{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,23]],"date-time":"2026-10-23T19:46:49Z","timestamp":1792784809390,"version":"4.5.0"},"reference-count":35,"publisher":"American Chemical Society (ACS)","issue":"36","license":[{"start":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T00:00:00Z","timestamp":1788393600000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-029"},{"start":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T00:00:00Z","timestamp":1788393600000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-037"},{"start":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T00:00:00Z","timestamp":1788393600000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-045"}],"funder":[{"name":"National Institutes of Health (NIH)","award":["GM127090"],"award-info":[{"award-number":["GM127090"]}]},{"name":"National Institutes of Health (NIH)","award":["GM131839"],"award-info":[{"award-number":["GM131839"]}]},{"name":"National Institutes of Health (NIH)","award":["OD036329"],"award-info":[{"award-number":["OD036329"]}]},{"name":"National Institutes of Health (NIH)","award":["OD20012"],"award-info":[{"award-number":["OD20012"]}]},{"name":"University of Oxford subaward of United Kingdom Medical Research Council","id":[{"id":"https:\/\/ror.org\/052gg0110","id-type":"ROR","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,9,16]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>To effectuate RNA interference (RNAi), Argonaute (AGO) proteins recognize and load RNA guide strands by their requisite 5\u2032-phosphate (5\u2032-P). However, the structural features that govern AGO2 tolerance for chemically modified 5\u2032-P architectures remain poorly defined, despite their importance for small interfering RNA (siRNA) drug design. Moreover, degradative enzymes also recognize 5\u2032-P, yet few stereoelectronic features are known to differentiate AGO2 recognition from competing degradation pathways. Here, we show that AGO2 accommodates a substantially broader range of 5\u2032-P derivatives than previously recognized, and that select 5\u2032-P mimics can exploit novel interactions with the protein to reinforce guide anchoring. Through systematic diversification of 35 organic substituents, we interrogated structural determinants of guide anchoring and enzymatic susceptibility. Our analysis identified previously uncharacterized 5\u2032-P mimics, including methyl and phenylpropargyl 5\u2032-phosphates (5\u2032-PO-Me, 5\u2032-PO-PhPrp), phenylpropargyl 5\u2032-phosphorothioate (5\u2032-PS-PhPrp), and 5\u2032-mesylphosphoramidate (5\u2032-MsPA), that preserve efficient AGO2 loading in cells while inhibiting phosphatase-mediated degradation. We further found that 5\u2032-exonuclease susceptibility depends strongly on canonical 5\u2032 phosphate chemistry rather than steric accessibility. Structural analysis of a 5\u2032-PO-PhPrp guide:AGO2 complex shows that the phenylpropargyl substituent engages a \u03c0-stacking network within a cluster of aromatic side chains (Y529\/F811\/Y815), while extended 5\u2032-P substituents are accommodated through rotation of the 5\u2032-P and coordinated rearrangement of water molecules within the 5\u2032-P binding pocket. Collectively, these findings reveal the structural basis for plasticity of guide anchoring in AGO2 and establish chemically programmable 5\u2032-end interactions as a general strategy for modulating oligonucleotide stability and activity.<\/jats:p>","DOI":"10.1021\/jacs.6c03647","type":"journal-article","created":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T20:47:15Z","timestamp":1788468435000},"page":"38608-38618","source":"Crossref","is-referenced-by-count":0,"title":["Programmable Recognition\nof Organyl 5\u2032-Phosphates\nby Human Argonaute-2"],"prefix":"10.1021","volume":"148","author":[{"given":"Theodore","family":"Carrigan-Broda","sequence":"first","affiliation":[{"name":"RNA Therapeutics Institute, UMass Chan Medical School , 368 Plantation Street , , ,","place":["Worcester, Massachusetts, United States, 01605"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Luca F. R.","family":"Gebert","sequence":"additional","affiliation":[{"name":"Scripps Research Institute , MB-45, 10550 N. Torrey Pines Road , , ,","place":["La Jolla, California, United States, 92037"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Samuel","family":"Hildebrand","sequence":"additional","affiliation":[{"name":"RNA Therapeutics Institute, UMass Chan Medical School , 368 Plantation Street , , ,","place":["Worcester, Massachusetts, United States, 01605"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nozomi","family":"Yamada","sequence":"additional","affiliation":[{"name":"RNA Therapeutics Institute, UMass Chan Medical School , 368 Plantation Street , , ,","place":["Worcester, Massachusetts, United States, 01605"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eric","family":"Luu","sequence":"additional","affiliation":[{"name":"RNA Therapeutics Institute, UMass Chan Medical School , 368 Plantation Street , , ,","place":["Worcester, Massachusetts, United States, 01605"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jillian","family":"Caiazzi","sequence":"additional","affiliation":[{"name":"RNA Therapeutics Institute, UMass Chan Medical School , 368 Plantation Street , , ,","place":["Worcester, Massachusetts, United States, 01605"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nicholas","family":"McHugh","sequence":"additional","affiliation":[{"name":"RNA