Sequencing Glycosaminoglycans using Recognition Tunneling Nanopores

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 disaccharide 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 conjugated to a DNA primer. In this application, we will develop such a GAG-ratcheting RTP device for GAG sequencing. 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 tethered 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-ratcheting 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.