Glycosaminoglycans (GAGs) are large, linear, sulfated polysaccharides found in many organisms, including all mammals. Interest 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 struc-tures, 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 com-posed of a recognition tunneling junction embedded in a nanopore. It sequentially read a mono- or di-saccharide unit when it forms a transient complex with recognition molecules attached to two tunneling elec-trodes as a polysaccharide chain is translocates 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 recogni-tion tunneling (RT) signals from disaccharide building blocks of GAGs possess unique signatures that can be used in distinguishing different stereoisomers. We also improved the process of manufacturing of RTPs and showed that conductance of the RT signals alone was sufficient to determine GAG types. Finally, we demon-strated 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 F29 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 se-quencing. In particular, we will complete the following aims: (1) Build a RT reference database for RTP sequenc-ing 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 F29 DNA polymerase to upper rim of the nanopore, so it can per-form rolling circle extension using a circular template and a DNA primer whose 5 end is conjugated to the reduc-ing end of the GAG chain to be sequenced. As the F29 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 monosac-charide for recording of sufficient electrical signals. Our goal is to complete the two aims in the first two years, allowing us to perform the GAG sequencing and cross validating the device in the final year.
|Effective start/end date||8/1/17 → 7/31/20|
- HHS-NIH: National Cancer Institute (NCI): $1,351,584.00
DNA-Directed DNA Polymerase