An international research team, including scientists from the Institute of Organic Chemistry and Zhejiang University, has uncovered how a specific change in an RNA molecule can activate fluorescent dyes used to track RNA inside living cells. This discovery could improve the way scientists observe RNA activity in real time, without the need for complex modifications. The study centers on a small RNA molecule called RhoBAST, which helps activate fluorescent dyes, enabling a technique known as super-resolution imaging. This method allows researchers to see structures at a much higher resolution than traditional microscopes, without adding extra proteins to the RNA molecules being studied. Fluorescent light-up aptamers, or FLAPs, are short RNA sequences that bind to and activate small dye molecules. These dyes typically emit very weak light on their own, but when bound to FLAPs, they become much brighter. This allows scientists to "tag" specific RNA molecules for observation within living cells, with minimal background noise that could interfere with the results. The research team studied the structure of RhoBAST both on its own and when it was bound to two types of dyes—TMR-DN and similar compounds. They found that RhoBAST forms a shape resembling an inverted "V," with two RNA loops that hold the dye in place. A key finding was that when the dye binds to RhoBAST, a single RNA building block—a guanosine residue known as G38—flips from a position facing inward to one facing outward. This movement creates space for the dye and allows it to bind more effectively. The researchers used several techniques, including fluorescence spectroscopy, surface plasmon resonance, and 2-aminopurine kinetics, to confirm that this flipping motion enables the dye to rapidly exchange places with other molecules. This rapid exchange is crucial for the "blinking" effect seen in super-resolution imaging, where the dye alternates between bright and dark states, allowing for precise imaging at a very high resolution. Ronald Micura, a researcher from the Institute of Organic Chemistry at the University of Innsbruck, explained that the flipping of this single nucleotide is essential for the quick exchange of the dye, which in turn creates the blinking effect needed for super-resolution microscopy. This discovery reveals a previously unknown mechanism and provides a foundation for developing new tools to study RNA at high resolution in living cells. The findings have been published in the journal Nature Communications, marking an important step in the field of RNA imaging.