Institute of Pharmacy and Molecular Biotechnology (IPMB), Heidelberg University, 69120 Heidelberg, Germany.
ACS Chem Biol. 2023 Aug 18;18(8):1838-1845. doi: 10.1021/acschembio.3c00258. Epub 2023 Aug 2.
The development of fluorescent probes for visualizing endogenous RNAs in living cells is crucial to understand their complex biochemical roles. Recently, we developed RhoBAST, one of the most photostable and brightest fluorescence light-up aptamers (FLAPs), as a genetically encoded tag for imaging messenger RNAs (mRNAs). Here, we describe programmable RhoBAST sequences flanked by target-binding hybridization arms that light up only when bound to the untagged target RNA in . As part of the hybridization arm, we introduced a modular transducer sequence that switches the secondary structure of RhoBAST and renders it incapable of binding to its fluorogenic ligand TMR-DN. Only the specific binding of the hybridization arms to the target RNA triggers the correct folding of RhoBAST and fluorescence light-up after binding to TMR-DN. We characterized the structural switching of programmable RhoBAST sequences extensively and applied them to visualize untagged mRNAs in live bacteria.
开发用于在活细胞中可视化内源性 RNA 的荧光探针对于理解它们复杂的生化作用至关重要。最近，我们开发了 RhoBAST，它是最稳定和最亮的荧光点亮适体（FLAP）之一，作为一种用于成像信使 RNA（mRNA）的基因编码标签。在这里，我们描述了可编程 RhoBAST 序列，其两侧是靶结合杂交臂，只有当与未标记的靶 RNA 结合时才会发光。作为杂交臂的一部分，我们引入了一个模块化转导序列，该序列会切换 RhoBAST 的二级结构，并使其无法与荧光配体 TMR-DN 结合。只有杂交臂与靶 RNA 的特异性结合才能触发 RhoBAST 的正确折叠，并在与 TMR-DN 结合后发出荧光。我们广泛表征了可编程 RhoBAST 序列的结构切换，并将其应用于活细菌中未标记的 mRNA 的可视化。
