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j.crmeth.2022.100301.pdf | 11.24 MB | Adobe PDF | 見る/開く |
タイトル: | Engineered fast-dissociating antibody fragments for multiplexed super-resolution microscopy |
著者: | Zhang, Qianli Miyamoto, Akitoshi Watanabe, Shin Arimori, Takao Sakai, Masanori Tomisaki, Madoka Kiuchi, Tai https://orcid.org/0000-0002-1102-3094 (unconfirmed) Takagi, Junichi Watanabe, Naoki https://orcid.org/0000-0001-8492-200X (unconfirmed) |
著者名の別形: | 張, 千里 宮本, 章歳 渡辺, 慎 有森, 貴夫 冨﨑, 円香 木内, 泰 ⾼⽊, 淳⼀ 渡邊, 直樹 |
キーワード: | super-resolution microscopy IRIS antibody engineering nanobody Fv-clasp mutagenesis fast-dissociating probe multiplexed imaging synaptic connection |
発行日: | 24-Oct-2022 |
出版者: | Elsevier BV |
誌名: | Cell Reports Methods |
巻: | 2 |
号: | 10 |
論文番号: | 100301 |
抄録: | Image reconstruction by integrating exchangeable single-molecule localization (IRIS) achieves multiplexed super-resolution imaging by high-density labeling with fast exchangeable fluorescent probes. However, previous methods to develop probes for individual targets required a great amount of time and effort. Here, we introduce a method for generating recombinant IRIS probes with a new mutagenesis strategy that can be widely applied to existing antibody sequences. Several conserved tyrosine residues at the base of complementarity-determining regions were identified as candidate sites for site-directed mutagenesis. With a high probability, mutations at candidate sites accelerated the off rate of recombinant antibody-based probes without compromising specific binding. We were able to develop IRIS probes from five monoclonal antibodies and three single-domain antibodies. We demonstrate multiplexed localization of endogenous proteins in primary neurons that visualizes small synaptic connections with high binding density. It is now practically feasible to generate fast-dissociating fluorescent probes for multitarget super-resolution imaging. |
記述: | モノクローナル抗体の用途を広げる革新技術 --多重超解像可視化プローブへの迅速変換法--. 京都大学プレスリリース. 2022-09-21. |
著作権等: | © 2022 The Authors. This is an open access article under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International license. |
URI: | http://hdl.handle.net/2433/276855 |
DOI(出版社版): | 10.1016/j.crmeth.2022.100301 |
PubMed ID: | 36313806 |
関連リンク: | https://www.kyoto-u.ac.jp/ja/research-news/2022-09-21-0 |
出現コレクション: | 学術雑誌掲載論文等 |
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