Nakadate Mamiko, Kojima Ryosuke, Seike Naoki, Tachibana Ryo, Fujita Kyohhei, Tsuchiya Reiko, Kamiya Mako, Plückthun Andreas, Urano Yasuteru
Graduate School of Medicine, The University of Tokyo, Tokyo 113-0033, Japan.
Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan.
J Am Chem Soc. 2025 Aug 27;147(34):30684-30693. doi: 10.1021/jacs.5c04193. Epub 2025 Jul 23.
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.
用于抗原的荧光探针可用于多种目的，包括医学诊断和成像，但要实现快速、大幅的荧光增强却很困难。在此，我们报告了一类基于结合了位点特异性掺入半胱氨酸的模拟抗体DARPin和硅芘（SiP）的新型抗原荧光探针，SiP可与硫醇发生可逆反应。通过文库筛选方法，我们发现与DARPin偶联的SiP的荧光通过DARPin中安装的半胱氨酸与SiP的π-去共轭加成反应而猝灭。抗原结合后，该反应的平衡向解离方向移动，恢复SiP中的π-共轭，导致荧光大幅增加。作为这种化学设计原理的概念验证，我们构建了靶向绿色荧光蛋白（GFP）和上皮细胞黏附分子（EpCAM）的荧光探针，它们在结合时分别显示出25倍和12倍的荧光增强。后一种探针能够在低背景下实现免洗癌细胞成像。
