Bioconjugation of Au25 Nanocluster to Monoclonal Antibody at Tryptophan

Katarzyna Joanna Malawska; Shinjiro Takano; Kounosuke Oisaki; Haruaki Yanagisawa; Masahide Kikkawa; Tatsuya Tsukuda; Motomu Kanai

doi:10.1021/acs.bioconjchem.3c00069

Bioconjugation of Au₂₅ Nanocluster to Monoclonal Antibody at Tryptophan

Bioconjug Chem. 2023 Mar 9. doi: 10.1021/acs.bioconjchem.3c00069. Online ahead of print.

Authors

Katarzyna Joanna Malawska¹, Shinjiro Takano², Kounosuke Oisaki¹, Haruaki Yanagisawa³, Masahide Kikkawa³, Tatsuya Tsukuda², Motomu Kanai¹

Affiliations

¹ Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
² Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
³ Department of Cell Biology and Anatomy, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.

PMID: 36893358
DOI: 10.1021/acs.bioconjchem.3c00069

Abstract

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.