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.

项目概要 糖胺聚糖 (GAG) 是一种大型、线性、硫酸化多糖，存在于许多生物体中，包括所有生物体。 对 GAG 结构的兴趣源于 GAG 在以下现象中的多种生物活性。 除了组织发育/再生、炎症、凝血和淀粉样斑块形成之外。 由于其治疗用途，GAG 也被用作生物标志物。 在过去的两年里，我们一直在开发 GAG 测序技术，即使不是不可能，也是很困难的。 使用识别隧道纳米孔 (RTP) 的单分子方法对 GAG 进行测序是一种 RTP 装置。 由嵌入纳米孔中的识别隧道结组成，它依次“读取”单或双结构。 当糖与连接到两个隧道的识别分子形成瞬时复合物时，糖单元 多糖通过纳米孔移位过程中的电极。 该方法包括规避获得均质 GAG 样品的需要以及分析完整的能力 GAG链，这是大多数现有分析技术无法做到的，在R21阶段，我们已经展示了。 来自 GAG 的二糖构建块的识别隧道 (RT) 信号具有独特的特征 我们还改进了 RTP 的制造并展示了它可用于区分不同的立体异构体。 仅 RT 信号的电导就足以确定 GAG 类型。 GAG链可以在没有辅助的情况下移位固态纳米孔，但是移位的速度太快了。 收集足够数量的各个结构单元的 RT 信号，为了降低易位速度，我们有 设计了 Φ29 DNA 聚合酶介导的棘轮机制来控制 GAG 结合的易位 在本应用中，我们将开发一种用于 GAG 序列的 GAG 棘轮 RTP 装置。 具体来说，我们将完成以下目标：（1）建立RTP测序的RT参考数据库。 GAG。我们将使用最新的 RTP 设备来分析 GAG 构建块的 RT 签名。 该设置模拟了实际测序过程中的条件，并且应该产生以下数据： (2) 我们将开发一种制造GAG-ratch-的方法 我们将单个 Φ29 DNA 聚合酶固定到纳米孔的上边缘，因此它可以执行 使用圆形模板和 DNA 引物进行滚环延伸，该引物的 5' 端与还原性缀合 当 Φ29 聚合酶延伸 DNA 引物时，它将推动 GAG 进行测序。 链以足够慢的速度通过 RT 连接点，以便 RT 连接点与单个 GAG 单糖相互作用 我们的目标是在头两年内完成这两个目标，从而允许 我们在最后一年对设备进行 GAG 测序和交叉验证。