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Adaptamers Thrombin-binding domain Thrombin Streptavidin-binding domain Linking region Streptavidin Why develop adaptamers? • To offer a new way to target substrates to cells. • To create a tool for studying cell-cell interactions. • To provide a foundation for engineered catalysts. Adaptamer testing streptavidin adaptamer Adaptamer = A15 thrombin anti-thrombin antibody Control: no thrombin Bead surface Adaptamer quenching… Displacement strand + Results: 4000 3500 Relative Fluorescence 3000 2500 2000 1500 1000 500 0 + No displacement strand added Displacement strand added Background fluorescence Streptavidin on the cell surface Streptavidin • Binds strongly to biotin molecule • Used to bind biotinylated nucleic acids or peptides McDevitt, 1999 Big idea: Streptavidin protein expressed on the cell surface can be used to target any biotinylated DNA/protein to the cell surface. Lpp-OmpA surface display J. Francisco, C. Earhart, G. Georgiou, 1992 Surface protein extracellular Lpp (lipoprotein) signal peptide outer membrane periplasm OmpA (outer membrane protein A) transmembrane domains (1 or 5) BioBricks assembly of protein domains Standard BioBrick Protein domain BioBrick 5’…TCTAGAG XbaI TACTAGT…3’ SpeI 5’…TCTAGA_ XbaI _ACTAGT…3’ SpeI 6-bp mixed site Reading frame maintained Promoter RBS Lpp TCTAGT Mixed OmpA O1 O5 Streptavidin SM (Ting) SD (Aslan) I. Phillips P. Silver 2006 Expression of construct Anti-His Anti-Streptavidin
