The single-stranded RNA genome of SARS-CoV-2 is highly structured. Numerous helical stem-loop structures interrupted by mismatch motifs are present in the functionally important 5'- and 3'-UTRs. These mismatches modulate local helical geometries and feature unusual arrays of hydrogen bonding donor and acceptor groups. However, their conformational and dynamical properties cannot be directly inferred from chemical probing and are difficult to predict theoretically. A mismatch motif (SL1-motif) consisting of three consecutive U•U base pairs is located in stem-loop 1 of the 3'-UTR. We combined NMR-spectroscopy and MD-simulations to investigate its structure and dynamics. All three U•U base pairs feature two direct hydrogen bonds and are as stable as Watson-Crick A:U base pairs. Plasmodium falciparum 25S rRNA contains a triple U•U mismatch motif (Pf-motif) differing from SL1-motif only with respect to the orientation of the two closing base pairs. Interestingly, while the geometry of the outer two U•U mismatches was identical in both motifs the preferred orientation of the central U•U mismatch was different. MD simulations and potassium ion titrations revealed that the potassium ion-binding mode to the major groove is connected to the different preferred geometries of the central base pair in the two motifs.

Center for Biomolecular Magnetic Resonance Goethe University Frankfurt Max von Laue Str 9 60438 Frankfurt Germany

Institute of Biophysics of the Czech Academy of Sciences Kralovopolska 135 612 00 Brno Czech Republic

Institute of Molecular Biosciences Goethe University Frankfurt Max von Laue Str 9 60438 Frankfurt Germany

Institute of Organic Chemistry and Chemical Biology Max von Laue Str 7 60438 Frankfurt Germany

Institute of Pharmaceutical Chemistry University of Marburg Marbacher Weg 6 35037 Marburg Germany

Volkswagen AG Brieffach 1617 0 38436 Wolfsburg Germany

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