Figure 6.

Small DNAs stabilize KmAgo. (A) KmAgo preloaded with gDNA tolerates higher treated temperature. KmAgo preloaded with gDNA, KmAgo and gDNA were incubated at 37°C for 10 min before treated at indicated temperature for 30 min, and then incubated at 37°C for 10 min; KmAgo not preloaded with gDNA, KmAgo was incubated at 37°C for 10 min before treated at indicated temperature for 30 min, and then incubated with gDNA at 37°C for 10 min. (B) Effects of gDNA loaded temperatures on KmAgo activity. All cleavage experiments in (A) and (B) were performed at the 4:5:1 KmAgo:guide:target molar ratio in reaction buffer containing Mn²⁺ ions at 37°C for 15 min. (C) Schematic of the sequences of the 5′ phosphorylated DNA guides. (D) The effects of KmAgo–gDNA duplex loaded temperature on plasmid cleavage activity. Plasmid cleavage assay performed by loading KmAgo with the indicated 5′P-gDNAs at 37°C or 55°C for 30 min, followed by incubation with the target plasmid at 37°C for 2 h, and analysis of the target plasmid by electrophoresis. gDNA loaded temperature, the temperature of KmAgo–gDNA duplex formation stage. M, molecular weight marker; Lin, linearized plasmid; SC, supercoiled plasmid; OC, open circular plasmid. See also Supplementary Figure S8A.