01/09/2025
New chemistry for the engineering of covalent RNA complexes
Researchers from the University of Innsbruck have developed a new method for covalently labeling RNA in the cell. In the journal Nature Chemical Biology, they show how it can be used to map RNA movements in the cell.
The specific labeling of RNA in living cells poses many challenges. In the journal Nature Chemical Biology, researchers from the University of Innsbruck describe a structure-guided approach to the formation of covalent (i.e. irreversibly tethered) RNA-ligand complexes.
The key to this is the modification of the original ligand with a reactive "handle" that allows it to react with a nucleobase at the RNA binding site. This was first demonstrated in vitro and in vivo using the example of an RNA riboswitch. The versatility of the approach is highlighted by the first covalent "fluorescent light-up RNA aptamer" (coFLAP).
This system retains its strong fluorescence during imaging in living cells even after washing, can be used for high-resolution microscopy and is particularly suitable for FRAP (fluorescence recovery after photobleaching) for monitoring intracellular RNA dynamics. "In addition, we were able to develop a fluorophore with a second handle for bioorthogonal chemistry, which enables a simple pull-down of the covalently bound target RNA (traceable by fluorescence spectroscopy)," explains chemist Ronald Micura from the Department of Organic Chemistry and the Center for Molecular Biosciences Innsbruck (CMBI) at the University of Innsbruck. Finally, we have shown the suitability of this strategy for the development of potential drug lead structures that bind covalently to an RNA target.
Joint success
One key to the success of the study lies in the willingness to overcome seemingly insurmountable experimental hurdles with complementary methods. A total of four working groups from the Innsbruck universities, which are jointly based at the Center for Chemistry and Biomedicine, have made a decisive contribution to bringing together the chemical synthesis of the fluorophores and the RNA (team led by Ronald Micura), their mass spectrometric sequencing (team led by Kathrin Breuker), the crucial in vivo applications (team led by Alexandra Lusser) and finally the necessary high-resolution microscopy.
"We benefit greatly from working together in a joint research center, where we can exchange ideas between the working groups regardless of institutional affiliation and quickly try out new approaches," says Alexandra Lusser from the Institute of Molecular Biology am Biocenter at the Medical Universityt Innsbruck.
Source: University of Innsbruck