A. The translocation assay was performed on 5′-triphosphate containing substrate which consists of 86mer ssRNA from in vitro transcription annealed to complementary 20mer ssDNA with 3′ Cy3 and 5′ biotin.
B, D. Activity of wtRIG was greatly stimulated by the presence of 5′-triphosphate. The data shown was taken at room temperature due to extremely fast kinetic rate observed at 37°C. RIGh also showed robust translocation activity on this substrate. C, E. Dwell time analysis indicates that wtRIG showed slightly higher rate of translocation than RIGh.