Figure 6. ExoN is inhibited by the Rp diastereomer at the phosphorothioate-substituted scissile bond.

(A) The Sp diastereomer of a nucleoside-5’-O-thiotriphosphate is preferentially incorporated by RNA polymerases. Nucleophilic attack at the alpha phosphorous atom leads to inversion of configuration and creation of Rp diastereomer at the phosphorothioate bond. (B) Schematic of assay. Primer extension is initiated by adding an RNA polymerase in the presence of either UTP and CTPαS or ATPαS. CTPαS is a mixture of both the Rp and Sp diastereomers. ATPαS was obtained as the pure Sp diastereomer. Incorporation yields extended products. The last nucleotide to be incorporated is either CMP(αS) or AMP(αS). Once 50–75% of the primers were extended to the end, ExoN was added to the reaction. The reaction was monitored over time for hydrolysis. Unextended primer in reactions served as a useful control to demonstrate the presence of active ExoN in the reaction. (C, D) Analysis of reaction products by denaturing PAGE. Incorporation of CMP and AMP results in removal of the incorporated nucleotide by ExoN. Incorporation of CMPαS and AMPαS (Rp diastereomers) results in a terminated primer that cannot be cleaved by ExoN. (E,F) Kinetics of excision of CMP, CMPαS (Rp diastereomer) and AMP and AMPαS (Rp diastereomer) by ExoN. Data were fit to a single exponential. Rates are provided in Table 2.