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 TMR₂ and silicon rhodamine SiR₂). Red fluorescent biRhoBAST–TMR₂ and near-infrared fluorescent biSiRA–SiR₂ 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.

荧光发光适体 (FLAP) 已成为可视化 RNA 的宝贵工具，但主要受限于它们在活细胞中的亮度差、光稳定性差和荧光背景高。利用亲和力概念，我们在这里展示了两个最亮的 FLAP，它们具有最强的适体-染料相互作用、高荧光性和显着的光稳定性。它们由嵌入 RNA 支架的二聚荧光团结合适体（biRhoBAST 和 biSiRA）及其二价荧光团配体（二价四甲基罗丹明 TMR ₂和硅罗丹明 SiR ₂）组成。红色荧光 biRhoBAST–TMR ₂和近红外荧光 biSiRA–SiR ₂彼此正交，促进活细胞中两种不同 RNA 种类的同时可视化。一份 biRhoBAST 允许简单而强大的 mRNA 成像，其信号背景比明显高于其他 FLAP。此外，八个 biRhoBAST 重复使单分子 mRNA 成像和跟踪成为可能，同时对其定位、翻译和降解的扰动最小，证明了亲合力增强的 FLAP 对 RNA 动力学成像的潜力。