FIGURE 1.

Conditions to examine nascent RNA synthesis using the RCAP assay. (A) Schematic of the reversible cross-linking affinity purification (RCAP) strategy used to identify nascent RNA contact sites on the HCV NS5B protein. (B) Sequences of the nascent RNA mimic (5P) and template (12T) used in this study. The alkyn modification is indicated by the Ξ symbol on the 5′ end of 5P. (C) Autoradiogram of a urea PAGE to illustrate walking NS5B down the template. (☆) Radiolabeled NTPs; (+) presence of cold NTPs. In the presence of only CTP or ATP, no RNA synthesis is observed. In the presence of UTP and of both ATP and UTP, synthesis of a 7-nt and 10-nt product is observed, respectively. In the presence of ATP, UTP, and CTP, full-length 12-nt product formation is detected. The product lengths were assigned by electrophoresis of chemically synthesized RNAs of known lengths in the same gel. These control RNAs were then visualized by staining with Toluidine Blue (data not shown).