Ribonucleic acid (RNA) plays a key role in various fundamental biological processes. It transmits genetic information, translates it into proteins, or supports gene regulation. To gain a more detailed understanding of the exact functions it performs, researchers at the University of Heidelberg and the Karlsruhe Institute of Technology (KIT) have developed a new fluorescence imaging technique that allows RNA to be visualized in living cells with unprecedented resolution.
The method is based on a new molecular marker called the rhodamine-binding aptamer for ultra-high resolution imaging (RhoBAST) techniques. This RNA fluorescence marker is used in combination with the dye rhodamine. Due to their distinctive properties, the marker and dye interact in a very specific way, which causes the individual RNA molecules to glow. They can then be made visible using single molecule localization microscopy (SMLM), an ultra-high resolution imaging technique. Due to the lack of suitable fluorescent markers, direct observation of RNA by optical fluorescence microscopy has been severely limited to date.
RhoBAST is developed by researchers at the Institute of Pharmacy and Molecular Biotechnology (IPMB) at the University of Heidelberg and the Institute for Applied Physics (APH) at KIT. The marker they created is genetically encoded, which means that it can be fused with the gene of any RNA produced by the cell. RhoBAST itself is not fluorescent, but illuminates the cell-permeable rhodamine dye by binding to it in a very specific manner.
“This results in the dramatic increase in fluorescence achieved by the RhoBAST complex, which is a key requirement for superior fluorescence imaging. However, to visualize ultra-high-resolution RNA, the marker needs additional properties. “Murat Zünbühl from IPMB
The researchers found that each rhodamine dye molecule remains bound to RhoBAST for only about one second before detaching again. After a few seconds, this procedure is repeated with a new dye molecule. It is quite rare to find strong interactions, for example, between RhoBAST and rhodamine, combined with extremely fast metabolic kinetics. Since rhodamine only lights up after binding to RhoBAST, the constant sequence of re-emerging interactions between the marker and the dye leads to continuous “blinking”. This “on-off” is exactly what you need for rendering.
At the same time, the RhoBAST system solves another important problem. Fluorescent images are collected by exposure to laser light, which over time breaks down the dye molecules. Rapid dye change ensures that photobleached dyes are replaced with fresh ones. This means that individual RNA molecules can be observed for longer periods of time, which can significantly improve image resolution.
Researchers from Heidelberg and Karlsruhe were able to demonstrate the superior properties of RhoBAST by visualizing the RNA structures within intestinal bacteria (Escherichia coli) and cultured human cells with excellent localization accuracy. Scientists were able to uncover details of previously invisible subcellular structures and molecular interactions involving RNA using ultra-high-resolution fluorescence microscopy. This will provide a fundamentally new understanding of biological processes.