Mitochondrial G-quadruplex structures in health and disease

项目摘要 G-四链体结构（G4）出现在富含鸟嘌呤的序列中，并且在富含鸟嘌呤的序列中具有高的形成潜力。 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 先前发表的工作表明G4影响线粒体功能，但证据主要是间接的， 模糊了这些迷人的结构在正常和病理线粒体生物学中的作用。这 一项提案将通过定义在细胞中形成G4的mtDNA的特定区域来填补这一知识空白 以及促进G4出现和稳定的条件。这一建议意义重大，因为一个更全面的 理解线粒体DNA的维持和表达的调节在未来的研究中可能是重要的 包括遗传性线粒体疾病、代谢综合征和散发性癌症在内的多种病理。 该提案在开发检测线粒体G4的新型试剂方面具有创新性， 线粒体疾病的治疗方法。总体假设是生理G4 线粒体DNA内的形成是广泛存在的，并调节线粒体的转录和复制。第一 具体目标将采用一种创新的工具，即结合G4序列的脑靶向胞内抗体， 通过染色质免疫沉淀（ChIP）检测线粒体基质中的G4相互作用序列。的 将鉴定mtG 4序列，并在一定范围内评估其相对丰度。 条件条件包括基础，升高和抑制线粒体功能，并在G4-活化 或G4抑制条件。DNA解旋酶的作用也将进行评估。第二特定 目的将扩大最近的观察，诱导G4形成，使用G4结合剂，选择对 增强G4形成的特定致病性mtDNA变体。这种变体通常以已知的状态存在， 作为异质性，其中也存在健康的线粒体DNA以及致病型和野生型之间的比例 顺序决定了轻重。我们将使用患者细胞系和患者来源的胞质杂交体细胞， 评估可能对这种方法敏感的致病性变体的范围。我们亦会扩大 鉴定区分致病性和野生型等位基因的新型G4结合化合物。 总的来说，这些研究将为不同的细胞中特定的G4结构形成提供机制证据。 条件下，连接它们的形成，以调节线粒体DNA的复制和转录，以及发展 新的工具和试剂，在某些异质性条件下对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.