Figure 2. Optimal assay conditions for detecting unlinkase activity using full-length poliovirus virion RNA ³⁵S-methionine labeled substrate.

A protein chromatography fraction enriched for unlinkase activity was incubated with full-length ³⁵S-methionine radiolabeled W1-VPg 31 virion RNA substrate with either (A) increasing amounts of protein (0.01, 0.02, 0.04, 0.1, 0.2, 0.4, and 0.6 µg/µl) from an enriched source of unlinkase activity (Fraction SA) at 30°C for 30 minutes or (B) increasing incubation time (0, 1, 2, 5, 10, 15, 20, and 30 minutes) with 0.4 µg/µl of protein from a partially-purified fraction of unlinkase activity (Fraction SA) to determine optimal assay conditions for the full-length substrate. (C) ³⁵S-methionine radiolabeled W1-VPg 31 virion RNA substrate was mock-incubated (lane 1), incubated with 0.8 µg/µl RSW (lane 2), 0.4 µg/µl of protein from a partially-purified fraction of unlinkase activity (Fraction SA) (lane 3), or one unit of RNase A (lane 4) to differentiate between non-specific nuclease activity and authentic unlinkase activity.