@article {70003,
	title = {BPS2026 {\textendash} De novo-designed rhodamine binders for advanced fluorescence microscopy},
	journal = {bioRxiv},
	year = {2026},
	month = {2026 Feb 19},
	abstract = {<p>Fluorescence microscopy is a powerful tool for studying biomolecules in their native environments, offering high spatio-temporal resolution but requiring fluorescent labels. Current live-cell compatible labeling strategies repurpose natural systems, such as fluorescent proteins or proteins that bind fluorescent ligands. While advances have been made to engineer natural proteins into labels with minimal size, high brightness, as well as enhanced thermo- and photostability, these approaches often require trade-offs among desirable properties due to the inherent limitations of re-engineering natural proteins. In this work, we present rhodamine binder (Rhobin) tags{\textemdash}de novo-designed proteins that bind rhodamine-derived fluorophores. Unlike traditional approaches, Rhobin tags were developed by directly incorporating desirable features during the computational design process, which resulted in compact binders with outstanding thermostability. Their nanomolar substrate affinity, rapid labeling kinetics, and orthogonality to established labeling systems such as HaloTag and SNAP-tag facilitate versatile live and fixed-cell imaging of diverse subcellular targets in mammalian cells. Transient fluorophore binding further enables advanced imaging techniques, including live-cell super-resolution STED microscopy with reduced photobleaching and single-molecule localization microscopy in live and fixed cells. To the stability of Rhobin tags under extreme environmental conditions, we demonstrate showcase protein tagging and timelapse imaging in the extremophile\&nbsp;<em>Sulfolobus acidocaldarius</em>\&nbsp;living at 75{\textdegree}C, an application previously inaccessible with existing tags. We anticipate that Rhobin tags will become a central component in the toolbox of fluorescent labels and will pave the way for a new generation of modular protein tags and biosensors with tailor-made properties.</p>
},
	doi = {10.1016/j.bpj.2025.11.2270},
	url = {https://doi.org/10.1016/j.bpj.2025.11.2270},
	author = {Yserentant, Klaus and Chen, Yuda and Hong, Kibeom and Kuang, Yiming and Bhowmick, Arghya and Charles-Orszag, Arthur and Lord, Samuel J. and Lu, Lei and Hou, Kaipeng and Mann, Samuel I and Grimm, Jonathan B and Lavis, Luke D and Mullins, R. Dyche and DeGrado, William F and Huang, Bo}
}
