Sequencing Glycosaminoglycans using Single Molecule Enzyme Conductance Fluctuations

糖胺聚糖（GAG）是哺乳动物中产生的大的线性硫酸化多糖家族， 其他生物。GAG在组织发育/生长、炎症、血液凝固、病毒感染、免疫抑制和免疫调节中发挥不同的作用。 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剂。由于它们的生物活性， GAG的结构-活性关系一直很高。然而，由于其规模、复杂性和... 然而，由于遗传性，使用常规系综技术分析GAG结构一直是具有挑战性的。 目前还没有对这些重要的多糖进行测序的方法。我们一直在探索- 确定GAG结构的分子技术。在本提案中，我们希望探讨 使用GAG裂解酶的电导波动来阐明GAG结构的可能性。 这一想法源于我们对单个蛋白质电导测量的工作，该工作显示了许多非氧化还原 活性蛋白质可以导电。此外，蛋白质的电导往往对构象很敏感 由底物结合或催化活性触发的动力学，使它们能够作为单分子传感器， 印刷受体.我们已经将这样的测量应用于DNA聚合酶，并显示了DNA聚合酶中的电流波动。 聚合酶与DNA复制过程中的酶构象变化相关。这一点的概括 这一想法潜在地允许对任何生物聚合物进行测序，只要进行性代谢酶可以 发现聚合物。这类酶对于糖胺聚糖通常是稀缺的。然而，一类新的进行性外解 从还原末端降解GAG的细菌GAG裂解酶刚刚被鉴定。在本提案中，我们 我想把这种技术应用于这类酶，以确定是否波动的电导率 这些裂解酶反映了被处理的基底的结构。因为这种方法需要 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巨大改进。特别是，我们希望 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，我们将设计和生产裂解酶，可以连接到电极特异性和优化 锚定点以最大化传导性和对底物结合的敏感性，同时保持酶活性。 2)我们将准备一个结构确定的GAG配体库，并用配体探测酶，以使其脱乙酰化。 确定底物诱导的酶电导波动是否包含可用于 以识别衬底的结构。这些目标的实现将为真实的- 从而实现了开发用于测序GAG的通用方法的目标。

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.