与ALS/FTD相关联的C9orf72六核苷酸重复扩增形成G-四链体的结构研究

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are devastating neurological diseases. Abnormal expansion of a hexanucleotide repeat, (GGGGCC)n, is the most common genetic cause of familial and sporadic forms of ALS and FTD. This nucleotide repeat element is located in a non-protein-coding region of the C9orf72 gene. Previous studies suggested that the C9orf72 hexanucleotide repeat expansion (HRE), either as DNA or the transcribed RNA, can fold into G-quadruplexes with distinct structures. It could also form transcriptionally induced RNA-DNA hybrids known as R-loops. These structural polymorphisms lead to C9orf72 HRE-containing abortive transcripts, which may aggregate and form RNA foci in patient cells. Despite the critical connection of C9orf72 HRE G-quadruplexes to the pathogenic mechanism of C9orf72 HRE-linked ALS/FTD, the molecular basis of G-quadruplex formation by C9orf72 HRE remains elusive..In this project, we plan to perform systematic biochemical and structural characterizations for C9orf72 HRE G-quadruplexes. First, we will determine the 3D structure of G-quadruplex formed by the C9orf72 HRE DNA. In our preliminary study, we successfully obtained a homogenous DNA sample, d(GGGGCC)4, which favored a monomeric G-quadruplex fold. We have determined the topology of this G-quadruplex DNA using the nuclear magnetic resonance (NMR) approach. It forms a chair-type G-quadruplex with a four-layer antiparallel G-tetra core and three edgewise loops, which is distinct from known structures of chair-type G-quadruplexes. We will further resolve its detailed structure using NMR techniques. We have also successfully obtained a homogeneous C9orf72 HRE RNA sample with sufficient quality for NMR structure determination. Unlike the monomeric d(GGGGCC)4 DNA, this RNA, r(GGGGCC)GGGG, is an asymmetric dimer. We will perform more NMR studies to obtain its 3D structure. As no structure of the C9orf72 HRE G-quadruplex has ever been solved, the structural information of C9orf72 HRE DNA/RNA G-quadruplexes to be obtained in the current project will provide valuable insights into the molecular mechanism of abortive transcripts formation by C9orf72 HRE and RNA foci appearance in ALS/FTD patients. The outcome of the proposed research will not only provide insight into the molecular mechanism underlying the C9orf72 HRE G-quadruplex formation in the etiology of ALS/FTD, but may also lay the necessary basis for designing small molecules that therapeutically target C9orf72 HRE G-quadruplexes for the modulation of ALS/FTD pathogenesis.

肌萎缩性侧索硬化症（ALS）和额颞叶痴呆（FTD）是严重的神经退行性疾病。在家族性或散发性ALS/FTD患者中，位于9号染色体第72个开放阅读框基因（C9orf72）的GGGGCC六核苷酸重复扩增（HRE）是最常见的遗传致病因子。以往研究表明，C9orf72 HRE无论是作为DNA或转录的RNA产物，均可折叠成具有不同结构的G-四链体，并与ALS/FTD的致病机理密切相关。由于缺乏相关G-四链体的高分辨率结构信息，C9orf72 HRE形成G-四链体的分子机理仍不清楚。本研究将利用核磁共振技术首次获取C9orf72 HRE DNA及RNA G-四链体的高分辨率结构，对于在ALS/FTD患者中由于此种结构导致的异常转录产物诱发疾病的分子机理提供极有价值的信息，并且为针对C9orf72 HRE G-四链体设计小分子用以靶向调节ALS/ FTD的发病机制奠定必要的结构学基础。

肌萎缩性侧索硬化症（ALS）和额颞叶痴呆（FTD）是严重的神经退行性疾病。最近的研究发现，在家族性或散发性ALS/FTD患者中，位于9号染色体第72个开放阅读框基因（C9orf72）的GGGGCC(G4C2)六核苷酸重复扩增（HRE）是最常见的遗传致病因子。过往研究表明，C9orf72 HRE DNA或转录出的RNA产物，均可折叠成具有不同结构的G-四链体。 此种核酸结构及其与特定核酸结合蛋白的相互作用与ALS/FTD的致病机理密切相关。由于缺乏相关G-四链体的高分辨率结构信息，C9orf72 HRE形成G-四链体的分子机理及其与特定蛋白的相互作用机制仍不清楚。.本项目主要利用核磁共振和X射线晶体学技术研究C9orf72 HRE DNA/RNA的G-四链体结构。我们首先用核磁共振方法确定了G-四链体d(G4C2)4的反平行构象拓扑结构。之后通过核酸纯化方法我们发现并纯化得到具有平行构象的C9orf72 HRE DNA G-四链体。通过对d(G4C2)2的鸟嘌呤进行置换，我们得到了两个单一的具有平行和反平行构象的样品，并解析了其拓扑结构。在实验过程中，我们还发现钡离子可以稳定d(G4C2)2 G-四链体的平行构象，随后我们使用X射线晶体学技术得到了高分辨率结构。 运用分子置换方法，我们也得到了d(G4C2)2 G-四链体在钾离子结合下的平行构象的高分辨率结构。.建立在对C9orf72 HRE DNA的G-四链体结构成功解析基础上，我们对C9orf72 HRE RNA也进行了类似研究。 我们筛选了多个C9orf72 HRE RNA序列， 也对优化出的RNA G-四链体进行了NMR实验， 可惜多维核磁共振实验的效果均不能够满足结构解析条件。我们也同时尝试了X射线晶体学方法。 我们筛选结晶条件，长出单晶， 并得到了高分辨率的衍射数据。 后续结构解析工作仍然在进行中。.该项研究工作对于在ALS/FTD患者中由于此种结构导致的异常转录产物诱发疾病的分子机理提供极有价值的信息，并且为针对C9orf72 HRE G-四链体设计小分子用以靶向调节ALS/ FTD的发病机制奠定必要的结构学基础。项目资助期间，发表标注资助号的SCI 论文2 篇；与多个实验室开展合作研究，促进研究工作的进展；有5名硕士毕业，获得硕士学位，3名博士毕业，获得博士学位。