Design of Antigen-Targeting Fluorogenic Probes Utilizing Intramolecular Addition Reaction of Protein-Dye Hybrids

作者：Mamiko Nakadate, Ryosuke Kojima, Naoki Seike, Ryo Tachibana, Kyohhei Fujita, Reiko Tsuchiya, Mako Kamiya, Andreas Plückthun, Yasuteru Urano · 发表于：Journal of the American Chemical Society · 年份：2025 · DOI：10.1021/jacs.5c04193 · 被引用次数：5 · 研究领域：Click Chemistry and Applications、Advanced Biosensing Techniques and Applications、Monoclonal and Polyclonal Antibodies Research

Fluorogenic probes for antigens are useful for various purposes, including medical diagnostics and imaging, but achieving a rapid, large fluorescence increase is difficult. Here, we report a new class of fluorogenic probes for antigens based on a conjugate of an antibody-mimetic DARPin bearing a site-specifically incorporated cysteine and silicon-pyronine (SiP), which reacts reversibly with thiols. By using a library-screening approach, we found that the fluorescence of SiP conjugated to a DARPin is quenched via π-deconjugating addition reaction of the cysteine installed in the DARPin to SiP. Upon antigen binding, the equilibrium of this reaction is shifted to dissociation, restoring π-conjugation in the SiP and resulting in a large increase in fluorescence. As proof of concept of this chemical design principle, we constructed fluorogenic probes targeting GFP and EpCAM, which showed 25- and 12-fold fluorescence increases upon binding, respectively. The latter probe enabled wash-free cancer cell imaging with a low background.