Chen Yuda, Yserentant Klaus, Hong Kibeom, Kuang Yiming, Bhowmick Arghya, Charles-Orszag Arthur, Lord Samuel J, Lu Lei, Hou Kaipeng, Mann Samuel I, Grimm Jonathan B, Lavis Luke D, Mullins R Dyche, DeGrado William F, Huang Bo
Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA, USA.
Cardiovascular Research Institute, University of California San Francisco, San Francisco, CA, USA.
bioRxiv. 2025 Jun 25:2025.06.24.661379. doi: 10.1101/2025.06.24.661379.
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 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 living at 75°C, an application previously inaccessible by existing tags. Rhobin will serve as the basis for a new class of live cell fluorescent tags and biosensors.
蛋白质设计已成为一种强大的策略，有望创造新工具。然而，设计的荧光团结合剂的实际性能仍远未满足荧光显微镜的要求。在这里，我们设计了罗丹明结合剂（Rhobin）标签，它结合了理想的特性，包括大小、亮度，现在还增加了超稳定性。Rhobin可对哺乳动物细胞中的多种亚细胞靶点进行活细胞和固定细胞成像。其可逆的荧光团结合进一步实现了具有低光漂白的活细胞超分辨率受激发射损耗显微镜以及PAINT型单分子定位显微镜。我们在生活在75°C的嗜极端微生物中展示了Rhobin，这是现有标签以前无法实现的应用。Rhobin将成为一类新型活细胞荧光标签和生物传感器的基础。
