Function-based single molecule mapping of glycan monomers and motifs

There are several well-known examples where the sequencing of polymers has provided immense benefits. Today entire DNA genomes are being sequenced, bringing with them the prospect of a revolution in medicine and biology. Similarly, a new protein can have its polypeptide sequence read, with the result that the molecular detail of its structure can be determined and the detailed mechanism of its biological function revealed. The situation is very different for the third major class of biopolymers, the glycans (sugar-containing molecules - polysaccharides and glycosylated polymers in general), despite increasing recognition of their critical role in the biology of all life and their industrial importance. On the one hand, specific short sequences may be defined with submolecular precision, but on the other, beyond a dozen or so monomers the relationship between these sequences is lost and we must rely on bulk methods to describe the material. This often leaves those who wish to understand the multiple, critical roles played by polysaccharides with little of the molecular scale detail now taken for granted by molecular biologists. Examples of these beneficiaries include the food and pharmaceutical industries looking to develop new food and drug delivery formulations, medical researchers hoping to appreciate the role of glycopolymers in human health, or botanists and microbiologists studying the function of plant and bacterial cell walls in growth or pathogenic activity. This project exploits the recent development of a force-measuring microscope capable of, for the first time, mapping the distribution of defined oligomer (short polymer) sequences in single glycan polymers. It will do this by exploiting the phenomenon of rotaxanes - molecular rings threaded over a polymer chain. In this case, an atomic force microscope (AFM) probe picks up the ring (a cyclodextrin molecule) from its 'base' on a suitable polymer and slides it along and on to the glycan chain of interest, which is coupled to the rotaxane. Molecules known to recognise and bind to well-defined sequences within the polymer are allowed to interact with the polymer chain and form complexes; the ring is then passed along the chain and when it encounters a complex will 'unzip' it, removing the bound molecule. The mapping information comes from the magnitude of the interaction between the ring and each bound complex it encounters, along with the position along the chain at which the interaction occurs. By collecting this information from a large sample of individual polymers, a map of the distribution patterns of the known sequences is revealed. We have shown that this appealingly simple mechanical concept actually works for simple model polymers; now this project is designed to apply this entirely new sequencing tool to a medically and commercially highly significant glycan, alginate. Alginate is produced by seaweeds and also by bacteria, including Pseudomonas aeruginosa when it colonises the lung in cases of cystic fibrosis. Alginate produced by the bacteria in the lung forms a gel to protect the bacteria from immune responses and attacks but also contributes to obstructions in the airways of the lung which may be fatal. Median life expectancy of cystic fibrosis sufferers is 35 years. Alginate gels form in the presence of calcium and other divalent cations due to the formation of so-called 'egg box' junction zones between aligned pairs of guluronic acid (oligoG) sequences. The minimum length of oligoG required to form a stable junction zone is not known and thus this project aims to determine both this minimum length and its distribution within well-characterised samples of alginate polymers.

This project exploits the recent development of a force-measuring microscope capable of, for the first time, mapping the distribution of defined oligomer sequences in single glycan polymers, by exploiting the phenomenon of rotaxanes - molecular rings threaded over a polymer chain. In this case, an atomic force microscope (AFM) probe picks up the ring (a cyclodextrin molecule) from its 'base' on a suitable polymer and slides it along and on to the glycan chain of interest, which is coupled to the rotaxane. Molecules known to recognise and bind to well-defined sequences within the polymer are allowed to interact with the polymer chain and form complexes; the ring is then passed along the chain and when it encounters a complex will 'unzip' it, removing the bound molecule. The mapping information comes from the magnitude of the force of interaction between the ring and each bound complex it encounters, along with the position along the chain at which the interaction occurs. By collecting this information from a large sample of individual polymers, a map of the distribution patterns of the known sequence is revealed. We have shown that this appealingly simple mechanical concept works for simple model polymers; now this project is designed to apply this entirely new sequencing tool to a medically and commercially highly significant glycan, alginate. Alginate gels form in the presence of calcium and other divalent cations due to the formation of so-called 'egg box' junction zones between aligned pairs of guluronic acid (oligoG) sequences.The minimum length of oligoG required to form a stable junction zone is not known and thus this project aims to determine both this minimum length and its distribution within well-characterised samples of alginate polymers.

It is envisaged that this project will make very significant impacts on several different areas in the field of glycoscience. While the proposal focuses explicitly on mapping the distribution of a few well-defined oligosaccharide sequences in one class of polysaccharide, success in this endeavour will underline efforts to bring this approach to a level at which it becomes a mature tool for mapping and sequencing glycans. Thus the main impacts of this proposal will be (i) to provide entirely novel information on the detailed structure of a polysaccharide with significant applications in medicine and industry and (ii) to bring this novel approach to the necessary maturity for productive engagement with end-users in the communities identified below. The impact of generating distribution maps of molecularly defined oligosaccharide sequences in alginate will be felt strongest by those using alginate as a component of food and pharmaceutical formulations and as a medicine in its own right. The occurrence and distribution of the oligosaccharides targeted are known to be key factors in the performance of alginate as a gel while gelation is itself the critical parameter in its commercial and medical usage. By collaborating with an internationally leading group in the field, and one with commercial interests in an alginate-based therapy for cystic fibrosis, the impact of this new information will be maximised. The results of the work will be published and disseminated widely at the highest level, including at international conferences attended by key members of the glycoscience and single molecule research communities. The data generated will be examined for intellectual property exploitation prior to its dissemination and any IP identified will be appropriately protected in partnership with my collaborators and the University's Research, Enterprise and Engagement office. While this proposal focuses explicitly on mapping sequences in one glycan, alginate, success in this endeavour will underline efforts to bring this approach to a level at which it becomes a mature tool for mapping and sequencing glycans. Such a tool will be of great interest commercially. Pharmaceutical companies with whom I have engaged view SRFS as a highly promising approach to addressing the problems they encounter in characterising the performance of their cellulosic ether and modified starch products. Other specific targets in the immediate future beyond this proposal include pectin and heparin, both commercially important polysaccharides used (in the case of pectin) in food and (in both cases) health products and pharmaceuticals. Collaborative partners for each of the above examples have been identified and engaged. In all the cases highlighted above success in the current project will provide strong incentive for these partners to engage in collaborative exploitation of the new technique and of the new information it provides.Funding to pursue the sequencing of these polymers will be sought directly following the successful achievement of the aims of this proposal from and in partnership with these industrial sources as well as through Research Council routes. The PDRA appointed to this project will have the opportunity to conduct pioneering work in the field of glycopolymer sequencing and will benefit from the training to be received both in the PI's lab and while on secondment in the lab of Prof. Skjåk-Bræk. Finally, the technique used in this proposal lends itself to effective visual demonstration and as such may be utilised as a resource in public outreach activities in the area of biotechnology and nanoscience. Opportunities for such demonstrations will be explored with the University's Outreach coordinator.