Skip to main content
[Preprint]. 2022 Aug 11:2022.08.11.503614. [Version 1] doi: 10.1101/2022.08.11.503614

Figure 9. Viperin product (ddhCMP)-terminated RNA is a good substrate for ExoN but 3’-dCMP-terminated RNA is not.

Figure 9.

(A) Structure of nucleotide analogs used. (B) Schematic of the assay. Primer extension is initiated by adding an RNA polymerase in the presence of UTP and a CTP analog. Incorporation produces n+1 and n+2 products. The last nucleotide to be incorporated is the CTP analog. Once 50–75% of the primers were extended to n+2, 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) Analysis of reaction products by denaturing PAGE. Only the 3’-dCMP-terminated RNA exhibited a delay in excision. (D) Kinetics of excision of CMP analogs. The quantitation revealed that only 3’-dCMP-terminated RNA exhibited a significant delay in the rate of excision relative to the CMP control, even though ddhCMP also lacks a 3’-OH. Data were fit to a single exponential. Rates are provided in Table 4.