RNA viruses possess strictly conserved RNA segments that adopt local structures and act as regulatory cis-acting elements. In addition, long-range RNA-RNA interactions between distant parts of their genome add additional layers of regulatory control. In SARS-CoV-2, crosslinking experiments identified multiple transiently formed long-range interactions within its genome. The longest distance interaction occurs between nucleotides in the 5’- and the 3’-terminal, untranslated regions (UTR’s). Their interaction requires opening of the 5’SL3 element that contains the transcription regulatory core sequence (TRS-L) in the 5’-UTR and of the 3’SL3base element in the 3’-UTR. In this study, we investigate the minimal sequence context necessary for formation of this long-range interaction. We determine alternative secondary structures formed by these elements, their thermodynamic stability and the stability of individual base-pairs. Further, we quantify populations and kinetic parameters in a three-strand equilibrium between the circularized form and the alternative structures formed within the UTR’s. We demonstrate that the stability of circularization is significantly reduced in subgenomic RNAs, pointing at a role of circularization in differentiating between genomic and subgenomic RNA during the viral life cycle.

raw NMR and MST data and a script to fit EXSY data from the manuscript and the Supporting Information