ArticlePublished: 24 June 2026Sucharita Sarkar ORCID: orcid.org/0000-0002-0996-16471 na1,Luca F. R. Gebert ORCID: orcid.org/0000-0002-1830-119X1 na1 &Ian J. MacRae ORCID: orcid.org/0000-0002-5112-02941 Nature Structural & Molecular Biology (2026) Cite this articleSave articleView saved researchSubjectsCryoelectron microscopyRNAisiRNAsAbstractSmall interfering RNAs (siRNAs) are an expanding class of RNA therapeutics, with seven drugs approved by the US Food and Drug Administration and many more in development. Rational design, however, has been limited by incomplete understanding of how human Argonaute 2 (hAgo2) catalyzes target cleavage. Here we report high-resolution cryo-electron microscopy structures of hAgo2 bound to a target RNA in catalytic and noncatalytic conformations. The structures reveal that guide–target pairing alone is insufficient for slicing and catalysis requires deformation of the duplex through a coordinated network of RNA–protein interactions. Expansion of the central major groove positions the scissile phosphate, while compression toward the supplementary region docks the duplex into the hAgo2 cleft. A kink after guide nucleotide g6 disrupts seed-only pairing conformation and promotes the extended pairing required for catalysis. This rearrangement enables repositioning of K709 within the active site, while a pyrimidine at target position t10 optimally aligns R710 to accelerate cleavage. These findings provide a structural framework linking siRNA duplex geometry to catalytic efficiency and inform rational design of siRNAs with improved potency and specificity.This is a preview of subscription content, access via your institutionAccess optionsAccess Nature and 54 other Nature Portfolio journalsGet Nature+, our best-value online-access subscription27,99 € / 30 dayscancel any timeLearn moreSubscribe to this journalReceive 12 print issues and online access269,00 € per yearonly 22,42 € per issueLearn moreBuy this articlePurchase on SpringerLinkInstant access to the full article PDF.39,95 €Prices may be subject to local taxes which are calculated during checkoutFig. 1: Guide 3′-end release favors the catalytic conformation.Fig. 2: Human Ago2 adopts catalytic and noncatalytic conformations after guide–target pairing.Fig. 3: Target RNA docking requires distortion of the guide–target duplex.Fig. 4: The g6 kink senses target pairing and licenses K709 to active cleavage.Fig. 5: Target nucleotide t10 influences cleavage rates via R710 conformation.Fig. 6: The hAgo2 catalytic conformation is not perturbed by PAZ domain mutations.Data availabilityThe cryo-EM maps and corresponding atomic model coordinates were deposited to the EM Data Bank and Protein Data Bank, respectively, as follows: hAgo2-R315V;H316A–guide–target (EMD-46888, PDB 9DHX), hAgo2-R315V;H316A–guide–target (conformation 2) (EMD-75679, PDB 11GK), hAgo2-R315V;H316A–guide 10U–target 10A (EMD-75678, PDB 11GJ) and hAgo2 WT–guide(3′-amino)–target (EMD-75677, PDB 11GI). 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Web 3DNA 2.0 for the analysis, visualization, and modeling of 3D nucleic acid structures. Nucleic Acids Res. 47, W26–W34 (2019).Article CAS PubMed PubMed Central Google Scholar Download referencesFundingI.J.M. discloses support for the research of this work from the National Institute of General Medical Sciences (grant number R35GM127090) and Eli Lilly and Co.Author informationAuthor notesThese authors contributed equally: Sucharita Sarkar, Luca F. R. Gebert.Authors and AffiliationsDepartment of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA, USASucharita Sarkar, Luca F. R. Gebert & Ian J. MacRaeAuthorsSucharita SarkarView author publicationsSearch author on:PubMed Google ScholarLuca F. R. GebertView author publicationsSearch author on:PubMed Google ScholarIan J. MacRaeView author publicationsSearch author on:PubMed Google ScholarContributionsS.S. and L.F.R.G. prepared hAgo2 and RNA samples and collected cryo-EM data. S.S. processed cryo-EM data and built the hAgo2 RNA model with input and guidance from L.F.R.G. L.F.R.G. produced the hAgo2 mutants and conducted target cleavage experiments. I.J.M. and L.F.R.G. conceptualized the study and made major decisions regarding project direction. I.J.M. took the lead in writing the manuscript with input and help from S.S. and L.F.R.G.Corresponding authorsCorrespondence to Luca F. R. Gebert or Ian J. MacRae.Ethics declarationsCompeting interestsI.J.M. is a scientific advisor to City Therapeutics. The other authors declare no competing interests.Peer reviewPeer review informationNature Structural & Molecular Biology thanks Francois Major and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling Editor: Sara Osman, in collaboration with the Nature Structural & Molecular Biology team.Additional informationPublisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Extended dataExtended Data Fig. 1 Reconstructions of guide 3′-end binding pocket mutant hAgo2 proteins engaged with guide and fully complementary target RNA.Both the (A) F294A and (B) R315V;H316A hAgo2 mutants produced reconstructions with density for an intact guide–target duplex in the central cleft. Data for the hAgo2 R315V/H316A mutant were of higher quality and thus used for high-resolution reconstruction and analysis.Extended Data Fig. 2 Cryo-EM data processing of the hAgo2 F294A–guide–target complex.The workflow summarizes the number of