White Rose Chemical Biology Toolkits: Enabling the dissection of biomolecular interactions through chemistry

Lead Academic – Dr Martin A. Fascione (York)

Prof Jon Sayers (Sheffield)

Dr Deijan Zhou (Leeds)

Surface plasmon resonance (SPR) is a powerful technique to study label-free, real-time biomolecular interactions, it is widely applicable to protein-protein, protein-DNA and protein-ligand binding events. SPR use is limited in academic environments by the requirement that one binding partner must be immobilised on an expensive gold surface with limited reusability. Although reusable systems are available, these are prohibitively expensive. This collaboration aims to develop an improved DNA-hybridisation approach to biomolecule immobilisation using chemical biology techniques previously established by the applicants. Our method will address many of the drawbacks of currently available immobilisation strategies, reduce cost and improve researcher access to SPR facilities available across York, Sheffield and Leeds. Using the enzyme Sortase-A from Gram-positive bacteria we will covalently modify N-terminal glycine residues on recombinantly expressed proteins with depsipeptide tags (Angew. Chem., 2012, 51, 9377) which have previously been linked to single strands of DNA through azide-alkyne ‘click’ reactions. Subsequent facile attachment to an SPR surface will be achieved through hybridisation to the complementary biotin tagged-DNA strand, which will be immobilised on a standard streptavidin-coated chip surface. DNA hybridisation will provide an extremely tight anchor to the surface, but is also fully reversible through denaturation, thus allowing the same chip to be reused, potentially for many different interaction partners.

Co-researchers

Dr John Darby (York),

Dr Sandra Greive (York)

Dr Yuan Guo (Leeds)

Dr Jonathan Shaw (Sheffield)

Surface Plasmon Resonance Explained