以單分子螢光共振能量轉移技術檢測r(G4C2)重複序列之構形變化

Abstract

ALS和FTD的病因之一為C9ORF72基因中的GGGGCC重複序列異常擴增，致病機制與轉錄出的多重複G4C2 RNA有關，其結構多型性相當複雜，可能在細胞核內形成RNA foci以結合各種RNA-binding protein，導致蛋白質功能異常，而RNA foci的形成可能和G4C2 RNA的結構密切相關。此外，離子種類也會影響G4C2 RNA的結構。至今，G4C2 RNA的結構多型性使其折疊方式備受爭議，我們將藉由single-molecule技術研究G4C2 RNA結構的特性以理解相關疾病的潛在分子機制。 文獻表明d(G4C2)4（含有四重複序列的G4C2 DNA）於KCl鹽溶液中會形成G-quadruplex，而我們在實驗中也發現互補的antisense難以結合並展開該結構，然而在相同條件下，antisense得以部分展開r(G4C2)4（含有四重複序列的G4C2 RNA），顯示RNA形成之結構較DNA不穩定。而五重複的r(G4C2)5展開比例減少，揭示重複次數和結構穩定性相關。此外，RNA在MgCl2的溶液中幾乎完全被展開，表明離子種類會影響結構多型性和穩定性。後續以識別G-quadruplex的BG4抗體搭配photobleaching實驗進行初步檢測，結果顯示在MgCl2溶液中，r(G4C2)4可能形成tetramer G-quadruplex，而且flanking sequence也會影響其結構多型性。在初步解析G4C2 RNA的結構特性後，我們進一步研究分子間的交互作用，結果顯示G4C2 RNA於KCl中容易發生交互作用但不易維持，相對而言，於MgCl2中不易發生交互作用卻得以維持，於NH4Cl中不僅不易發生交互作用亦難以維持。此外，r(G4C2)4似乎形成某種特別的構形，而此構形不易發生分子間的交互作用。 綜上所述，我們推論G4C2 RNA將形成hairpin或G-quadruplex結構並且維持動態平衡，在MgCl2中有利於形成hairpin，而KCl中有利於形成G-quadruplex，此外，hairpin loop可能為分子間交互作用的潛在位點，而MgCl2有助於維持loop-loop interactions，文獻亦表明該結構可能導致RNA-binding protein結合，以上現象符合RNA foci的致病機制，值得未來研究並驗證。

The abnormal expansion of the GGGGCC tandem repeats in the human C9ORF72 gene is the most common genetic cause of neurological disorders Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD). One of the proposed disease mechanisms is the toxicity of bidirectionally transcribed repeat-containing RNAs, which result in the sequestration of RNA-binding proteins into RNA foci. The formation of RNA foci is closely related to the structure of G4C2 repeats, which is affected by the type of ions. However, the structural polymorphism of the G4C2 repeat sequence has elicited a considerable discussion regarding its folding variability; especially the more the repeat number, the higher the structural complexity. We aim to study the structural characteristics of G4C2 RNA by using single-molecule techniques to understand the potential molecular mechanisms of pathogenesis. It has been reported that d(G4C2)4 forms G-quadruplexes in KCl. Consistently, we found that the complementary antisense strand was hardly to form duplexes with d(G4C2)4. However, the antisense strand partially unfolded r(G4C2)4 under the same conditions, indicating that the structure of r(G4C2)4 is less stable than DNA. The extent of antisense-mediated unfolding of r(G4C2)5 is less than r(G4C2)4, suggesting that the number of repeats correlates with structural stability. Furthermore, RNA was nearly completely unfolded in MgCl2, indicating that the type of ions affects structural polymorphism and stability. Photobleaching experiments with the BG4 antibody, which recognizes G-quadruplexes, showed that r(G4C2)4 might form tetramer G-quadruplexes in MgCl2 and the formation was affected by flanking sequences. After preliminarily analyzing the structural characteristics of G4C2 RNA, we further studied their intermolecular interactions. The results showed that the binding between G4C2 RNA molecules was more frequent in KCl but the overall association duration was shorter, whereas the binding was less frequent in MgCl2 but was more stable. Besides, the dynamic interaction frequency and the intermolecular association of G4C2 RNA in NH4Cl were the least in all the three ions. Additionally, r(G4C2)4 seems to form a specific conformation that doesn't prefer to participate in intermolecular interactions. In conclusion, we propose that G4C2 RNA dynamically forms hairpin or G-quadruplex structures, with KCl favoring the formation of G-quadruplexes, and MgCl2 favoring the hairpins. In addition, hairpin loops may be potential sites for intermolecular interactions, and putative loop-loop interactions are relatively stable in MgCl2. It has been suggested that these structures may be the binding sites of proteins. Our observations shed light on the pathogenic mechanisms of RNA foci and are worthy of future research and verification.