Grün Franziska, van den Bergh Niklas, Klevanski Maja, Verma Mrigank S, Bühler Bastian, Nienhaus G Ulrich, Kuner Thomas, Jäschke Andres, Sunbul Murat
Institute of Pharmacy and Molecular Biotechnology (IPMB), Heidelberg University, 69120, Heidelberg, Germany.
Department of Nuclear Medicine, Heidelberg University Hospital, 69120, Heidelberg, Germany.
Angew Chem Int Ed Engl. 2024 Dec 16;63(51):e202412810. doi: 10.1002/anie.202412810.
Efficient labeling methods for protein visualization with minimal tag size and appropriate photophysical properties are required for single-molecule localization microscopy (SMLM), providing insights into the organization and interactions of biomolecules in cells at the molecular level. Among the fluorescent light-up aptamers (FLAPs) originally developed for RNA imaging, RhoBAST stands out due to its remarkable brightness, photostability, fluorogenicity, and rapid exchange kinetics, enabling super-resolved imaging with high localization precision. Here, we expand the applicability of RhoBAST to protein imaging by fusing it to protein-binding aptamers. The versatility of such bifunctional aptamers is demonstrated by employing a variety of protein-binding aptamers and different FLAPs. Moreover, fusing RhoBAST with the GFP-binding aptamer AP3 facilitates high- and super-resolution imaging of GFP-tagged proteins, which is particularly valuable in view of the widespread availability of plasmids and stable cell lines expressing proteins fused to GFP. The bifunctional aptamers compare favorably with standard antibody-based immunofluorescence protocols, as they are 7-fold smaller than antibody conjugates and exhibit higher bleaching-resistance. We demonstrate the effectiveness of our approach in super-resolution microscopy in secondary mammalian cell lines and primary neurons by RhoBAST-PAINT, an SMLM protein imaging technique that leverages the transient binding of the fluorogenic rhodamine dye SpyRho to RhoBAST.
单分子定位显微镜（SMLM）需要高效的蛋白质标记方法，以实现最小的标签尺寸和合适的光物理性质，从而在分子水平上深入了解细胞中生物分子的组织和相互作用。在最初为RNA成像开发的荧光点亮适体（FLAP）中，RhoBAST因其显著的亮度、光稳定性、荧光生成能力和快速交换动力学而脱颖而出，能够实现具有高定位精度的超分辨成像。在这里，我们通过将RhoBAST与蛋白质结合适体融合，将其应用扩展到蛋白质成像。通过使用多种蛋白质结合适体和不同的FLAP，证明了这种双功能适体的多功能性。此外，将RhoBAST与GFP结合适体AP3融合，有助于对GFP标记的蛋白质进行高分辨率和超分辨率成像，鉴于表达与GFP融合蛋白质的质粒和稳定细胞系广泛可用，这尤其有价值。双功能适体与基于标准抗体的免疫荧光方案相比具有优势，因为它们比抗体偶联物小7倍，并且具有更高的抗漂白性。我们通过RhoBAST-PAINT（一种利用荧光罗丹明染料SpyRho与RhoBAST的瞬时结合的SMLM蛋白质成像技术）证明了我们的方法在二级哺乳动物细胞系和原代神经元超分辨率显微镜中的有效性。
