Figure 5. RNA hydrolysis by ExoN is inhibited by a phosphorothioate bond.

(A) Structure of phosphorothioate. One of the non-bridging oxygens of the phosphodiester bond is replaced with a sulfur atom, creating Rp and Sp diastereomers. A 50:50 ratio of diastereomers were used in these experiments. (B) Schematic of phosphorothioate-substituted ssRNAs used. (C) Effect of phosphorothioate ExoN-catalyzed hydrolysis of dsRNA. Reactions contained 0.5 μM ExoN (1:1) and 0.1 μM of the indicated RNA, were incubated at the indicated times, then quenched. Product analysis is shown. Fraction of RNA remaining is indicated (% uncleaved). The unmodified RNA is completely degraded; however, ExoN is unable to hydrolyze beyond the phosphorothioate substitution, for half the RNA molecules at least. This observation is consistent with only a single diastereomer being inhibitory.