Sequencing Glycosaminoglycans using Single Molecule Enzyme Conductance Fluctuations

使用单分子酶电导波动对糖胺聚糖进行测序

• 批准号: 10568069
• 负责人: Xu Wang
• 金额: $ 19.02万
• 依托单位: ARIZONA STATE UNIVERSITY-TEMPE CAMPUS
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10568069
• 关键词: 2019-nCoV; Anticoagulants; Binding; Biological; Biological Markers; Biological Phenomena; Biopolymers; Biotin; Blood coagulation; Categories; Cells; Complex; DNA; DNA biosynthesis; DNA-Directed DNA Polymerase; Development; Disease; Electric Conductivity; Electricity; Electrodes; Electronics; Engineering; Enzymes; Event; Family; Foundations; Glycosaminoglycans; Goals; Grant; Growth and Development function; Heparin; Heparin Lyase; Heparitin Sulfate; Heterogeneity; Inflammation; Leukocyte Trafficking; Libraries; Ligands; Lyase; Machine Learning; Mammals; Measurement; Measures; Medicine; Methods; Microbe; Molecular Conformation; Natural regeneration; Organism; Physiological; Play; Polymerase; Polymers; Polysaccharides; Positioning Attribute; Process; Property; Proteins; Research Personnel; Resolution; Role; Sampling; Senile Plaques; Signal Transduction; Signaling Protein; Speed; Streptavidin; Structure; Structure-Activity Relationship; Sulfate; Surface; Techniques; Technology; Therapeutic; Time; Tissues; Virus Diseases; Work; analytical method; cost; design; enzyme activity; improved; interest; nanopore; novel; pathogenic bacteria; pathogenic virus; pharmacologic; polysulfated glycosaminoglycan; receptor; sensor; single molecule; solid state

Glycosaminoglycans (GAG) are a family of large, linear, sulfated polysaccharides produced in mammals and other organisms. GAGs play diverse roles in tissue development/growth, inflammation, blood coagulation, viral infection, and amyloid plaque formation. As a result, GAGs have been used as biomarkers for many diseases. They are also the most widely used anticoagulant in medicine. Because of their biological activities, interest in structure-activity relationships of GAGs has always been high. However, due to their size, complexity and het- erogeneity, analysis of GAG structures using conventional ensemble techniques has always been challenging. There is currently no method to sequence these important polysaccharides. We have been exploring single- molecule techniques for determining GAG structures for several years. In this proposal, we want to explore the possibility of using fluctuations in the electrical conductance of GAG lyases to elucidate the structures of GAGs. This idea originates from our work on single protein conductance measurements that showed many non-redox active proteins can conduct electricity. In addition, the conductance of proteins is often sensitive to conformation dynamics triggered by substrate binding or catalytic activity, allowing them to act as single-molecule sensors for substrates. We have applied such measurements to DNA polymerases and showed current fluctuations in the polymerase correlated with enzyme conformation changes during DNA replication. The generalization of this idea potentially allows any biopolymer to be sequenced as long as a processive metabolizing enzyme can be found for the polymer. Such enzymes were usually scarce for GAGs. However, a new class of processive exolytic bacterial GAG lyases that degrade GAGs from their reducing end has just been identified. In this proposal, we want to apply this technique to this class of enzymes to determine whether fluctuations in the conductance of these lyases are reflective of the structures of the substrates being processed. Because such a method requires no homogeneous samples, can sequence longer GAG polymers, and can provide high-resolution information, we think its realization will be a dramatic improvement over all existing techniques. In particular, we want to complete the following two aims: 1) Leveraging the technologies we developed to connect DNA polymerases to electrodes, we will design and produce lyases that can be attached to electrodes specifically and optimize the anchoring points to maximize conductance and sensitivity to substrate binding while retaining the enzyme activity. 2) We will prepare a library of structurally defined GAG ligands and probe the enzymes with the ligands to de- termine if the substrate-induced fluctuations in the enzymes’ conductance contain information that can be used to identify the structures of the substrates. Completion of these aims will provide the crucial foundation for real- izing the goal of developing a general method for sequencing GAGs.

糖胺聚糖 (GAG) 是哺乳动物中产生的大型、线性、硫酸化多糖家族 其他生物体。 GAG 在组织发育/生长、炎症、凝血、病毒等方面发挥着不同的作用 感染和淀粉样蛋白斑形成。因此，GAG 已被用作许多疾病的生物标志物。 它们也是医学上使用最广泛的抗凝剂。由于它们的生物活性，人们对它们感兴趣 GAG 的构效关系一直很高。然而，由于它们的尺寸、复杂性和异质性 由于异构性，使用传统集成技术分析 GAG 结构一直具有挑战性。 目前还没有方法对这些重要的多糖进行测序。我们一直在探索单 多年来一直致力于确定 GAG 结构的分子技术。在本提案中，我们希望探讨 利用 GAG 裂解酶电导的波动来阐明 GAG 结构的可能性。 这个想法源于我们对单一蛋白质电导测量的工作，该测量显示了许多非氧化还原 活性蛋白质可以导电。此外，蛋白质的电导通常对构象敏感 由底物结合或催化活性触发的动力学，使它们能够充当单分子传感器 基材。我们已将此类测量应用于 DNA 聚合酶，并显示了当前的波动 聚合酶与 DNA 复制过程中酶构象的变化相关。对此的概括 只要持续代谢酶可以被测序，这个想法就有可能允许对任何生物聚合物进行测序。 发现为聚合物。对于 GAG 来说，此类酶通常很稀缺。然而，一类新的进行性离析 刚刚鉴定出能够从还原端降解 GAG 的细菌 GAG 裂解酶。在这个提案中，我们 想要将该技术应用于此类酶，以确定电导是否波动 这些裂合酶反映了正在处理的基材的结构。因为这样的方法需要 无需均质样品，可以对更长的 GAG 聚合物进行测序，并且可以提供高分辨率信息， 我们认为它的实现将是对所有现有技术的巨大改进。特别是，我们想要 完成以下两个目标：1）利用我们开发的技术将 DNA 聚合酶连接到 电极，我们将设计和生产可以专门附着在电极上的裂合酶，并优化 锚定点可最大限度地提高对底物结合的电导和敏感性，同时保留酶活性。 2) 我们将准备一个结构明确的 GAG 配体文库，并用配体探测酶以脱除 终止底物引起的酶电导波动是否包含可以使用的信息 识别基材的结构。这些目标的完成将为实现真正的目标奠定重要基础。 制定了开发 GAG 测序通用方法的目标。