Fig. 3: Extracted ion chromatograms and MS spectra of Nsp15 RNA cleavage products.

a Extracted ion chromatograms of the 3′-TAMRA labeled AA-TAMRA RNA cleavage product. 5′-HO-AA-TAMRA is readily observed in the presence of Nsp15 (+Nsp15) as a singly charged ion at m/z 1463.42, but is undetectable in the absence of enzyme (-Nsp15). NL stands for normalized level. b MS spectrum confirms the identity of 5′-HO-AA-TAMRA and does not detect the presence of alternative 5′-cleavage products. c Extracted ion chromatograms of the 5′-fluorescein labeled (FI) FI-AAAU cleavage product. FI-AAAU is only detected in the presence of Nsp15. d MS spectrum confirms the identity of FI-AAAU. The 3′-product is observed primarily as a doubly charged ion at m/z 914.14, which corresponds to a 2′3′-cP terminated moiety. A doubly charged ion at m/z 923.14 is also present and corresponds to a 3′-P terminated species. MSMS spectra of m/z 1463.14 and m/z 914.14 unambiguously confirm the identity of these ions (Fig. S5) e Extracted ion chromatograms of the 5′-FI-AAAU-2′3′-cP cleavage product (top) and the 5′-FI-AAAU-3′-P cleavage product (bottom) set to the same scale to demonstrate that under the conditions employed, the majority of the cleavage product is terminated by a cyclic phosphate. The graphical representation of the areas under the curve of the extracted ion chromatograms shows that approximately 80% of the cleavage product is the cyclic phosphate (assuming similar ionization efficiencies of the two species). f Cartoon schematic of RNA cleavage by the uridine-specific Nsp15 endoribonuclease. Nsp15 catalyzes a transesterification reaction that results in 2′3′-cyclic phosphate (2′3′-cP) and 5′-hydroxyl (5′-OH) RNA ends. Nsp15 can further catalyze a subsequent hydrolysis reaction resulting in the conversion of the 2′3′-cyclic phosphate to a 3′-phosphate (3′-P), however, the rate of hydrolysis is slow. R denotes the 5′ and 3′-ends of the phosphodiester backbone.