第十八期科研小助手supplementary data.pdfVIP

Supplementary Figure 1 NgAgo binds ssDNA guide in a one-guide-faithful manner. NgAgo-expressing plasmid was transfected to 293T cells and then NgAgo was purified and used in an in vitro plasmid cleavage assay. The NgAgo derived from the cells co-transfected with target-complementary guide (FW, Lane 4) but not that derived from the cells co- transfected with a random guide (NC, Lane 5) could cause DSBs and linearize the plasmid. The NgAgo derived from the cells without guide c ivery could not cleave the target even if the purified NgAgo was later co-incubated with the FW guide (Lane 3). After NgAgo binds to an ssDNA, it will not swap its bound guide (here is NC) to another guide (here is FW) at 37℃ (Lane 6). Representative figure of 3 independent experiments. Nature Biotechnology: doi:10.1038/nbt.3547 Supplementary Figure 2 Guide reloading process at 55℃impairs endonuclease activity of NgAgo. NgAgo was purified from E.Coli. and then co-incubated with 5’ phosphorylated ssDNA guide at 37 ℃ or 55℃ for 1 hour or 72 hours. NgAgo was then subject to a plasmid cleavage assay to test i donuclease activity. It shows that 55℃ for 1 hour changes the activity of NgAgo to a ase and 55℃ for 72 hours completely deprived of it ivity. SC, supercoiled; Lin, linearized; OC, open circular. Representative figure of 3 independent experiments. Nature Biotechnology: doi:10.1038/nbt.3547 Supplementary Figure 3 NgAgo has no activity to cleave linearized target or single strand target. (a) pACYCDeut-eGFP plasmid was first linearized by BamHI restriction cleavage and then co-incubated with or without the purified NgAgo preloaded with FW guide in 293T cells. NgAgo was not found to further cleave the target sequence. (b) An 86nt ssDNA co-