Sequencing Glycosaminoglycans using Recognition Tunneling Nanopores

使用识别隧道纳米孔对糖胺聚糖进行测序

• 批准号: 9752985
• 负责人: Xu Wang
• 金额: $ 40.62万
• 依托单位: ARIZONA STATE UNIVERSITY-TEMPE CAMPUS
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-01 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9752985
• 关键词: Biological; Biological Markers; Biological Phenomena; Blood; Blood coagulation; Cells; Charge; Complex; DNA; DNA Primers; DNA biosynthesis; DNA polymerase A; DNA-Directed DNA Polymerase; Data; Data Analyses; Databases; Development; Devices; Disaccharides; Electrodes; Electrons; Enzyme Kinetics; Enzymes; Event; Funding; Genome; Glycosaminoglycans; Goals; Grant; Heterogeneity; High-Throughput DNA Sequencing; Immobilization; Individual; Infection; Inflammation; Leukocyte Trafficking; Mammals; Mediating; Mediator of activation protein; Methods; Modeling; Monosaccharides; Natural regeneration; Neoplasm Metastasis; Oligonucleotides; Oligosaccharides; Organism; Pathogenicity; Pharmacology; Phase; Physiological; Polymerase; Polysaccharides; Positioning Attribute; Preparation; Procedures; Property; Proteins; Sampling; Scheme; Senile Plaques; Side; Signal Transduction; Signaling Protein; Site; Speed; Stereoisomer; Structure; Techniques; Technology; Therapeutic; Therapeutic Uses; Time; Tissues; Unspecified or Sulfate Ion Sulfates; Validation; Work; amyloid formation; analytical method; base; cancer cell; cost; density; design; electric field; extracellular; improved; interest; monomer; nanoparticle; nanopore; novel; pathogenic microbe; polysulfated glycosaminoglycan; programs; single molecule; solid state; stem; sugar; sulfation

Project Summary Glycosaminoglycans (GAGs) are large, linear, sulfated polysaccharides found in many organisms, including all mammals. Interests in GAG structures stem from GAGs’ diverse biological activities in phenomena such as tissue development/regeneration, inflammation, blood coagulation and amyloid plaque formation. In addition to their therapeutic use, GAGs have also been used as biomarkers. Due to complexity and heterogeneity of their structures, GAG sequencing has been difficult, if not impossible. For the last two years, we have been developing a single molecule method to sequence GAGs using recognition tunneling nanopore (RTP). A RTP device is composed of a recognition tunneling junction embedded in a nanopore. It sequentially “read” a mono- or di- saccharide unit when the sugars form a transient complex with recognition molecules attached to two tunneling electrodes during translocation of a polysaccharide through the nanopore. Advantages of a single molecule method include circumvention of the need to obtain homogeneous samples of GAGs and ability to analyze intact GAG chains, which most of the existing analytical techniques are unable to do. In the R21 phase, we have shown that recognition tunneling (RT) signals from disaccharide building blocks of GAGs possess unique signatures that can be used in distinguishing different stereoisomers. We also improved manufacturing of RTPs and showed that conductance of the RT signals alone was sufficient to determine GAG types. Finally, we demonstrated that GAG chains can translocate solid-state nanopore unaided. However, the speed of translocation is too fast to collect sufficient amount of RT signals of individual structure units. To reduce the translocation speed, we have designed a Φ29 DNA polymerase mediated ratcheting mechanism to control the translocation of GAGs conju- gated to a DNA primer. In this application, we will develop such a GAG-ratcheting RTP device for GAG sequenc- ing. In particular, we will complete the following aims: (1) Build a RT reference database for RTP sequencing of GAGs. Using the most up-to-date RTP devices, we will analyze the RT signatures of GAG building blocks teth- ered to nanoparticles. This set-up mimics the conditions during actual sequencing and should produce data that more accurately reflect those collected during sequencing. (2) We will develop a method to fabricate GAG-ratch- eting RTPs. We will immobilize a single Φ29 DNA polymerase to the upper rim of the nanopore, so it can perform rolling circle extension using a circular template and a DNA primer whose 5’ end is conjugated to the reducing end of the GAG chain to be sequenced. As the Φ29 polymerase extends the DNA primer, it will push the GAG chain pass the RT junction at a rate slow enough for RT junction to interact with individual GAG monosaccharide for recording of sufficient electrical signals. Our goal is to complete the two aims in the first two years, allowing us to perform GAG sequencing and cross validation of the device in the final year.

项目摘要 糖胺聚糖（插科打）是在许多生物中发现的大型，线性的，硫酸的多糖，包括所有生物 哺乳动物。对插科打结构的兴趣源于插科打s的潜水生物学活动，例如 组织发育/再生，感染，血液凝结和淀粉样斑块形成。此外 他们的治疗用途，插科打s也被用作生物标志物。由于其复杂性和异质性 结构，插科打测序是困难的，即使不是不可能的。在过去的两年中，我们一直在发展 使用识别隧道纳米孔（RTP）对插科打ag的单分子方法。 RTP设备是 由嵌入在纳米孔中的识别隧道连接组成。它依次“读取”单声道或单词 当糖形成瞬态复合物，并带有识别分子，糖糖单位连接到两个隧道 多糖通过纳米孔转运过程中的电极。单分子的优点 方法包括规定获得插科打的均匀样本的需求和完整的能力 插科打链，大多数现有的分析技术无法做到。在R21阶段，我们显示了 来自二糖的识别隧道（RT）信号具有独特的签名 可以用来区分不同的立体异构体。我们还改进了RTP的制造 仅RT信号的电导就足以确定GAG类型。最后，我们证明了 插科打链可以将固定状态纳米孔易位。但是，易位速度太快了 收集足够数量的单个结构单元的RT信号。为了降低易位速度，我们有 设计了φ29DNA聚合酶介导的赛车机制，以控制堵嘴的易位。 门控到DNA底漆。在此应用程序中，我们将开发这样的GAG骑行RTP设备用于GAG序列 - ing。特别是，我们将完成以下目的：（1）构建用于RTP测序的RT参考数据库 插科打。使用最新的RTP设备，我们将分析堵嘴构建块的RT签名TETH- 纳入纳米颗粒。此设置模仿实际测序期间的条件，并应产生数据 更准确地反映了在测序过程中收集的那些。 （2）我们将开发一种制造Gag束缚的方法 Etuning RTP。我们将将单个φ29DNA聚合酶固定到纳米孔的上边缘，以便它可以执行 使用圆形模板和DNA底漆的滚动圆延伸，其5'端与还原相结合 插科打链的末端要测序。随着φ29聚合酶延伸DNA底漆，它将推动GAG 链条以足够慢的速率通过RT连接，以使RT连接与单个GAG单糖相互作用 用于记录足够的电信号。我们的目标是在头两年完成两个目标，允许 我们在最后一年对设备进行插科打测序和交叉验证。