movies collected, a representative micrograph, representative 2D classes, 3D classes, and the step-by-step data-processing pipeline. The 3D classes corresponding to the catalytic conformation are shown in cyan, the 3D class lacking visible RNA duplex density is shown in purple, and all other classes that were not included in the final 3D reconstruction are shown in gray.Extended Data Fig. 3 Kinetic target cleavage data in this study.a Cleavage of target RNA over time by Ago2 wild type, F294A mutant, and R315V/H316A mutant under single turnover conditions at 37 °C(left) and multiple turnover conditions at 37 °C (right). b Cleavage of target RNA over time by Ago2 wild type and R710A with a g10A or g10U target at 37 °C under multiple turnover conditions (left); detail view of the R710A curves (right). All points represent the mean value from 3 experiments. Error bars, when visible, indicate SEM. The data were fit to a simple one phase decay equation.Source dataExtended Data Fig. 4 Cryo-EM data processing of the hAgo2 R315V/H316A–guide–target complex.The workflow summarizes the number of movies collected, a representative micrograph, representative 2D classes, 3D classes, and the step-by-step data-processing pipeline. The 3D classes corresponding to the catalytic conformation are shown in blue, non-catalytic conformations are shown in orange and brown, 3D classes of hAgo2 lacking visible RNA duplex density are shown in green, and all other classes that were not included in the final 3D reconstruction are shown in gray. Transparent volume represents the mask used for local refinement.Extended Data Fig. 5 Map quality of hAgo2 R315V;H316A–guide–target complex.(a) RNA density quality was often high enough to unambiguously visualize nucleobase identity. Map quality in the central region (right), which contains the scissile phosphate and an extended major groove, is shown. (b) Density for individual domains of hAgo2 and RNAs fit into the cryo-EM map. The map is shown as a mesh; protein models are shown in cartoon representation, with side chains shown as sticks; RNAs are shown in stick representation.Extended Data Fig. 6 Helical distortion data.(a) Plotting the delta (δ) torsion angle, which describes sugar pucker, for all nucleotides in the guide-target duplex shows that only g6 adopts the C2’ endo conformation. (b) Hydrophobic interactions between Ile-365 and the sugar face at positions 6-7 connect the guide-target duplex to helix-7. (c) Plotting the Chi (χ) torsion angle, which describes the relationship between the ribose and nucleobase, for guide and target nucleotides shows local distortions in the guide strand at the g6-kink and the target strand at positions 16-17, associated with compression of the major groove. (d) Plotting helical rise between stacked bases reveals local duplex compressions at the g6-kink and position 16.Source dataExtended Data Fig. 7 Cryo-EM data processing of the hAgo2 R315V/H316A–guide10U–target10A complex.The workflow summarizes the number of movies collected, a representative micrograph, representative 2D classes, 3D classes, and the step-by-step data-processing pipeline. The 3D classes corresponding to the catalytic conformation are shown in blue, non-catalytic conformations are shown in orange and brown, 3D classes of hAgo2 lacking visible RNA duplex density are shown in green, and all other classes that were not included in the final 3D reconstruction are shown in gray. Transparent volumes covering the protein region represent the masks used for local refinement.Extended Data Fig. 8 Cryo-EM data processing of the hAgo2 WT–guide(3′-amino)–target complex.The workflow summarizes the number of movies collected, a representative micrograph, representative 2D classes, 3D classes, and the step-by-step data-processing pipeline. The 3D classes corresponding to the catalytic conformation are shown in blue, non-catalytic conformations are shown in orange and brown, 3D class with of hAgo2 lacking visible RNA duplex density is shown in purple and green, and all other classes that were not included in the final 3D reconstruction are shown in gray. Transparent volumes represent the masks used for local refinement.Extended Data Fig. 9 Comparison of TtAgo Catalytic tetrad with human Ago2 structures.Superposition of the TtAgo catalytic tetrad (gray, PDB: 4NCB) with the human Ago2 structures in this study (A,B) and with previously published inactive structures (PDB: 9CMP, 9K6T, and 9K6S)14,15,18. Alignments are best fits to the Cα atoms in structural elements that create the TtAgo active site (residues 473–483, 507–517, 541–551, and 655–665). Catalytic metal ions in the TtAgo structure are shown as spheres (labeled A and B).Supplementary informationSupplementary Information (download PDF )Supplementary Figs. 1–4 and Data files.Reporting Summary (download PDF )Peer Review File (download PDF )Source dataSource Data Fig. 1 (download XLSX )Kinetics assay data.Source Data Fig. 3 (download XLSX )RNA geometry data.Source Data Fig. 4 (download XLSX )Kinetics assay data.Source Data Fig. 5 (download XLSX )Kinetics assay data.Source Data Fig. 6 (download XLSX )Kinetics assay data.Source Data Extended Data Fig. 3 (download XLSX )Kinetics assay data.Source Data Extended Data Fig. 6 (download XLSX )RNA geometry data.Rights and permissionsSpringer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.Reprints and permissionsAbout this article