Fig. 5.
5′-BrdU-gRNA MpAgo RNP ssRNA substrate binding specificity at the sixth and seventh positions. Filter-binding assays were performed using 5′-BrdU-gRNA MpAgo RNPs programmed with A, G, U, or C gRNA at the sixth (A) or at the seventh (B) positions and ssRNA substrates containing U, C, A, G, or I at the corresponding position. The average Kds were extracted and are represented in the heat maps. (C) A filter-binding assay to isolate the A-to-I edited substrate (black), but not the nonedited substrate (red), from 500 ng total RNA purified from HEK239T cells, in the presence of 200 ng/μL yeast tRNA. For this assay MpAgo RNP programmed with a 5′-BrdU gRNA containing either A at its sixth or C at its seventh position was used. The fraction of the ssRNA substrate bound to MpAgo RNP was quantified and plotted as the mean ± SD from three independent experiments.