Extended Data Fig. 6: EMSA and nucleic acid cleavage experiments.
a, b, Binding of single- and double-stranded oligonucleotides by wt GsSir2/Ago and wt CcSir2/Ago, respectively. A radiolabelled strand indicated by the asterisk. c, Binding of complementary DNA targets by GsSir2/Ago binary complexes pre-loaded with RNA guide containing 5’-phosphate terminus in the presence or absence of heparin. To show that no displacement of the radiolabelled guide by the target strand is observed, a control (Cg*) equivalent to the experimental lane marked by a black triangle, but with the guide, rather than the target, bearing the radioactive label, was performed. d, Control EMSA experiments of ssRNA guide binding by wt GsSir2/Ago (left) and non-complementary DNA target binding by the binary wt GsSir2/Ago-gRNA complex in the presence and absence of heparin. e, Representative binding fit curves of several independent replicates used to calculate Kd of ssRNA guide binding by wt GsSir2/Ago (left) and of target DNA binding by wt GsSir2/Ago-gRNA complex (right). f, (No) cleavage activity of various DNA and RNA oligonucleotides by wt GsSir2/Ago and wt CcSir2/Ago. Reaction products were resolved on a 21% denaturing polyacrylamide gel. In heteroduplexes, the asterisk indicates the radiolabelled strand. For panels A-D and F, at least three independent replicates were performed for each experiment.
