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. Author manuscript; available in PMC: 2013 Sep 1.
Published in final edited form as: Nat Struct Mol Biol. 2013 Jan 27;20(3):380–386. doi: 10.1038/nsmb.2487

Figure 6.

Figure 6

AfC3PO duplex RNA cleavage assays. (a) Denaturing urea-PAGE analysis of duplex RNA cleavage by increasing concentrations of D114A mutant or wild-type AfC3PO. The duplex was as crystallized (with 5′-labeling). Incubation was at 20°C for 60 min. Protein concentrations were 20 nM, 200 nM and 2 μM (AfTrax monomer concentrations), or no protein (0). Primary and secondary cleavage products are indicated. (b) Timecourse of RNA duplex cleavage (duplex as in panel a) by wild-type AfC3PO (200 nM AfTrax monomer, 30°C incubation). RNA size markers are shown at the right. (c) Cleavage of RNA substrates containing dual phosphorothioate modifications (positions between nucleotides indicated). Incubation was at 30°C for 30 min, with or without AfC3PO as indicated (200 nM AfTrax monomer). (d) Cleavage of RNA substrates containing single phosphorothioate modifications (positions between nucleotides indicated). Incubation was at 30°C for the times indicated (200 nM AfTrax monomer). (e) Schematic showing primary cleavage positions on the assay duplex. The dashed box shows a 13 bp segment as observed in the crystal structure. * indicates 5′ labeling.