Mitochondrial G-quadruplex structures in health and disease

项目概要 G-四链体结构 (G4) 出现在富含鸟嘌呤的序列中，并且在 线粒体 DNA (mtDNA) 由于其核苷酸含量的链特异性偏差。初步研究和 之前发表的研究表明 G4 会影响线粒体功能，但证据在很大程度上仍然是间接的， 模糊了这些迷人结构在正常和病理线粒体生物学中的作用。这个 该提案将通过定义细胞中形成 G4 的 mtDNA 特定区域来解决这一知识空白 以及促进G4出现和稳定的条件。该提案意义重大，因为更全面 了解 mtDNA 维持和表达的调节可能对未来的方法很重要 多种疾病，包括遗传性线粒体疾病、代谢综合征和散发性癌症。 该提案的创新之处在于开发了检测线粒体 G4 的新型试剂及其新颖的方法。 线粒体疾病的治疗方法。总体假设是生理 G4 mtDNA 内的形成广泛存在并调节线粒体转录和复制。第一个 具体目标将采用一种创新工具，一种与 G4 序列结合的线粒体靶向胞内抗体， 通过染色质免疫沉淀 (ChIP) 探针检测线粒体基质中的 G4 相互作用序列。的 mtG4 的序列将被鉴定，并且它们的相对丰度将在一系列 条件。这些条件包括线粒体功能基础、升高和抑制，以及 G4 激活下 或 G4 抑制条件。 DNA 解旋酶的作用也将得到评估。第二具体 目标将扩展最近的观察结果，即使用 G4 结合剂诱导 G4 形成，选择反对 增强 G4 形成的特定致病 mtDNA 变异。此类变体通常以已知的状态存在 作为异质性，其中也存在健康的 mtDNA，以及致病型和野生型之间的比例 顺序决定外显率和严重程度。我们将使用患者细胞系和患者来源的细胞杂种细胞 评估可能对该方法敏感的致病变异的范围。我们还将扩大 鉴定区分致病性和野生型等位基因的新型 G4 结合化合物。 总体而言，这些研究将为不同细胞中特定 G4 结构的形成提供机制证据。 条件，将它们的形成与 mtDNA 复制和转录的调节联系起来，并开发 新工具和试剂可在某些异质条件下对 mtDNA 含量产生积极影响。

PROJECT SUMMARY G-quadruplex structures (G4) arise in guanine-rich sequences and have a high potential for formation in the mitochondrial DNA (mtDNA) due to its strand specific biases in nucleotide content. Preliminary studies and prior published work suggest that G4s impact mitochondrial function, but the evidence remains largely indirect, obscuring the role of these fascinating structures in normal and pathological mitochondrial biology. This proposal will address this gap in knowledge by defining the specific regions of mtDNA that form G4 in the cell and the conditions that promote G4 emergence and stability. The proposal is significant because a fuller understanding of the regulation of mtDNA maintenance and expression may be important in future approaches to diverse pathologies including heritable mitochondrial diseases, metabolic syndromes and sporadic cancers. The proposal is innovative in its development of novel reagents to detect mitochondrial G4s and in its novel approach to the therapy of mitochondrial disorders. The overarching hypothesis is that physiological G4 formation within mtDNA is widespread and regulates mitochondrial transcription and replication. The first specific aim will employ an innovative tool, a mitochondrial-targeted intrabody that binds to G4 sequences, to probe for G4-interacting sequences in the mitochondrial matrix by chromatin immunoprecipitation (ChIP). The sequences of mtG4s will be identified and their relative abundance will be evaluated under a range of conditions. The conditions include basal, elevated and inhibited mitochondrial function, and under G4-activated or G4-inhibited conditions. The role of DNA unwinding enzymes will also be evaluated. The second specific aim will expand upon a recent observation that induced G4 formation, using G4 binding agents, selects against specific pathogenic mtDNA variants that enhance G4 formation. Such variants typically exist in a state known as heteroplasmy, where healthy mtDNA is also present and the ratio between pathogenic and wild type sequence determines penetrance and severity. We will use patient cell lines and patient-derived cybrid cells to evaluate the range of pathogenic variants that may be susceptible to this approach. We will also expand the identification of novel G4 binding compounds that discriminate between pathogenic and wild type alleles. Overall, these studies will contribute mechanistic evidence for specific G4 structure formation in different conditions, connect their formation to the regulation of mtDNA replication and transcription, as well as develop new tools and reagent to positively impact mtDNA content in certain heteroplasmic conditions.