Scientists have discovered that a single nucleotide flip in the RhoBAST RNA molecule triggers the activation of fluorescent dyes, enabling high-resolution live-cell imaging.

An international research team led by Ronald Micura of the University of Innsbruck and Aiming Ren of Zhejiang University has identified the structural mechanism behind RhoBAST, an RNA molecule used to track cellular processes. By determining the structure of RhoBAST in both bound and unbound states, researchers found that a specific nucleotide flip controls the activation of fluorescent dyes. The findings, published in Nature Communications, explain how this process facilitates super-resolution imaging.

Mechanism of Fluorescence Activation

RhoBAST functions as a fluorescent light-up aptamer, a short RNA sequence that binds to and activates dye molecules that are otherwise weakly fluorescent. The researchers determined that the RNA adopts an inverted V-shaped structure, holding the dye between two loops. Upon binding, a single guanosine residue, G38, flips from an inward-facing to an outward-facing position to create the necessary space for the dye.

Implications for Imaging Technology

The study utilized structure-guided mutagenesis and biophysical assays, including fluorescence spectroscopy and surface plasmon resonance, to analyze the system. The team found that the dynamic flipping of G38 allows for rapid ligand exchange and fluorescence 'blinking,' which is a requirement for super-resolution microscopy. This discovery provides a molecular basis for developing future tools to image RNA in living cells.