Sequencing Glycosaminoglycans using Recognition Tunneling Nanopores

项目摘要 糖胺聚糖（GAG）是在许多生物体中发现的大的线性硫酸化多糖，包括所有 哺乳动物对GAG结构的兴趣源于GAG在以下现象中的多种生物活性， 组织发育/再生、炎症、血液凝固和淀粉样斑块形成。除了 除了它们的治疗用途之外，GAG还被用作生物标志物。由于其复杂性和异质性， 由于结构复杂，GAG测序即使不是不可能，也是困难的。在过去的两年里，我们一直在开发 使用识别隧道纳米孔（RTP）对GAG进行测序的单分子方法。RTP设备 由嵌入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- 当糖与连接到两个隧道的识别分子形成瞬时复合物时， 在多糖移位通过纳米孔期间，电极。单个分子的优点 方法包括避免需要获得均匀的GAG样品，并能够分析完整的 GAG链，这是大多数现有的分析技术无法做到的。在R21阶段，我们已经证明 来自GAG二糖结构单元的识别隧道（RT）信号具有独特的特征， 可用于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， GAG链可以独立地移位固态纳米孔。然而，易位的速度太快， 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 设计了一种Φ29 DNA聚合酶介导的棘轮机制来控制GAG的转运， 门控DNA引物。在这个应用中，我们将开发这样一个GAG棘轮RTP设备的GAG序列， ing.具体而言，我们将完成以下目标：（1）建立一个RT参考数据库， 搞笑使用最新的RTP设备，我们将分析GAG构建块teth的RT签名， 纳米粒子。该设置模拟了实际测序期间的条件，并应产生 更准确地反映测序期间收集的那些。(2)我们将开发一种制造GAG棘轮的方法， 制定区域贸易方案。我们将单个Φ29 DNA聚合酶固定在纳米孔的上边缘，因此它可以执行 使用环状模板和DNA引物进行滚环延伸，所述DNA引物的5’末端与还原性引物缀合， 待测序的GAG链末端。当Φ29聚合酶延伸DNA引物时，它将推动GAG 链以足够慢的速率通过RT接头，使RT接头与单个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， 我们将在最后一年对器械进行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.