, S.;Ha, T., A Practical Guide to Single-Molecule FRET. Nat. Methods 2008, 5 (6), 507-16.
[30] McKinney, S. A.;Joo, C.;Ha, T., Analysis of Single-Molecule FRET Trajectories Using Hidden Markov Modeling. Biophys. J. 2006, 91 (5), 1941-51.
[31] 黃郁筑與侯明宏, 未發表資料.
[32] OligoAnalyzer Tool - Integrated DNA Technologies, Inc.
<https://sg.idtdna.com/calc/analyzer>