a, b, The nucleic acid binding canyon changes width on proceeding from the binary (a) to the ternary (b) complex. The Ago protein is shown in a space-filling representation with labelled domains and linkers colour-coded as in Fig. 1. The guide DNA (red) and target RNA (blue) are shown in stick representation with backbone phosphorus atoms in yellow. c, View of alignment of binary (cyan) and ternary (magenta) Ago complexes, after superpositioning of their PIWI-containing modules. The red arrows indicate the magnitude of the conformational changes on proceeding from binary to ternary complexes.

a, Sequence of the guide DNA-target RNA duplex. The traceable segments of the bases of the guide DNA and target RNA in the structure of the ternary complex are shown in red and blue, respectively. Disordered segments of the bases on both strands that cannot be traced are shown in grey. b, Stereo view of the 3.0 Å crystal structure of the Ago ternary complex. The Ago protein is colour-coded by domains (N in cyan, PAZ in magenta, Mid in orange and PIWI in green) and linkers (L1 and L2 in grey). The bound 21-nucleotide guide DNA is in red and traced for bases of the 1–10 and 19–21 segments, whereas the bound 20-nucleotide target RNA is in blue and traced for bases of the 1′ to 9′ segment. Backbone phosphorus atoms are in yellow. c, An alternate view of the complex.

a, Expanded view of the ternary complex highlighting the guide DNA (1–10)-target RNA (1′–9′) duplex and Mg²⁺-coordinated catalytic residues (D478, D546 and D660) of the RNase H fold of the PIWI domain. Intermolecular hydrogen bonds between the Ago protein and the DNA guide strand in red are shown by dashed lines. b, Positioning of the sugar-phosphate backbone of the target RNA strand spanning the mismatch-containing 10–11-step relative to the catalytic residues of the PIWI domain. c, Comparison of the trajectory of traceable bound guide DNA in the binary (bases 1–11 and 18–21 in silver) and ternary (bases 1–10 and 19–21 in red) Ago complexes after superposition of their 5′-phosphate-binding pockets. d, Superposition of the guide DNA (red)-target RNA (blue) duplex spanning the 2–8 seed segment on A-form (left panel) and B-form (right panel) helices (silver) after best-fit superposition of the target RNA strand of the ternary Ago complex with one strand of the A/B-form helices. e, Positioning of stacked residues 6–10 of the DNA guide strand relative to R548, with emphasis on intermolecular interactions involving the sugar-phosphate backbone. f, Relative positioning of the 6 to 10/11 segment of the bound guide DNA strand and R548 in the binary (guide strand in silver, protein in cyan) and ternary (guide strand in red, protein in magenta) Ago complexes. The conformational change in the protein on proceeding from binary to ternary Ago complexes is indicated by a red arrow.

Besides Ago-mediated target RNA cleavage, extensive chemical hydrolysis is observed over the entire RNA substrate length, except for a 12-nucleotide (nt) region across guide DNA positions 2 to 13, which was protected from hydrolysis by the interaction with Ago and its guide DNA. Mismatches or bulges in the 5′ region reduce protection, and frequently catalysis, because of reduced base-pairing stability and deviation from hydrolysis-protecting helical geometry. The black bar to the left of the images defines the region of the cleavage substrate complementary with the 21-nucleotide (nt) guide DNA (a–d) or the region complementary with the 7-nucleotide guide DNA with the dotted line indicating pairing for 3′-extended guide DNAs (e). Arrows indicate the cleavage site. Del, deletion; H, hydrolysis ladder of substrate RNA; T1, partial RNase T1 digest of substrate RNA. a, Single mismatches were placed in the 21-nucleotide guide, at positions 1 to 19. b, Double, quadruple, sextuple and octuple mismatches were introduced in the DNA guide 3′ region at indicated positions. c, Cleavage assay using DNA guides with a deletion or insertions of bulged nucleotides at indicated positions. d, Insertion of bulges in the target RNA. Insertion of nucleotides upstream of the cleavage site leads to shifts in the cleavage site by the number of inserted nucleotides. e, DNA guide length was increased from 7 to 36 nucleotides. The sequences of DNA guides and RNA targets are listed in Supplementary Table 2. As evident from increased hydrolysis of the target region recognized by the guide, insertion of bulges seems to destabilize guide-target interactions.