Three-dimensional structure of RhoBAST RNA interacting with the fluorophore (yellow).
Image Credit: Christoph Mitteregger and Ronald Micura
Scientific Frontline: Extended "At a Glance" Summary: RhoBAST RNA
The Core Concept: RhoBAST is a small, specialized RNA molecule (a fluorescent light-up aptamer) that binds to and activates specific fluorescent dyes, allowing researchers to track individual RNA molecules inside living cells with high resolution.
Key Distinction/Mechanism: Unlike traditional tagging methods that require bulky protein fusions, RhoBAST functions through a simple "nucleotide flip." When the dye approaches, a single RNA building block (guanosine residue G38) flips outward, creating a pocket for the dye. This dynamic, localized movement enables rapid ligand exchange and fluorescence "blinking"—the key to super-resolution imaging. Background fluorescence remains low because the dye only emits strong light when bound to the RNA.
Major Frameworks/Components:
- Fluorescent Light-Up Aptamers (FLAPs): Short RNA sequences designed to bind small molecules and enhance their fluorescence.
- Inverted V-Shaped Structure: The specific three-dimensional conformation RhoBAST adopts to accommodate the dye between two RNA loops.
- Nucleotide Flipping: The critical structural rearrangement (specifically of the G38 residue) that allows dye binding and the characteristic "blinking" effect.
- Biophysical Assays: Techniques such as fluorescence spectroscopy, surface plasmon resonance (SPR), and 2-aminopurine kinetics used to verify the mechanism.
Branch of Science: Biochemistry, Molecular Biology, Organic Chemistry, and Biophysics.
Future Application: The detailed understanding of this switching mechanism provides a foundation for engineering a new generation of more precise, efficient tools for super-resolution RNA imaging and tracking in living cells.
Why It Matters: Being able to observe individual RNA molecules in real-time within living cells is crucial for understanding gene expression, cellular processes, and the molecular basis of diseases, ultimately contributing to advancements in health and medicine.
RhoBAST is a tiny RNA molecule that activates fluorescent dyes, enabling researchers to track RNA molecules in living cells with super-resolution. An international collaboration, which includes Ronald Micura and his team from the Institute of Organic Chemistry, has now shown that a small, local “nucleotide flip” within the RNA controls this fluorescence activation.
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 in recent 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 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 provides a foundation for developing a new generation of tools for high-resolution imaging of RNA in living cells.
Published in journal: Nature Communications
Title: Nucleotide flipping correlates with fluorescence activation in the RhoBAST imaging platform
Authors: Xiaoqing Tai, Mengqi He, Christoph Mitteregger, Huiqin You, Zejia Hu, Peng Xiong, Xinyue Bao, Xin Shen, Hongcheng Li, Yu Li, Jinzhu Zhang, Ronald Micura, and Aiming Ren
Source/Credit: University of Innsbruck
Edited by: Scientific Frontline
Reference Number: bchm091326_01