Fig. 3

Effect of a range of reaction conditions on RNA-guided DNA cleavage activity of PsPIWI-RE. A Temperature dependence of the DNA target cleavage reaction with a 5′ phosphorylated (5′P) RNA guide. “–”: without PsPIWI-RE. B Quantitative analysis of the cleavage efficiency of PsPIWI-RE at different temperatures. Histogram were plotted from triplicate data. Mean values ± SD are shown, n = 3. C Dependence of the efficiency of DNA cleavage on the length of guide RNA. D DNA cleavage assays of PsPIWI-RE and mutants. PsPIWI-RE was loaded with a 21 nt guide RNA and was incubated with a 78 nt single stranded DNA (ssDNA) target in a 25:5:1 molar ratio (protein:guide:target). The target cleavage took place at 37 °C for 4 h with 10 mM Mg²⁺. Products were resolved on a 16% denaturing polyacrylamide gel. E–H Effect of the variation of the 5′ end nucleoside of the guide RNA on PsPIWI-RE cleavage efficiency. 0–2 μM of 21 nt RNA guides containing a different 5′ end nucleoside were incubated with PsPIWI-RE, followed by 78 nt target DNA. The 5′ nucleoside modifications were as follows: 5′ phosphorylated (5′P) DNA, 5′ hydroxyl (5′OH) DNA, 5′ phosphorylated (5′P) RNA, and 5′ hydroxyl (5′OH) RNA. Products of the reactions were resolved on denaturing polyacrylamide gels