Polysaccharide-DNA strings for single molecular polysaccharide studies : a thesis submitted in fulfillment of the requirements for the degree of Doctor of Philosophy in Biophysics and Soft Matter, School of Natural Sciences, Massey University, Palmerston North, New Zealand

Abstract

For several decades DNA has been the workhorse of single molecule experiments, owing to its large controllable size and simplicity of end group attachment. The use of DNA handles to study DNA-protein conjugates has also previously been employed to understand the behaviour of proteins at the single molecular level. In contrast, single molecule studies of polysaccharides are not widely known. This project attempts to develop a methodology in order to facilitate single molecule polysaccharide studies with optical tweezers (OT). Homogalacturonan (HG), a polysaccharide component extracted from pectin, a key component in plant cell walls, was chosen to be the subject of this study. The proposed strategy was to utilise DNA strands as "handles" with one end attached onto HG, and the other coupled to beads, to allow for stretching of HG, and other single molecule studies. In order to attach HG between different DNA handles, the chemistry present at the reducing and non-reducing ends of the polysaccharide which can be used to form bonds with end functionalised DNA strands was the point of focus. Ultimately the DNA-polysaccharide connection was mediated by streptavidin moieties linking biotin-functionalised ends.

Streptavidin is a tetrameric protein, renowned for its strong binding to biotin that has to led to multitudinous applications. By separating streptavidin species that have differing numbers of binding sites plugged, "linking hubs" with trivalent, divalent and monovalent functionality were obtained. Species identity, and the plugging process were studied with capillary electrophoresis, which in this case provides several advantages over traditional gels. Subsequently, divalent linkers were used to concatenate two biotin-terminated 5 kb pieces of double stranded DNA, and the resulting string stretched in an optical tweezers experiment, demonstrating the "plug-and-play" potential of the methodology for coupling and extending molecules for use in single molecule biophysical experiments.