Self-assembly of DNA-streptavidin nanostructures and their use as reagentsin immuno-PCR

The self-assembly of bis-biotinylated double-stranded DNA and the tetravale nt biotin-binding protein streptavidin (STV) have been studied by non-denat uring gel electrophoresis and atomic force microscopy (AFM), The rapid self -assembly reproducibly generated populations of individual oligomeric compl exes. Most strikingly, the oligomers predominantly contained bivalent STV m olecules bridging two adjacent DNA fragments to form linear nanostructures, Trivalent STV branch points occurred with a lower frequency and the presen ce of tetravalent STV was scarce. However, valency distribution, size and t he exchange dynamics of the supramolecular aggregates were highly sensitive to stoichiometric variations in the relative molar coupling ratio of bis-b iotinylated DNA and STV, The largest aggregates were obtained from equimola r amounts while excess STV led to the formation of smaller oligomers appear ing as fingerprint-like band patterns in electrophoresis. Excess DNA, howev er, induces a complete breakdown of the oligomers, likely a consequence of the instability of STV conjugates containing more than two biotinylated DNA fragments. It was demonstrated that the oligomers can further be functiona lized, for instance by the coupling of biotinylated immunoglobulins, Both p ure and also antibody-modified DNA-STV oligomers were used as reagents in i mmuno-PCR (IPCR), a highly sensitive detection method for proteins and othe r antigens, Employment of the supramolecular reagents led to an similar to 100-fold enhanced sensitivity compared to the conventional IPCR procedure.