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

Abstract

The self-assembly of bis-biotinylated double-stranded DNA and the tetravalent biotin-binding protein streptavidin (STV) have been studied by non-denaturing gel electrophoresis and atomic force microscopy (AFM). The rapid self-assembly reproducibly generated populations of individual oligomeric complexes. Most strikingly, the oligomers predominantly contained bivalent STV molecules bridging two adjacent DNA fragments to form linear nanostructures. Trivalent STV branch points occurred with a lower frequency and the presence of tetravalent STV was scarce. However, valency distribution, size and the exchange dynamics of the supramolecular aggregates were highly sensitive to stoichiometric variations in the relative molar coupling ratio of bis-biotinylated DNA and STV. The largest aggregates were obtained from equimolar amounts while excess STV led to the formation of smaller oligomers appearing as fingerprint-like band patterns in electrophoresis. Excess DNA, however, 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 functionalized, for instance by the coupling of biotinylated immunoglobulins. Both pure and also antibody-modified DNA-STV oligomers were used as reagents in immuno-PCR (IPCR), a highly sensitive detection method for proteins and other antigens. Employment of the supramolecular reagents led to an approximately 100-fold enhanced sensitivity compared to the conventional IPCR procedure.

Cytokine detection by antibody-based proximity ligation

“…The latter reagents can form multivalent complexes due to the tetrameric structure of STV and the multiple biotins groups coupled per antibody (11,12). Accordingly, each proximity probe can involve several antibodies and STV molecules, increasing the apparent affinity by cooperative effects.…”

Controlled hierarchical assembly of switchable DNA–multiprotein complexes

“…As in nature, synthetic assemblies comprised of biological macromolecules can be constructed through highly selective biomolecular interactions (previous references and Padilla et al, 2001;Smith et al, 1997), with the streptavidin-biotin system used most broadly (Moll et al, 2002;Niemeyer, 2001). However, the rigid and nearly covalent binding between streptavidin and biotin, and the inherently non-selective protein biotinylation that results in non-uniform complexation (Niemeyer et al, 1999) limit the design of responsive, smart nanomaterials. Conversely, TF-TFBS recognition is highly controllable, and therefore ideal to serve as the first level of a multicomponent assembly.…”

Nanoscience enables ultrasensitive detection of Alzheimer's biomarker

“…IPCR provides substantial increases in sensitivity as compared with ELISA but requires conjugation of the secondary antibodies to DNA strands as well as the thermocycling and enzymatic amplification common to PCR. Variations have been reported in which improved reagents or other forms of enzymatic amplification provide improvements in sensitivity, quantification, or ease of use (e.g., by avoiding thermocycling) (14)(15)(16)(17)(18)(19)(20)(21). For example, IPCR using oligovalent streptavidin-DNA assemblies has been shown to provide 1,000-fold increases in sensitivity as compared with traditional ELISA (18,19).…”