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)是一类大的、线性的、硫酸盐化的多糖家族，存在于哺乳动物和 其他生物。GAGs在组织发育/生长、炎症、凝血、病毒等方面发挥着不同的作用 感染和淀粉样斑块形成。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血剂。由于它们的生物活性，人们对 GAG的结构-活性关系一直很高。然而，由于它们的大小、复杂性和Het- 然而，使用传统的集成技术分析Gag结构一直是一个具有挑战性的问题。 目前还没有方法对这些重要的多糖进行测序。我们一直在探索单曲- 几年来用于确定Gag结构的分子技术。在这项提案中，我们想要探索 利用Gag裂解酶电导的波动来阐明Gag的结构的可能性。 这个想法源于我们在单蛋白电导测量方面的工作，它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很敏感。 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 底物。我们已经将这种测量应用于DNA聚合酶，并显示出目前在 聚合酶与DNA复制过程中酶的构象变化有关。这一点的泛化 IDEA潜在地允许对任何生物聚合物进行测序，只要过程代谢酶可以 为聚合物找到的。这样的酶通常很少用来作弊。然而，一类新的过程性解离剂 细菌裂解酶从其还原端降解GAG的作用刚刚被鉴定出来。在这项提案中，我们 想要将这项技术应用于这类酶来确定电导的波动是否 这些裂解酶反映了被加工的底物的结构。因为这样的方法需要 没有均匀的样品，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戏剧性改进。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特定连接到电极上的裂解酶，并优化 锚定点最大限度地提高了电导率和底物结合的敏感性，同时保持了酶的活性。 2)我们将准备一个结构定义的Gag配体的文库，并用这些配体探测酶来降解 如果底物引起的酶的电导波动包含可以使用的信息，则终止 以确定底物的结构。这些目标的实现将为真正-- 实现了开发一种通用的GAG测序方法的目标。