Pseudouridine is the most frequently naturally occurring RNA modification, found in all classes of biologically functional RNAs. Compared to uridine, pseudouridine contains an additional hydrogen bond donor group and is therefore widely regarded as a structure stabilizing modification. However, the effects of pseudouridine modifications on the structure and dynamics of RNAs have so far only been investigated in a limited number of different structural contexts. Here, we introduced pseudouridine modifications into the U-turn motif and the adjacent U:U closing base pair of the neomycin-sensing riboswitch (NSR)-an extensively characterized model system for RNA structure, ligand binding, and dynamics. We show that the effects of replacing specific uridines with pseudouridines on RNA dynamics crucially depend on the exact location of the replacement site and can range from destabilizing to locally or even globally stabilizing. By using a combination of NMR spectroscopy, MD simulations and QM calculations, we rationalize the observed effects on a structural and dynamical level. Our results will help to better understand and predict the consequences of pseudouridine modifications on the structure and function of biologically important RNAs.

CEITEC Central European Institute of Technology Masaryk University 625 00 Brno Czech Republic

Institute of Biophysics of the Czech Academy of Sciences 612 65 Brno Czech Republic

Institute of Molecular Biosciences and Center for Biomolecular Magnetic Resonance Goethe University Frankfurt 60438 Frankfurt Germany

National Centre for Biomolecular Research Faculty of Science Masaryk University 625 00 Brno Czech Republic

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