Structural analysis of large polysaccharides remains challenging in glycobiology. The problem is especially acute when polysaccharides in question are glycosaminoglycans (GAGs). GAGs are large, linear, sulfated polysaccharides ubiquitous to all mammals. Interests in GAG structures stem from GAGs' diverse biological activities that govern phenomena such as tissue development/regeneration, inflammation, blood coagulation and amyloid plaque formation. Abnormal GAG structures have also been associated with the development of a number of diseases, notably cancer and inflammation. As a result, there has been a desire to understand how GAG structures correlate with their biological activities, especially how the distribution of sulfate groups along the chain influence their interactions with GAG-binding proteins. However, GAGs' large size and complex sulfation patterns make analysis of intact GAG chains by conventional ensemble analytical techniques difficult, if not impossible. Here we propose to develop a single molecule sequencer for analysis of polysaccharides using the recognition tunneling nanopore (RTP) device currently under development for $1000 genome project as a template. With the R21 grant, we will demonstrate the feasibility by carrying out pre-requisite work needed to achieve single molecule sequencing of intact GAG chains using RTP. A RTP device incorporates a nanopore with a tunneling nanogap that contains two electrodes functionalized with recognition molecules capable of forming transient complexes with functional groups on a polymeric chain as it translocates the nanopore, thus generating electrical signals. Single molecule sequencing of GAG chains proposed here circumvents the need to obtain homogeneous samples of GAGs, greatly reducing complexity of sample preparation. GAG analysis by RT devices also does not have the size limitations of most of the existing analytical techniques, and the solid state device planned here are economical to manufacturer and operate. In this application, we aim to carry out pilot studies needed to make GAG sequencing by RTPs feasible: (1) we will investigate the translocation of size defined sulfated GAG fragments through nanopores to optimize the translocation efficiency of GAG ligands as well as to understand the influence of GAG sulfation density and GAG size on their translocation efficiency and speed; (2) we will carry out recognition tunneling experiments on sulfated GAG disaccharides as well as trisaccharides so these signals of GAGs can be analyzed using machine learning algorithms to identify unique signatures needed to detect the presence of these sulfation motifs in longer GAG chains. Completion of these aims will provide all the knowledge required for correct interpretations of RT signals produced by GAG translocation and sets the stage for sequencing of intact GAG chains by RT devices.
Work proposed here will allow single molecule sequencing of glycosaminoglycan polysaccharides using an electronic chip with a high speed and low cost for the first time. Glycosaminoglycans have important pharmacological properties and are modulators of critical biological phenomena such as tissue development/regeneration and inflammation. Determination of their sequence structures will allow better understanding of how organisms control these physiological events through glycosaminoglycans.
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