Fig. 4: A U’s susceptibility to cleavage by Nsp15 parallels its tendency to spontaneously flip.

A dsRNA substrate design, with observable target U* (in black). For cleavage assays, U* = 2′-OH, non-target Us in target strand = 2′-F-U. Target strand has 5′ Cy5 and 3′ Fl labels, as in Fig. 1. For NMR, U* = 2′-F-U, all other Us = 2′-OH. From this design, a set of substrates were created by changing 2 nt on both 5′ and 3′ sides of U* (gray, marked with?) to different sequences, shown in (B). For each sequence, U* was either unpaired or engaged in a U•A pair (orange). C Percent of target strand cleaved over time, quantified via intensity of the uncleaved RNA band and normalized to the 2 min timepoint. Each point with error bars represents average and standard deviation for at least three independent reactions, each using a distinct protein preparation (N = 3 biological replicates). Images of each gel and quantification data are provided in the Source Data file. Data for substrate 1 (“AAUUU”) in panel C is reproduced from Fig. 1C for clarity and ease of comparison. D 1D ¹⁹F NMR spectra for each dsRNA substrate in the absence of Nsp15. Each peak is labeled with its shift (ppm, top value) and linewidth (Hz, bottom value, in parentheses). E Scatter plot of shift (peak position) from 1D ¹⁹F NMR spectra vs. % cleaved at 60 min from our enzymatic assays. For each point, x position represents the average of three independent reactions for a single substrate (error bars represent standard deviation), y position represents one spectrum.