以單分子螢光共振能量轉移研究造成肌萎縮性脊髓側索硬化症與額顳葉癡呆症的GGGGCC重複序列其結構間的動力學

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2018

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肌萎縮性脊髓側索硬化症 (Amyotrophic lateral sclerosis, ALS) 與額顳葉癡呆症 (frontotemporal dementia, FTD) 皆為嚴重的神經退化性疾病,並且兩者在基因序列中被發現有高度的相關性。於C9orf72基因中的內顯子中發現含有GGGGCC六核苷酸重複序列擴張是家族性遺傳ALS與FTD常見的病因。在正常人的基因中,能在內顯子中發現的重複次數約在25次以下,但卻能在ALS或FTD的患者中發現數十至數百個重複序列。GGGGCC序列可能以不同G-四聯體結構的方式摺疊成二級結構,這樣的結構多元性可能導致轉錄失敗的RNA產物,或是產生錯誤摺疊的二肽重複蛋白。此種G-四聯體的結構隨著重複序列次數的不同,可能產生不同的形式。d(GGGGCC)4可形成反平行的G-四聯體結構,然而其他重複次數亦可能產生平行的G-四聯體結構。在本實驗中,我們利用單分子螢光共振能量轉移 (smFRET) 研究d(GGGGCC)4構形的動態變化。因而發現d(GGGGCC)4 於鉀離子的環境中容易形成G-四聯體結構,但若在無鉀離子的情況下易形成類髮夾結構,而此兩結構間的相互轉換在我們的研究中極為鮮少。為了解兩結構間的活化能,我們建立一套恆溫系統以利觀測,最終求得的活化能其數值極高,能說明兩者在室溫下鮮見相互轉換的原因,有了這些熱力學及動力學的資訊,能幫助我們建立結構間轉換反應坐標的圖像。最後,我們也嘗試研究 (GGGGCC)n重複序列5次以上的結構,並發現在重複次數較多時,較容易以類髮夾結構的形式存在。
Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are severe neurodegenerative diseases with common genetic cause. GGGGCC hexanucleotide repeat expansions (HRE) located in the intronic region of chromosome 9 open reading frame 72 (C9orf72) are the most common cause of familial ALD and FTD. In normal people, there are fewer than 25 intronic GGGGCC repeats, whereas patients with ALS/FTD have tens to hundreds repeats. GGGGCC repeats can fold into different conformations of G-quadruplex secondary structures. These structural polymorphism of GGGGCC repeats leads to transcription of abortive transcripts and translation of misfolded dipeptide-repeat proteins. The form of G-quadruplex varies with the fold number of GGGGCC repeats. d(GGGGCC)4 forms intramolecular anti-parallel G-quadruplex structure while other times of repeats tend to have mixed or parallel G-quadruplex structure. We use single-molecule fluorescence resonance energy transfer (smFRET) to study the structural dynamics of d(GGGGCC)4. We found that d(GGGGCC)4 will form G-quadruplex structure in the presence of potassium ion, but form hairpin-like structure in the absence of potassium ion. The interconversion of these two states is hardly found in our experiments. In order to find out the activation energy between these two states, we set up an environment in which the temperature is alterable. The estimated activation energy is as high as 33.4 kcal mol-1 and it’s in accordance with the result that the interconversion event is rare. With the aid of these thermodynamic and kinetic information, we can build a reaction coordinate of the two states. Finally, we also tried to study the d(GGGGCC)n sequence with n≥5, and we noticed that the d(GGGGCC)n prefer hairpin-like structure when the number of repeats increase.

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肌萎縮性脊髓側索硬化症, 額顳葉癡呆症, 六核苷酸重複序列擴張, G-四聯體, 單分子螢光共振能量轉移, ALS, FTD, hexanucleotide repeat expansions, G-quadruplex, smFRET

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