De novo designed bright, hyperstable rhodamine binders for fluorescence microscopy

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bioRxiv. 2025 Jun 25;. doi: 10.1101/2025.06.24.661379

De novo designed bright, hyperstable rhodamine binders for fluorescence microscopy Lavis Lab

Chen Y, Yserentant K, Hong K, Kuang Y, Bhowmick A, Charles-Orszag A, Lord SJ, Lu L, Hou K, Mann SI, Grimm JB, Lavis LD, Mullins RD, DeGrado WF, Huang B

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Abstract

De novo protein design has emerged as a powerful strategy with the promise to create new tools. The practical performance of designed fluorophore binders, however, has remained far from meeting fluorescence microscopy demands. Here, we design de novo Rhodamine Binder (Rhobin) tags that combine ideal properties including size, brightness, and now adding hyperstability. Rhobin allows live and fixed cell imaging of a wide range of subcellular targets in mammalian cells. Its reversible fluorophore binding further enables live super-resolution STED microscopy with low photobleaching, as well as PAINT-type single-molecule localization microscopy. We showcase Rhobin in the extremophile Sulfolobus acidocaldarius living at 75 degrees Celsius, an application previously inaccessible by existing tags. Rhobin will serve as the basis for a new class of live cell fluorescent tags and biosensors.

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De novo designed bright, hyperstable rhodamine binders for fluorescence microscopy

Abstract

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