RESUMO
We report the first bioconjugation of Au25 nanocluster to a monoclonal antibody at scarcely exposed tryptophan (Trp) residues toward the development of high-resolution probes for cryogenic electron microscopy (cryo-EM) and tomography (cryo-ET). To achieve this, we improved the Trp-selective bioconjugation using hydroxylamine (ABNOH) reagents instead of previously developed N-oxyl radicals (ABNO). This new protocol allowed for the application of Trp-selective bioconjugation to acid-sensitive proteins such as antibodies. We found that a two-step procedure utilizing first Trp-selective bioconjugation for the introduction of azide groups to the protein and then strain-promoted azide-alkyne cycloaddition (SPAAC) to attach a bicyclononyne (BCN)-presenting redox-sensitive Au25 nanocluster was essential for a scalable procedure. Covalent labeling of the antibody with gold nanoclusters was confirmed by various analytical methods, including cryo-EM analysis of the Au25 nanocluster conjugates.
