以單分子螢光共振能量轉移技術探討G4C2 RNA重複序列的分子內與分子間的交互作用

Abstract

肌萎縮側索硬化症（Amyotrophic Lateral Sclerosis, ALS）與額顳葉失智症（Frontotemporal Dementia, FTD）為嚴重的神經退化性疾病，其主要的致病原因與C9orf72基因中六核苷酸（G4C2）序列的異常擴增密切相關。該序列轉錄生成的G4C2 RNA聚集現象，被認為是引發神經毒性的重要因素，然而其具體的致病機制仍有待釐清。因此，本研究利用螢光共振能量轉移（FRET）技術，探討G4C2 RNA重複序列的結構特性與其分子間的交互作用。 首先，為了探究不同鹽類與重複次數對G4C2 RNA結構穩定性的影響，我們設計了多種能與G4C2 RNA重複序列互補的反義寡核苷酸，藉由與r(G4C2)2的結合效率測定，顯示dC2G2C4為較合適的DNA探針。在此偵測系統中，就鹽類效應而言，r(G4C2)2的結構穩定度依序遞減：100 mM NaCl, 100 mM KCl, 100 mM NH4Cl ≫ 10 mM MgCl2。就重複次數（n = 2 ～ 5）而言，結果顯示在10 mM MgCl2和100 mM KCl的條件下，G4C2 RNA可能摺疊成不同結構，並且在100 mM KCl中，G4C2 RNA的結構穩定性呈現出重複次數依賴性。接著，利用凝膠電泳定量與螢光漂白實驗，結果顯示分別在10 mM MgCl2和100 mM KCl條件下製備的r(G4C2)2複合物，傾向形成RNA雙鏈（Duplex）或二聚體（dimer）結構，且在100 mM KCl的條件下更為穩定。最後，團簇模擬實驗顯示在10 mM MgCl2條件下，r(G4C2)25可以形成分子間相互作用。 本篇研究藉由評估反義寡核苷酸與G4C2 RNA的結合效率，顯示G4C2 RNA結構穩定性受重複次數與離子環境共同調控，且在較長重複序列中觀察到分子間相互作用，進而為在神經細胞內RNA團簇形成的起始機制提供線索。

Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD) are severe neurodegenerative diseases, with their primary pathogenic cause closely linked to abnormal expansions of the hexanucleotide (G4C2) repeat sequence in the C9orf72 gene. The aggregation of G4C2 RNA transcribed from this sequence is considered a key factor triggering neurotoxicity; however, the precise pathogenic mechanisms remain to be clarified. Therefore, this study employs Förster Resonance Energy Transfer (FRET) technology to investigate the structural characteristics of the G4C2 RNA repeat sequence and its intermolecular interactions. Initially, to examine the influence of different salts and repeat numbers on the structural stability of G4C2 RNA, we designed various antisense oligonucleotides complementary to the G4C2 RNA repeat sequences. Binding efficiency assays conducted with r(G4C2)2 identified dC2G2C4 as the most suitable DNA probe. In the detection system, regarding the effect of salts, the structural stability of r(G4C2)2 follows the order: 100 mM NaCl, 100 mM KCl, 100 mM NH4Cl ≫ 10 mM MgCl2. In terms of repeat number (n = 2 – 5), the results indicate that under conditions of 10 mM MgCl2 and 100 mM KCl, G4C2 RNA may fold into distinct conformations, with a more pronounced positive correlation between repeat numbers and structural stability observed in 100 mM KCl. Subsequently, using gel electrophoresis quantification and fluorescence bleaching assays, the results show that r(G4C2)2 complexes formed under 10 mM MgCl2 and 100 mM KCl conditions predominantly assemble into RNA duplexes or dimeric structures, with greater stability under 100 mM KCl. Finally, cluster simulation experiments demonstrate that r(G4C2)25 can form intermolecular interactions under 10 mM MgCl2 conditions. This study evaluates the binding efficiency between antisense oligonucleotides and G4C2 RNA, demonstrating that the structural stability of G4C2 RNA is jointly regulated by the number of repeats and the ionic environment. Intermolecular interactions were observed in longer repeat sequences, offering potential insights into the initiation mechanism of RNA cluster formation in neuronal cells.