DNA-directed immobilization: Efficient, reversible, and site-selective surface binding of proteins by means of covalent DNA-streptavidin conjugates

Covalent DNA-streptavidin conjugates have been utilized for the reversible and site-selective immobilization of various biotinylated enzymes and antib odies by DNA-directed immobilization (DDI). Biotinylated alkaline phosphata se, beta-galactosidase, and horseradish peroxidase as well as biotinylated anti-mouse and anti-rabbit immunoglobulins have been coupled to the DNA-str eptavidin adapters by simple, two-component incubation and the resulting pr econjugates were allowed to hybridize to complementary, surface-bound captu re oligonucleotides. Quantitative measurements on microplates indicate that DDI proceeds with a higher immobilization efficiency than conventional imm obilization techniques, such as the binding of the biotinylated proteins to streptavidin-coated surfaces or direct physisorption. These findings can b e attributed to the reversible formation of the rigid, double-stranded DNA spacer between the surface and the proteins. Moreover, BIAcore measurements demonstrate that DDI allows a reversible functionalization of sensor surfa ces with reproducible amounts of proteins. Ultimately, the simultaneous imm obilization of different compounds using microstructured oligonucleotide ar rays as immobilization matrices demonstrate that DDI proceeds with site sel ectivity due to the unique specificity of Watson-Crick base pairing. (C) 19 99 Academic Press.