Multifunctional streptavidin-biotin conjugates with precise stoichiometries

Streptavidin is ubiquitously used to link different biotinylated molecules thanks to its tetravalent binding to biotin. An unwanted side-effect is the resulting statistical mixtures of products. Here, a general approach to form multifunctional streptavidin conjugates with precise stoichiometries and number of open binding pockets is reported. This method relies on an iminobiotin-polyhistidine tag, which allows separating streptavidin conjugates with different numbers of tags, and later reopening binding pockets at lowered pH to introduce a second functionality. Pure fluorescently labelled mono-, di- and trivalent streptavidin-biotin conjugates prepared in this way were used for imaging biotinylated cell surface molecules with controlled clustering. Furthermore, these conjugates were functionalized with a second biotinylated molecule, folic acid-biotin, to investigate the importance of multivalent binding in targeted delivery of cancer cells. These streptavidin-biotin conjugates with precise stoichiometries combined with a variety of biotinylated molecules render this method a diverse and powerful tool for molecular biology and biotechnology.