【作者】 王杨；

【导师】 郭沛；

【作者基本信息】 华南理工大学 ， 医药生物学， 2023， 硕士

【摘要】 神经退行性疾病是由大脑和脊髓的神经元细胞功能丧失而引起的一类不可逆转的神经系统疾病,以发作迟缓和选择性神经元功能障碍为表现特征。在人类基因组中,由1至6个核苷酸作为重复单元所形成的短串联重复序列（short tandem repeats,STRs）具有高度遗传不稳定性,在个体发育或传代的过程中会发生扩增突变（即重复单元数目的异常增加）,导致50多种神经退行性疾病的产生,如亨廷顿病、阿尔兹海默症和肌萎缩侧索硬化症等。大多数由STRs扩增引起的神经退行性疾病的致病机理尚不明确,且目前无可行的治疗方法。许多研究报道显示,这些在神经退行性疾病中异常扩增的STRs能形成非B型DNA结构,如发夹、三链体和四链体等。这些结构普遍含有异常的磷酸骨架折叠和错配碱基,会干扰机体重要的生理过程如DNA复制和转录等。因此,加强对其进一步的结构研究可以为其致病机理和药物研发的科学难题提供重要的参考意义。AAGGG五核苷酸重复序列位于复制因子C亚基1基因的内含子2上,目前已报道与迟发性共济失调、小脑共济失调/神经病变/前庭无反射综合征、多系统萎缩、慢性特发性轴索性多神经病变和帕金森病的发生相关。在本论文中我们研究了AAGGG重复序列的溶液结构,发现其在K⁺条件下极易堆积形成多聚平行G-四链体。通过在3′端引入悬挂基来扰乱G-四链体平面间的堆积作用,得到具有典型代表性的（AAGGG）₂AA二聚平行G-四链体结构,并结合约束型分子动力学计算测定其高分辨率三维空间结构。此外,还探索了AAGGG重复序列G-四链体与小分子配体的相互作用,并验证了其在体外能够有效抑制DNA的复制。CCTG四核苷酸重复序列位于细胞核酸结合蛋白基因内含子1中,该重复序列的扩增突变导致强直性肌营养不良。在病人体内CCTG重复是连续出现的,并且重复数目范围在～55-11000个,而正常人体内CCTG重复往往会被干扰序列（NCTG,N=A/T/G）所隔开,因此干扰序列被诸多文献报道与阻碍CCTG重复扩增相关。在本论文中我们探索了干扰序列对CCTG重复序列非B型DNA结构的影响,结果发现当在末端引入悬挂基后（CCTG）₂不能继续维持其迷你哑铃构象,并且（CCTG）₄的哑铃结构也发生了较大变化,基于此我们提出了CCTG重复序列扩增突变的潜在分子机制。本论文的研究结果为重复序列扩增疾病的遗传不稳定性带来了新见解,为靶向核酸结构的药物研发和基因治疗手段提供了理论基础。更多还原

【Abstract】 Neurodegenerative diseases are irreversible neurological disorders caused by the loss of functional neuronal cells in the brain and spinal cord,characterized by episodic retardation and selective neuronal dysfunction.In the human genome,short tandem repeats（STRs）,long repetitive sequences composed of 1 to 6 nucleotides as repeating units,are highly genetically unstable.Expansion mutations of STRs,i.e.abnormal increases in the number of repeat units,occur during ontogenetic or passage processes and cause more than 50 neurodegenerative diseases,such as the Huntington’s disease and amyotrophic lateral sclerosis.The pathogenesis of most repeat expansion diseases is unclear,and there is currently no viable treatment.Many studies have reported that these abnormally expanded STRs in neurodegenerative diseases can form non-B DNA structures such as hairpins,triplexes,and quadruplexes.These structures generally contain abnormal phosphate backbone folding and mismatched bases,which interfere with important physiological processes such as DNA replication,transcription,and translation.Therefore,understanding the structures and functions of STRs are key to pathogenic mechanism and drug development.The AAGGG pentanucleotide repeats,located on the intron 2 of the replication factor C subunit 1 gene,have been reported to be associated with five neurodegenerative diseases,namely late-onset ataxia,cerebellar ataxia/neuropathy/vestibular areflexia syndrome,multiple system atrophy,chronic idiopathic axonal polyneuropathy and Parkinson’s disease.In this thesis,we study the solution structure of AAGGG repeats and find that they are easily stacked to form multimeric parallel G-quadruplexes under K⁺conditions.By introducing overhangs at the 3′end to disturb the mutual stacking between the planes of the G-quadruplex,a typical representative（AAGGG）₂AA dimer parallel G-quadruplex structure was obtained,and its high-resolution three-dimensional structure was calculated by restrained molecular dynamics.In addition,the interaction of the AAGGG repeat G-quadruplex with small molecule ligands was also explored,and its effective inhibition of DNA replication in vitro was verified.The CCTG tetranucleotide repeats are located in intron 1 of the cellular nucleic acid-binding protein gene,and the expanded mutation of the repeats leads to myotonic dystrophy type 2（DM2）.In patients,CCTG repeats are usually continuous,and the number of replicates ranges from～55 to 11000,while CCTG repeats in normal humans are often separated by interrupted sequences（NCTG,N=A/T/G）,so interrupted sequences have been reported to hinder CCTG repeat expansion.In this thesis,we explored the effect of interrupted sequences on non-B DNA structures of CCTG repeats.When overhangs are introduced at the end of（CCTG）₂,it can no longer maintain its mini-dumbbell conformation,and the dumbbell structure of（CCTG）₄ also changes greatly.We propose a potential molecular mechanism for expanded mutations in CCTG repeats.The result of this thesis brings new insights into genetic instability of repeat expansion diseases,and provide theoretical basis for drug development targeting nucleic acid structures and gene therapy.更多还原