開發具有膜親和性之核醣核酸架構

Abstract

核醣核酸的多功能性隱含其可能於生命之初的重要性。核醣核酸不只可儲存遺傳訊息，更可在原始生命中處理許多任務，例如：執行催化反應（核酶）與結合分子（適體）。儘管自我複製的核醣核酸常被作為一個似生命的模型，但是整個系統－更特定的來說－細胞的區隔，還是要依賴物理性的擾動如攪拌來達成。在細胞中，分裂依靠與細胞膜有關的生物大分子協調各反應物間的行動。因此，核醣核酸能否與細胞的區隔互動及造成變形顯得相當重要。在先前以去氧核醣核酸摺紙讓膜變形研究中，使用能與膜結合，且可行低聚合作用的重複單體設計。然而，此策略需要多步驟及使用純化的複雜單體組裝過程阻礙了單體於合成細胞中的直接產生。另一方面，共轉錄摺疊的單股核醣核酸摺紙已被證實可藉由分子間結合，做為蛋白質與螢光分子的鷹架。本研究進而以該單股核糖核酸磚塊為基礎，加入了來自非編碼核糖核酸，且具有與磷脂質（磷脂醯肌醇，三、四、五，三磷酸）結合力的二級結構，希望創造一個膜交互作用架構。核醣核酸的折疊以原子力及螢光顯微鏡鑑定。核糖核酸與膜的交互作用藉由將試管內轉錄的產物純化後對由凝膠膨潤法或電滲透法製作之脂質體來驗證。結果顯示具磷脂質親合力之核醣核酸磚塊可在具有二價陽離子溶液中與膜結合。此外，不具有與磷脂質結合結構的核醣核酸磚塊也出乎預期地可以與膜結合，而無磚塊結構之核醣核酸則未發現相似的結合。這顯示了有結構的核醣核酸可能由二價陽離子媒介與磷脂質靜電結合。此膜與核醣核酸的框架供仿生分子設計一個替代方案，也提供基於核醣核酸的最簡人造細胞分裂機制可行方向。

The versatility of RNAs implies their plausible dominant role in a prebiotic scenario. Abilities to catalyze reactions (ribozymes) and bind molecules (aptamers) enable RNAs not only to bear genetic information but also to tackle multiple tasks for primitive systems. While self-replicating RNA had been demonstrated to be a life-like model, the division of the whole system, i.e., the compartment, relies on physical disturbance such as agitation. In cells, the division depends on coordinated actions of membrane-associated biomacromolecules. Therefore, it is crucial to examine whether the RNA can interact with surrounding compartments and deform them. In previous DNA origami studies, oligomerization of repetitive membrane-anchoring structures had been reported to deform the membrane. However, the complex monomer assembly process, which requires multiple synthetic and purifying steps, hindered them from being produced directly in synthetic cells. Co-transcriptional folding of single-stranded RNA origami structures, meanwhile, had been demonstrated to be capable of scaffolding molecules, including proteins and fluorophores, by forming intermolecular binding. This study further introduces a membrane-binding motif derived from a non-coding RNA that binds to phospholipid phosphatidylinositol 3,4,5 trisphosphate (PIP3) to a single-stranded RNA scaffold to create a membrane-interacting platform. The design of basic RNA tile structures is based on a previous study and de novo design by the RNA Origami Automated Design software. The folding and the function of the RNA tiles are characterized using atomic force microscopy, confocal microscopy, and fluorescence microscopy. The RNA-membrane interactions are assessed either by incorporating purified RNA or in vitro transcription system in liposomes via microfluidic jetting or by incubating with liposomes made of gel-assisted swelling or electroformation. The results showed that the binding of RNA tiles to the membrane could be achieved in a buffer that contains mainly divalent ions. Intriguingly, unexpected binding of RNA tiles without lipid-binding motif to the membrane are also observed, which implies a divalent cation-mediated electrostatic binding of structured RNA to the membrane, while a random protein-coding RNA did not show similar binding. The membrane-RNA scaffold may provide an alternative way for biomimetic molecule design and shed light on the simplest RNA-based artificial cell dividing mechanism.