Bühler Bastian, Schokolowski Janin, Benderoth Anja, Englert Daniel, Grün Franziska, Jäschke Andres, Sunbul Murat
Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Heidelberg, Germany.
Nat Chem Biol. 2023 Apr;19(4):478-487. doi: 10.1038/s41589-022-01228-8. Epub 2023 Jan 19.
Fluorescent light-up aptamers (FLAPs) have emerged as valuable tools to visualize RNAs, but are mostly limited by their poor brightness, low photostability, and high fluorescence background in live cells. Exploiting the avidity concept, here we present two of the brightest FLAPs with the strongest aptamer-dye interaction, high fluorogenicity, and remarkable photostability. They consist of dimeric fluorophore-binding aptamers (biRhoBAST and biSiRA) embedded in an RNA scaffold and their bivalent fluorophore ligands (bivalent tetramethylrhodamine TMR and silicon rhodamine SiR). Red fluorescent biRhoBAST-TMR and near-infrared fluorescent biSiRA-SiR are orthogonal to each other, facilitating simultaneous visualization of two different RNA species in live cells. One copy of biRhoBAST allows for simple and robust mRNA imaging with strikingly higher signal-to-background ratios than other FLAPs. Moreover, eight biRhoBAST repeats enable single-molecule mRNA imaging and tracking with minimal perturbation of their localization, translation, and degradation, demonstrating the potential of avidity-enhanced FLAPs for imaging RNA dynamics.
荧光点亮适配体（FLAPs）已成为可视化RNA的重要工具，但在活细胞中，它们大多受到亮度低、光稳定性差和荧光背景高的限制。利用亲和力概念，我们在此展示了两种最亮的FLAPs，它们具有最强的适配体与染料相互作用、高荧光性和出色的光稳定性。它们由嵌入RNA支架中的二聚体荧光团结合适配体（biRhoBAST和biSiRA）及其二价荧光团配体（二价四甲基罗丹明TMR和硅罗丹明SiR）组成。红色荧光的biRhoBAST-TMR和近红外荧光的biSiRA-SiR相互正交，便于在活细胞中同时可视化两种不同的RNA种类。一个拷贝的biRhoBAST就能实现简单且稳健的mRNA成像，其信号与背景比显著高于其他FLAPs。此外，八个biRhoBAST重复序列能够实现单分子mRNA成像和追踪，对其定位、翻译和降解的干扰最小，证明了亲和力增强型FLAPs在成像RNA动态方面的潜力。
