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

通过生物物理和计算相结合的方法，使用 PNA 阐明 G-四链体和发夹结构在 ALS/FTD 中的作用

• 批准号: 9513644
• 负责人: JEFFREY D EVANSECK
• 金额: $ 17.76万
• 依托单位: DUQUESNE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-01 至 2020-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9513644
• 关键词: Address; Adopted; Affect; Amyotrophic Lateral Sclerosis; Anterior; Antisense RNA; Behavior; Binding; Biophysics; Brain; C9ORF72; Cell physiology; DNA; DNA Sequence Alteration; Degenerative Disorder; Dependence; Development; Dipeptides; Disease; Drug Design; Equilibrium; FMR1; Familial Amyotrophic Lateral Sclerosis; Frontotemporal Dementia; G-Quartets; Genes; Genetic; Genetic Transcription; Label; Language; Length; Letters; Link; Methods; Modeling; Molecular; Motor Neurons; N-glycylalanine; Neurodegenerative Disorders; Nucleic Acid Binding; Pathology; Peptide Nucleic Acids; Personality; Play; Polymers; RNA; RNA-Binding Protein FUS; RNA-Binding Proteins; Research; Role; Sodium Chloride; Spinal Cord; Structure; System; Temporal Lobe; Testing; Therapeutic Agents; Time; Transcript; Translations; alanylglycine; alanylproline; arginylarginine; arginylproline; base; biophysical chemistry; biophysical techniques; computational chemistry; design; experimental study; glycylproline; in vivo; molecular diagnostics; neuron loss; neurotoxic; novel; prevent; prolylarginine; protein TDP-43; sarcoma; therapeutic development; tool

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.

肌萎缩性侧索硬化症（ALS），这是一种致命的神经退行性疾病，导致运动神经元流失 大脑和脊髓，以及额颞痴呆（FTD），大脑额叶的退化状况 前临时爱是具有重叠功能和遗传原因的多系统疾病。在2011年 被发现C9ORF72基因中的六核苷酸GGGGCC扩展是常见的遗传原因 但是，对于ALS和FTD，这种重复扩展导致ALS/FTD的机制尚不清楚 理解。拟议的研究将有助于我们理解这些分子机制 提供有关可变长度的感官/反义RNA所采用的结构的直接信息 通过C9orf72 GGGGCC扩展重复序列产生的，因为这些成绩单具有 有人建议他在这些疾病的病理学中扮演核心角色。这项研究推进了假设 可以开发辣核酸作为分子工具，以表征由 C9ORF72 Sense（G4C2）/反义（G2C4）转录本RNA，并可能破坏其与RNA的相互作用 结合蛋白质，并具有以下特定目的： 特定目标I. C9ORF72六核苷酸扩展感（G4C2）和反义的表征 （G2C4）成绩单结构。我们的初步结果表明感觉（G4C2）扩展重复RNA 形成G四链体结构，与发夹结构平衡并存。我们将使用一个数组 生物物理方法来表征C9orf72 Sense（G4C2）扩展重复的结构 RNA，专门分析重复长度和其他因素（盐依赖性，折叠）的影响 条件和时间）与它们之间的等效相同。另外，以确定RNA如何结合 蛋白质会影响这种相等的brium，我们将通过生物物理和计算化学方法分析 感官（G4C2）膨胀的相互作用与脆弱的X智力低下蛋白以及FUS相互重复 其他人和我们显示的蛋白质结合了g四链体RNA结构。随后，类似的生物物理 方法将用于表征由具有反义（G2C4）RNA转录本形成的结构 以前没有在结构上表征。 具体目标II。开发特异性与G四链体或特异性结合的肽核酸或 由C9ORF72扩展感（G4C2）和反义（G2C4）转录本形成的发夹结构。 有机合成方法将用于合成γ修饰的肽核酸（γPNA）分子 旨在专门识别C9ORF72扩展感觉/反义形成的结构 扩展重复转录本。 γPNA与指定目标的相互作用将被表征 通过生物物理和计算化学方法，随后它们可以防止相互作用 将研究带有RNA结合蛋白的C9ORF72扩展感/反义转录本。