Click-electrochemistry for the rapid labeling of virus, bacteria and cell surfaces

Abstract

The remodeling of microorganism surfaces with biomolecules is a powerful tool to study the role of membrane receptors in chemical biology and to develop drug delivery systems in gene therapy using viral vectors and cell-based therapies. Methods for direct covalent ligation of these surfaces remain poorly reported, and mostly based on metabolic engineering for bacteria and cells functionalization. In the latter case, a tagged precursor must first be enzymatically metabolized and delivered to the outer cell membrane to become available for chemo-selective labeling. While effective, a faster method avoiding the bio-incorporation step would be highly complementary. This would also need to be compatible with organisms showing poor levels of precursor assimilation or lacking the metabolic function. Here, we used N-methylluminol (NML), a fully tyrosine-selective protein anchoring group after one-electron oxidation, to label the surface of viruses, living bacteria and cells. The functionalization was performed electrochemically and in situ by applying a 750 mV vs Ag/AgCl electric potential to aqueous buffered solutions of tagged NML containing the viruses, bacteria or cells. The electro-coupling was performed with NML anchors bearing a bioorthogonal azide, biotin, or carbohydrate (mannose and N-acetyl galactosamine) handles. The broad applicability of the click-electrochemistry method was explored on recombinant adeno-associated viruses (rAAV2), E. coli (Gram-) and S. epidermis (Gram+) bacterial strains, and HEK293 and HeLa eukaryotic cell lines. Surface electro-conjugation was achieved in minutes to yield functionalized rAAV2 that conserved both structural integrity and infectivity properties, and living bacteria and cell lines that were still alive and able to divide. As NML activation immediately stops if there is no current, the method offers reproducible temporal control on the degree of surface functionalization. Thus, click-electrochemistry should significantly expand the scope of bioconjugation methods.

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