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Ultra-high-resolution RNA imaging in live cells

RNA is the key to various basic biological processes. It transmits genetic information, translates it into proteins, or supports gene regulation. To achieve a more detailed understanding of the exact functions they perform, resident researchers at Heidelberg University and the Karlsruhe Institute of Technology (KIT) have devised a new fluorescence imaging method that enables RNA imaging of live cells with unprecedented accuracy.

The method is based on a novel molecular marker called the Rhodamine-Binding Aptamer for Ultra High Resolution Imaging Technologies (RhoBAST). An RNA-based fluorescence marker is used in combination with the rhodamine dye. Due to their distinct properties, the marker and dye interact in a very specific way, causing individual RNA molecules to glow. They can then be made visible using Single-Molecular Localization Microscopy (SMLM), a super-resolution imaging technique. Due to the lack of suitable fluorescence markers, direct monitoring of RNA via fluorescence optical microscopy has been very limited thus far.

RhoBAST was developed by researchers from the Institute of Pharmacology and Molecular Biotechnology (IPMB) at the University of Heidelberg and the Institute for Applied Physics (APH) at KIT. The tag they have created is genetically encodable, which means it can be incorporated into the gene of whatever RNA the cell produces. RhoBAST itself is non-fluorescent, but it illuminates the cell-permeable rhodamine dye by binding to them in a very specific way. “This leads to a significant increase in the fluorescence achieved by the RhoBAST dye complex, which is a prerequisite for excellent fluorescence images,” explains Dr. Murad Senbol of the IPMB, adding: “However, for ultra-fine RNA imaging, it needs additional properties.”

Researchers discovered that each molecule of rhodamine remained bound to RhoBAST for about one second before separating again. Within seconds, this procedure repeats itself with a new dye molecule. Prof. Dr. Gerd Ulrich Nienhaus of APH says: “It is very rare to find strong interactions – as is the case between RhoBAST and rhodamine – along with exceptionally fast exchange kinetics.” Because rhodamine only lights up after binding to RhoBAST, the steady chain of newly emerging interactions between the marker and the dye leads to a continuous “flash”. “This” on-off switch “is exactly what we need to photograph the SMLM, says Professor Nienhouse.

At the same time, the RhoBAST system solves another important problem. The fluorescence images are collected under the radiation of laser light, which destroys the dye particles over time. The rapid exchange of the dye ensures that the light-bleached pigments are replaced by new ones. This means that individual RNA molecules can be observed for longer periods of time, which may improve the resolution of the image dramatically, explains Prof. Dr. Andres Jaishki, a scientist at the IPMB.

Researchers from Heidelberg and Karlsruhe were able to demonstrate the remarkable properties of RhoBAST as an RNA marker by visualizing RNA structures within gut bacteria (Escherichia coli) and cultured human cells with excellent localization accuracy. “We can reveal details of previously unseen subcellular structures and molecular interactions involving RNA using ultra-fine fluorescence microscopy. This will allow a fundamental new understanding of biological processes,” says Professor Jaysky.

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The research conducted by Murat Sunbul and Andres Jaeschke in the context of the study was supported by the German Research Foundation (DFG) and the work by Gerd Ulrich Nienhaus was supported by the DFG and the Helmholtz Association. The results have been published in the journal Nature Biotechnology.

https: //www.Yoni Heidelberg.From/at/Newsroom /Ultra-accurate RNA imaging in live cells

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