CAG三核苷酸重複序列擴增的單分子力學研究

Abstract

重複序列的擴增會影響DNA複製、轉錄、RNA結構以及蛋白質異常結合、聚集。然而序列和結構的不穩定性，使我們難以透過傳統巨量的生物化學方式來進行研究。在本研究中，透過單分子技術，我們想了解CAG重複序列的摺疊結構與展開方式，並與普通的雙股螺旋比較。利用光鉗，將帶有雙股DNA handles的CAG重複序列連結在兩個珠子之間，以機械應力展開其結構再放鬆使其摺疊。結果顯示，在力-延展量的作圖中，CAG重複序列呈現明顯的「高原區」，力的大小相對延展量增加並無太大改變。顯示CAG重複序列在打開或摺疊中的每一小步中不斷的局部重組，同時CAG重複序列最終能夠形成極為緊密的結構。此外，CAG重複摺疊與打開呈現了準可逆過程的特徵，與雙股螺旋結構解開的不可逆過程形成強烈對比。但是較高的離子濃度也會升高CAG重複序列的結構強度，降低可逆性。從固定光阱的實驗也佐證了CAG重複序列的結構具有結構的多型性。本研究揭示了CAG重複序列的摺疊結構與潛在的分子致病機制，可能協助未來能針對其特異的結構特性找到應用，或是其疾病的治療方式。

Trinucleotide expansion can affect DNA replication, transcription, RNA structure and abnormal protein binding or aggregation. Due to the instability, the structure is difficult to study by traditional bulk biochemical experiments. In this study, we apply the single molecule techniques to study the structure of CAG repeats and how they fold and unfold. On optical tweezers, DNA handles with CAG repeats are tethered to beads for mechanical unfolding. Here, we show the force-extension plots of the CAG repeats exhibit a distinct force plateau during unfolding. Our results suggested that CAG repeats form stable, compact structures which unfold and refold in continual substeps. Moreover, such unfolding/refolding resembled as a quasi-reversible process, contrary to irreversible unfolding of Watson-Crick helices. We also found high concentrations of metal ions also increase the structural strength of the CAG repeats and reduce the reversibility. This study reveals the structure and the potential pathogenic mechanism of CAG repeats. The characteristic feature of the repeats may be used in medical treatment or other applications.