Sequencing Glycosaminoglycans using Recognition Tunneling Nanopores

项目摘要 糖胺聚糖(GAG)是一种大的、线性的、硫酸盐化的多糖，存在于许多生物体中，包括所有 哺乳动物。对Gag结构的兴趣源于Gag在以下现象中的不同生物学活动 组织发育/再生、炎症、血液凝固和淀粉样斑块的形成。除了……之外 它们的治疗用途，GAG也被用作生物标志物。由于它们的复杂性和异构性 结构，插嘴测序一直很困难，如果不是不可能的话。在过去两年中，我们一直在发展 一种使用识别隧道纳米孔(RTP)对GAG进行排序的单分子方法。RTP设备是 由嵌入在纳米孔中的识别隧道结组成。它顺序地读取单声道或双声道 糖单位当糖与识别分子形成瞬时络合物时，识别分子连接到两个隧道 多糖通过纳米孔转移过程中的电极。单分子的优点 方法包括避免需要获得GAG的均质样本和完整分析的能力 封口链，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的双糖构建块的识别隧道(RT)信号具有独特的特征 可以用来区分不同的立体异构体。我们还改进了RTP的制造，并展示了 仅RT信号的电导就足以确定GAG类型。最后，我们演示了 GAG链可以在没有辅助的情况下转移固态纳米孔。然而，移位的速度太快了，以至于 采集足够量的单个结构单元的RT信号。为了降低移位速度，我们有 设计了一种Φ29DNA聚合酶介导的棘轮机制来控制GAGS结合蛋白的易位。 连接到一个DNA引子上。在本应用中，我们将开发这样一种用于GAG序列的插口棘轮RTP装置-- 英。特别是，我们将完成以下目标：(1)建立RTP序列测定的RT参考数据库 恶作剧。使用最新的RTP设备，我们将分析GAG构建块的RT签名- 被掺入纳米颗粒。这种设置模拟了实际测序过程中的条件，应该会产生 更准确地反映在测序过程中收集的数据。(2)我们将开发一种制造断口棘轮的方法-- 正在安排RTP。我们将单个Φ29DNA聚合酶固定在纳米孔的上缘，这样它就可以 使用环状模板和其5‘端连接到还原的DNA引物的滚环延伸 待测序的封口链的末端。当Φ29聚合酶延伸dna引物时，它将推动Gag。 链以足够慢的速度通过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这两个目标，允许 美国将在最后一年对该设备进行GAG测序和交叉验证。

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.