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（Glycosaminoglycans， GAG）是一类大型的、线性的、硫酸酸化的多糖，产生于哺乳动物和植物中