Fig. 2: HrAgo1 mediates RNA-guided RNA cleavage.
From: RNA-guided RNA silencing by an Asgard archaeal Argonaute
a HrAgo1 associates with 5’ phosphorylated (5’ P) small RNAs in vivo from E. coli. Nucleic acids that co-purified with HrAgo1 were [γ-32P] labeled, treated with RNase A or DNase I, and resolved on a denaturing gel (15% polyacrylamide 7 M urea). nt: nucleotides. b Length distribution of small RNAs associated with HrAgo1 as determined by small RNA sequencing. c Small RNAs associated with HrAgo1 have a bias for uracil bases at the 5’ end. d Sequences of guide and target oligonucleotides used in in vitro cleavage assays. e HrAgo1 cleaves ssRNA (but not ssDNA) targets with ssRNA guides, and ssDNA guides at lower efficiency, in the presence of Mg2+. HrAgo1 was incubated with ssDNA or ssRNA guides and Cy5-labeled ssDNA or RNA targets. Cy5-labeled cleavage products were resolved through denaturing (7 M urea) polyacrylamide gel electrophoresis and visualized by fluorescence imaging. Both ssRNA and ssDNA targets are 45nt. The HrAgo1-bound RNA extraction and digestion was carried out once; the results of the cleavage assays were confirmed by at least three repetitions.
