Fig. 5: Nsp15 prefers to cleave unpaired U in 1 nt bulge in stem loop RNA.
From: Spontaneous base flipping helps drive Nsp15’s preferences in double stranded RNA substrates
A Design of substrates with sequence from the SARS-CoV-2 genome’s Stem Loop 4 (SL4) used for 19F NMR. Key positions are marked where U was substituted for 2′-F-U to enable 19F NMR. Nts are labeled according to position in the positive strand viral genomic RNA. B Overlay of top ten structures of SL4 determined by Vögele et al.52, with key Us color coded (PDBID 8CQ1). Note that U95 is consistently flipped out, while U104 and other Us are consistently stacked. C 1D 19F NMR spectra of five different SL4 derivatives, each with a single 2′-F-U at the indicated position. Each peak is labeled with its shift (top value) and linewidth (bottom value, in parentheses). D Design of SL4 substrate used for enzyme assay, with 5′ Cy5 and 3′ A4-Fl. E Representative denaturing PAGE gel of time-course nuclease assays, with Cy5 (red) and Fl (blue) channels separated to facilitate band identification. Three independent reactions (each using a distinct protein preparation) were performed with each substrate. Images of each gel are provided in the Source Data file. F Mass spectrum showing multiply charged ions representing the major product of cleavage of SL4 by Nsp15 (at U95), corresponding to Cy5-C84:U95.
