Molecular probe for high-performance cryo-EM

2022 Fiscal Year Final Research Report

• Project/Area Number: 20K21472
• Research Category: Grant-in-Aid for Challenging Research (Exploratory)
• Allocation Type: Multi-year Fund
• Review Section: Medium-sized Section 47:Pharmaceutical sciences and related fields
• Research Institution: The University of Tokyo
• Principal Investigator: Kanai Motomu 東京大学, 大学院薬学系研究科(薬学部), 教授 (20243264)
• Project Period (FY): 2020-07-30 – 2023-03-31
• Keywords: 抗体修飾 / トリプトファン / 金ナノ粒子 / クライオ電顕

Outline of Final Research Achievements

We achieved 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). We improved the Trp-selective bioconjugation by using hydroxylamine reagents. 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 bioconju-gation for introduction of azide groups to the protein and then strain-promoted azide-alkyne cycloaddition (SPAAC) to attach bicyclononyne (BCN)-presenting, redox-sensitive Au25 nanocluster, was essential for scalable procedure. Covalent labeling of the antibody with gold nanoclusters was confirmed by cryo-EM analysis of the Au25 nanocluster conjugates.

Free Research Field

有機合成化学

Academic Significance and Societal Importance of the Research Achievements

抗体に金ナノ粒子を結合させたimmunogoldは、抗体の高い標的分子識別能と金粒子の高い電子線反射能の双方を併せ持つことから、高次の生命現象の分子機構解明への適用が期待される。Immunogoldのその他の用途として、光照射による金ナノ粒子の活性酸素生成を利用するがん光線力学療法や、吸光係数が大きいことから極低濃度でも視認できることを活かした抗体検査プローブなどが想定される。