Fluorescent light-up aptamers (FLAPs) are short RNA sequences that bind and activate small dye molecules that otherwise exhibit only weak fluorescence. This enables genetic tagging of RNAs for live-cell imaging without the need for protein fusion markers. Because the dye is only “switched on” upon binding to the RNA, background fluorescence remains low. Systems such as Spinach, Broccoli, Mango, and Pepper have continuously advanced this principle over the past years. RhoBAST additionally enables high-resolution imaging of individual RNA molecules in living cells.
Structure Revealed
To understand the molecular basis of these remarkable properties, scientists led by Ronald Micura from the Institute of Organic Chemistry at the University of Innsbruck and Aiming Ren from Zhejiang University determined, for the first time, the structure of RhoBAST without a bound dye, as well as its structures in complex with TMR-DN and related dyes. The results have now been published in Nature Communications.
The researchers discovered a surprisingly simple mechanism: RhoBAST adopts an inverted V-shaped structure and accommodates the dye between two RNA loops. When the dye binds, a single RNA building block—the guanosine residue G38—flips from an inward-facing to an outward-facing position, thereby creating space for the dye. Structure-guided mutagenesis and biophysical assays, including fluorescence spectroscopy, surface plasmon resonance (SPR), and 2-aminopurine kinetics, demonstrate that this dynamic flipping enables rapid ligand exchange and fluorescence “blinking”—a hallmark of super-resolution imaging. The findings reveal a previously unrecognized mechanism.
Advancing RNA Imaging
“It is fascinating that such a small local movement within the RNA is crucial for the remarkable properties of the entire system,” explains Ronald Micura. “The flipping of a single nucleotide enables the rapid exchange of the dye and thus the characteristic blinking that is essential for super-resolution microscopy.” This understanding of the molecular switching mechanism thus provides a foundation for developing a new generation of tools for high-resolution imaging of RNA in living cells.
Publication: Tai, X., He, M., Mitteregger, C. et al. Nucleotide flipping correlates with fluorescence activation in the RhoBAST imaging platform. Nat Commun 17, 8927 (2026). DOI: 10.1038/s41467-026-75573-w