鹽類濃度對神經退化性疾病相關的 CAG 與 CTG 重複序列之DNA髮夾結構動態轉換的影響

Salt concentration dependence on DNA hairpin conformational dynamics of CAG and CTG repeats associated with neurodegenerative diseases

王鴻仁(Wang Hong-Ren)

指導教授 ： 李以仁

國立臺灣師範大學/理學院/化學系/碩士(2020年)

https://doi.org/10.6345/NTNU202001277

摘要

三核苷酸重複序列 (Trinucleotide repeats; TNR) 的不正常擴張會引發多種神經退化性遺傳疾病，其中有許多疾病是由 CAG/CTG 重複序列不正常擴張引起的，例如亨丁頓舞蹈症 (Huntington’s Disease)。由於 TNR 會形成髮夾結構且具有構型間滑動重組的特性，使 DNA 在複製、修復和重組過程中發生錯誤而造成序列擴張。本研究利用單分子螢光共振能量轉移光譜，以氯化鈉濃度為操縱變因，探討奇數重複次數 (CAG)n 與 (CTG)n 序列之動態結構轉換。在低氯化鈉濃度下，可觀察到 (CAG)n 序列傾向形成尾端單組 CAG 序列突出、由四個核苷酸組成環的懸垂髮夾結構，且可能因髮夾結構尾端不穩定而造成其部分打開，但並未觀察到兩端對齊、由三個核苷酸組成環的鈍端髮夾結構。而在較高氯化鈉濃度下，除了使 (CAG)n 序列髮夾結構局部打開之速率常數明顯下降，還出現類似於 (CTG)n 序列之懸垂與鈍端髮夾結構動態結構轉換。我們認為鈉離子可以中和 DNA 磷酸根的負電荷，使鹼基配對作用力更穩定，因此氯化鈉濃度越高，(CAG)n 序列髮夾結構越不容易打開。另外由於 A-A 錯誤配對較 T-T 錯誤配對不穩定，在低氯化鈉濃度下 CAG-CAG 配對無法支撐由三核苷酸組成且較不穩定的環狀結構，因此較不傾向形成鈍端結構，而隨著氯化鈉濃度越高，CAG-CAG 配對穩定性越高，使其有機會生成鈍端結構，因此可觀察到類似於 (CTG)n 序列的動態結構轉換，且在 (CTG)n 序列中亦觀察到當氯化鈉濃度增高，構型間的平衡偏向鈍端結構的現象。因此我們結論 TNR 髮夾結構的莖 (stem) 穩定性可由氯化鈉濃度調控，而影響其動態髮夾構型重組。

關鍵字

單分子螢光共振能量轉移 ； CAG 重複序列 ； DNA 髮夾結構 ； 三核苷酸重複序列擴張疾病

並列摘要

Trinucleotide repeat (TNR) is responsible for several neurodegenerative diseases. Among them, CAG and CTG causes the most diseases, such as Huntington’s Disease. The general pathogenesis of these diseases is that TNR repeats form the hairpin structures with the capability of slippage hairpin configuration, result in errors that lead to abnormal expansions in DNA replication, repair, and recombination processes. In this work, we used single-molecule fluorescence resonance energy transfer (smFRET) to study sodium chloride (NaCl) concentration dependence on the dynamic conformational changes of odd-numbered CAG and CTG repeats. The result reveals that at low NaCl concentration, CAG repeats tend to fold into an overhang hairpin structure with a tetranucleotide loop and a single CAG repeat protruding unit. Occasionally, the termini of hairpin transiently and partially open due to the instability of the stem of the hairpin. Unlike CTG repeats, the blunt-end hairpin configuration of CAG repeats was not observed. However, at high NaCl concentration, the rate constant of partial opening of hairpin decrease dramatically, and the dynamic conformational changes between the overhang and blunt-end configuration was observed. We proposed that sodium ion neutralizes the phosphate groups in the DNA backbone, resulting in lowering the repulsions between the backbones of two antiparallel pairing strands (CAG:CAG) and stabilizing this pairing interaction in the stem of the TNR hairpins. Since the A-A mismatch is less stable than the T-T mismatch, at low NaCl concentration, the CAG:CAG pairs are unable to hold the relatively unstable trinucleotide loop and make the blunt-end structure unfavored. In contrast, at higher NaCl concentration, the stem consisting of CAG:CAG pairs is stabilized, making the blunt-end hairpin thermodynamically reachable, and therefore, transitions between blunt-end and overhang configuration were observed. Moreover, as the increase of sodium chloride concentration, the equilibrium between two hairpin configurations in (CTG)n also leans to the blunt-end configuration due to the strengthening of the stem. In conclusion, stem stability can be modulated by sodium chloride concentration and change the TNR reconfiguration dynamics.

並列關鍵字

Single-molecule fluorescence resonance energy transfer (smFRET)Single-molecule fluorescence resonance energy transfer (smFRET) ； CAG tandem repeats ； DNA hairpin structures ； trinucleotide repeat expansion diseases

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