Optimized avidin nucleic acid nanoassemblies by a tailored PEGylation strategy and their application as molecular amplifiers in detection

Avidin was recently found to display the ability to interact with high affinity with nucleic acids. In this work, we investigated how this property is affected by the protein modification with poly(ethylene glycol) (PEG). More precisely, we studied the influence of the size and geometry of the polymer and of the mode of anchorage to the protein surface. To this end, we synthesized five PEG derivatives capable of PEGylating avidin either through covalent attachment to its lysine primary amines or by exploiting its biotin binding pockets. Several differently PEGylated avidin derivatives were then obtained, which were later tested for their affinity for plasmid DNA by means of the electrophoretic mobility assay. The results show that covalent PEGylation reduces the affinity for DNA in a dose-dependent manner, whereas PEG anchoring through the biotin binding sites does not, even when bulky and high MW biotin-PEG derivatives are used. We then investigated how the size and molecular weight of the biotin-PEG affects the solubility and stability of avidin-nucleic acid nanoassemblies in physiological buffer. Among the biotin-PEG derivatives synthesized in this work, the branched forms were more efficient in protecting particle surface and preventing their aggregation. Full nanoparticle solubility was achieved by saturating 30% of the biotin binding sites with a 2 x 5 kDa branched derivative. the optimized avidin nucleic acid nanoassemblies (ANANAS) were employed in a model analytical test where they showed at least 40-fold higher efficiency than monomeric avidin in recognizing biotinylated surface immobilized IgGs. The results pave the way toward the application of this novel nanosystem in biomedicine.