Using PNAs to Elucidate the Role of G-quadruplex and Hairpin Structures in ALS/FTD through a Combined Biophysical and Computational Approach

肌萎缩侧索硬化症（ALS）是一种致命的神经退行性疾病，导致运动神经元丧失， 大脑和脊髓的退化性疾病，以及额颞叶痴呆（FTD），一种大脑额叶和 前颞叶是多系统疾病，具有重叠的功能和遗传原因。2011年 发现C9 ORF 72基因中的六核苷酸GGGGCC扩增是常见的遗传原因 然而，对于ALS和FTD，这种重复扩增导致ALS/FTD的机制尚不清楚 明白拟议的研究将有助于我们理解这些分子机制， 提供关于可变长度的有义/反义RNA所采用的结构的直接信息 通过C9 ORF 72 GGGGCC扩增重复序列的转录产生，因为这些转录物具有 被认为在这些疾病的病理学中起着核心作用。这项研究提出了假设 肽核酸可以作为分子工具来表征由蛋白质形成的结构， C9 ORF 72正义（G4 C2）/反义（G2 C4）转录RNA，并可能破坏其与RNA的相互作用 结合蛋白，并具有以下具体目标： 具体目标一。C9 ORF 72六核苷酸扩增正义（G4 C2）和反义的表征 （G2 C4）转录物结构。我们的初步结果表明，正义（G4 C2）扩展重复RNA 形成与发夹结构平衡共存的G四链体结构。我们将使用一个 生物物理方法来表征由C9 ORF 72正义（G4 C2）扩增重复序列形成的结构 RNA，具体分析重复长度和其他因素（盐依赖性、折叠 时间和条件，在他们之间的平衡。此外，为了确定RNA结合 蛋白质影响这种平衡，我们将通过生物物理和计算化学方法进行分析， 正义（G4 C2）扩增重复序列与脆性X智力低下蛋白和FUS的相互作用， 其他人和我们展示的蛋白质结合G四链体RNA结构。随后，类似的生物物理 方法将用于表征由反义（G2 C4）RNA转录物形成的结构， 在结构上以前没有被描述过。 具体目标二。开发特异性结合G四链体或 由C9 ORF 72扩增正义（G4 C2）和反义（G2 C4）转录物形成的发夹结构。 γ修饰肽核酸（γPNA）分子的合成将采用有机合成方法 旨在特异性识别C9 ORF 72扩展有义/反义形成的结构 扩增重复转录本。γPNAs与其指定靶标的相互作用将被表征 通过生物物理和计算化学方法，以及随后它们阻止相互作用的能力， 将研究C9 ORF 72扩增正义/反义转录物与RNA结合蛋白的关系。

Amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder resulting in motor neuron loss in the brain and spinal cord, and frontotemporal dementia (FTD), a degenerative condition of the brain frontal and anterior temporal lobes, are multisystem disorders with overlapping functional and genetic causes. In 2011 it was discovered that a hexanucleotide GGGGCC expansion in the C9ORF72 gene is a common genetic cause for ALS and FTD, however, the mechanisms by which this repeat expansion leads to ALS/FTD are not clearly understood. The proposed research will contribute to our understanding of these molecular mechanisms by providing direct information on the structures adopted by the sense/antisense RNA of variable lengths produced through the transcription of the C9ORF72 GGGGCC expansion repeats, as these transcripts have been proposed to play central roles in the pathology of these diseases. This study advances the hypothesis that peptide nucleic acids could be developed as molecular tools to characterize the structures formed by the C9ORF72 sense (G4C2)/antisense (G2C4) transcript RNAs and potentially to disrupt their interactions with RNA binding proteins, and has the following specific aims: Specific Aim I. Characterization of the C9ORF72 hexanucleotide expansion sense (G4C2) and antisense (G2C4) transcript structure(s). Our preliminary results indicate that the sense (G4C2) expansion repeat RNA forms G quadruplex structures that co-exist in equilibrium with hairpin structures. We will use an array of biophysical methods to characterize the structures formed by the C9ORF72 sense (G4C2) expansion repeat RNA, specifically analyzing the influence the repeat length and other factors (salt dependence, folding conditions and time) have upon the equilibrium between them. Additionally, to determine how RNA binding proteins affect this equilibrium we will analyze by both biophysical and computational chemistry methods the interactions of the sense (G4C2) expansion repeat with the fragile X mental retardation protein and with FUS, proteins that others and we showed bind G quadruplex RNA structures. Subsequently, similar biophysical methods will be used to characterize the structure(s) formed by the antisense (G2C4) RNA transcript that has not previously been structurally characterized. Specific Aim II. Development of peptide-nucleic acids that bind specifically to either G quadruplex or hairpin structures formed by the C9ORF72 expansion sense (G4C2) and antisense (G2C4) transcripts. Organic synthetic methods will be used to synthesize γ-modified peptide-nucleic acid (γPNA) molecules designed to recognize specifically the structures formed by the C9ORF72 expansion sense/antisense expansion repeat transcripts. The interactions of the γPNAs with their designated targets will be characterized by biophysical and computational chemistry methods, and subsequently their ability to prevent the interactions of the C9ORF72 expansion sense/antisense transcripts with RNA binding proteins will be investigated.