Key Research Interests and Expertise

Single polymer mapping and sequencing: Function-based single molecule mapping of glycan monomers and motifs
(Funded by BBSRC)

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.

To date, we have shown that we can use an AFM probe to pick up a cyclodextrin ring threaded over a PEG polymer chain incorporating different aromatic groups, and that sliding the ring over those groups gives a measurable response in the force spectrum. We have shown that the spectra can be interpreted simply in terms of a steric interaction between ring and thread, opening the possibility of using this technique as a general method for sequencing linear polymers.

Figure 1: cartoon representation of sliding ring force spectroscopy: (a) sketch of expected force spectrum for a polymer with two bulky groups and (b) experimental data obtained on such a system. [Dunlop et al 2008 Nanotechnology 19:345706]

Figure 2: distribution of rupture points (forces and distances) for control (open circles), PEO with aromatic groups at 100 and 200nm (red circles) and PEO with aromatic groups at 100 and 150nm (yellow circles). Graph at bottom right is a comparison of expected vs. experimental rupture positions for aromatic groups in PEO. [Dunlop et al 2008 Nanotechnology 19:345706]

We are currently working on applying this technique to polysaccharides, which requires the development of a specific end-conjugation method for attaching a PEG chain to the reducing end of a polysaccharide.

Following the hydrolysis of pectin at the single molecule scale
Coming soon

MUC2 mucin in the porcine small intestine trimerises to form flat, weakly interacting lamellae
Coming soon

Novel anticancer agents that bisintercalate DNA – a single molecule view
Coming soon

Research Funding

Molecular Recognition of Polymer Microstructure
04/04-03/06 IRC Exploratory Project in collaboration with Prof. Terence Cosgrove (Chemistry Dept., University of Bristol)

I was awarded an IRC exploratory project in collaboration with Prof. Terence Cosgrove (Bristol, School of Chemistry), providing funding for a 2 year postdoctoral research assistant to assist in the development of a novel force spectroscopy-based single molecule sequencing technique exploiting the spontaneous self-assembly of pseudorotaxanes. This project laid the groundwork for the following grant proposal and defined the approach we are now taking. Two papers resulting from this work are currently in preparation.

The AFM Lasso'- a new analytical tool for single molecule sequencing
10/05-09/08 EPSRC/RSC Analytical Chemistry Trust Fund Analytical Science Studentships
in collaboration with Prof. Terence Cosgrove and Prof. Tony Davis (Chemistry Dept., University of Bristol)

3 PhD studentships were awarded (starting October 2005) for this project, with the aim of developing the novel sequencing technique mentioned above for application to the sequencing of DNA using specifically designed macrocycles to form DNA-rotaxanes. After a successful first year we have produced a proof of the principle behind this project.

Function-based single molecule mapping of glycan monomers and motifs
10/10-10/13 BBSRC

Single Molecule Studies of Novel Bisintercalators for Pharmaceutical and Nanoscience Applications
07/10-06/13 UEA studentship award
in collaboration with Prof. Mark Searcey (PHA)
PhD studentship

Unravelling the Intricacies of Protein ‘Nanowires
10/09-09/12 UEA studentship award
in collaboration with Prof. Julea Butt (CHE/BIO) and Dr. Tom Clarke (BIO)
PhD studentship

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