Therapeutics Institute, UMass Chan Medical School , 368 Plantation Street , , ,","place":["Worcester, Massachusetts, United States, 01605"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dimas","family":"Echeverria","sequence":"additional","affiliation":[{"name":"RNA Therapeutics Institute, UMass Chan Medical School , 368 Plantation Street , , ,","place":["Worcester, Massachusetts, United States, 01605"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Atish","family":"Wagh","sequence":"additional","affiliation":[{"name":"RNA Therapeutics Institute, UMass Chan Medical School , 368 Plantation Street , , ,","place":["Worcester, Massachusetts, United States, 01605"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5706-1734","authenticated-orcid":true,"given":"Jonathan K.","family":"Watts","sequence":"additional","affiliation":[{"name":"RNA Therapeutics Institute, UMass Chan Medical School , 368 Plantation Street , , ,","place":["Worcester, Massachusetts, United States, 01605"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anastasia","family":"Khvorova","sequence":"additional","affiliation":[{"name":"RNA Therapeutics Institute, UMass Chan Medical School , 368 Plantation Street , , ,","place":["Worcester, Massachusetts, United States, 01605"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5112-0294","authenticated-orcid":true,"given":"Ian J.","family":"MacRae","sequence":"additional","affiliation":[{"name":"Scripps Research Institute , MB-45, 10550 N. Torrey Pines Road , , ,","place":["La Jolla, California, United States, 92037"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5714-8298","authenticated-orcid":true,"given":"Ken","family":"Yamada","sequence":"additional","affiliation":[{"name":"RNA Therapeutics Institute, UMass Chan Medical School , 368 Plantation Street , , ,","place":["Worcester, Massachusetts, United States, 01605"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,9,3]]},"reference":[{"issue":"3","key":"2026091609114307300_cit1","doi-asserted-by":"publisher","first-page":"238","DOI":"10.1038\/nbt.3765","article-title":"The Chemical Evolution of Oligonucleotide Therapies\nof Clinical Utility","volume":"35","author":"Khvorova","year":"2017","journal-title":"Nat. Biotechnol."},{"issue":"3","key":"2026091609114307300_cit2","doi-asserted-by":"publisher","first-page":"549","DOI":"10.1016\/S1097-2765(02)00652-4","article-title":"RNAi in Human Cells:\nBasic Structural and Functional\nFeatures of Small Interfering RNA","volume":"10","author":"Chiu","year":"2002","journal-title":"Mol. Cell"},{"issue":"5","key":"2026091609114307300_cit3","doi-asserted-by":"publisher","first-page":"341","DOI":"10.1038\/s41573-024-00912-9","article-title":"RNAi-Based\nDrug Design: Considerations and Future Directions","volume":"23","author":"Tang","year":"2024","journal-title":"Nat. Rev. Drug Discovery"},{"issue":"4","key":"2026091609114307300_cit4","doi-asserted-by":"publisher","first-page":"340","DOI":"10.1038\/nsmb918","article-title":"Purified Argonaute2\nand an SiRNA\nForm Recombinant Human RISC","volume":"12","author":"Rivas","year":"2005","journal-title":"Nat. Struct. Mol.\nBiol."},{"issue":"2","key":"2026091609114307300_cit5","doi-asserted-by":"publisher","first-page":"106","DOI":"10.1016\/j.tibs.2004.12.007","article-title":"SiRNA and\nMiRNA: An Insight into RISCs","volume":"30","author":"Tang","year":"2005","journal-title":"Trends Biochem.\nSci."},{"issue":"11","key":"2026091609114307300_cit6","doi-asserted-by":"publisher","first-page":"985","DOI":"10.1002\/cbic.201600130","article-title":"5\u2032-(E)-Vinylphosphonate:\nA Stable Phosphate\nMimic Can Improve the RNAi Activity of SiRNA\u2013GalNAc Conjugates","volume":"17","author":"Parmar","year":"2016","journal-title":"ChemBioChem"},{"issue":"6084","key":"2026091609114307300_cit7","doi-asserted-by":"publisher","first-page":"1037","DOI":"10.1126\/science.1221551","article-title":"The Crystal Structure\nof Human Argonaute2","volume":"336","author":"Schirle","year":"2012","journal-title":"Science"},{"issue":"6","key":"2026091609114307300_cit8","doi-asserted-by":"publisher","first-page":"2993","DOI":"10.1093\/nar\/gkv162","article-title":"Identification of\nMetabolically Stable\n5\u2032-Phosphate Analogs That Support Single-Stranded SiRNA Activity","volume":"43","author":"Prakash","year":"2015","journal-title":"Nucleic Acids Res."},{"issue":"6","key":"2026091609114307300_cit9","doi-asserted-by":"publisher","first-page":"3528","DOI":"10.1093\/nar\/gkw1171","article-title":"SiRNA Carrying\nan (E)-Vinylphosphonate Moiety at the 5\u2032 End of the Guide Strand\nAugments Gene Silencing by Enhanced Binding to Human Argonaute-2","volume":"45","author":"Elkayam","year":"2017","journal-title":"Nucleic Acids Res."},{"issue":"28","key":"2026091609114307300_cit10","doi-asserted-by":"publisher","first-page":"8694","DOI":"10.1021\/jacs.6b04454","article-title":"Structural\nAnalysis of Human Argonaute-2 Bound to a\nModified SiRNA Guide","volume":"138","author":"Schirle","year":"2016","journal-title":"J. Am. Chem. Soc."},{"issue":"6209","key":"2026091609114307300_cit11","doi-asserted-by":"publisher","first-page":"608","DOI":"10.1126\/science.1258040","article-title":"Structural Basis for MicroRNA Targeting","volume":"346","author":"Schirle","year":"2014","journal-title":"Science"},{"issue":"4","key":"2026091609114307300_cit12","doi-asserted-by":"publisher","first-page":"1036","DOI":"10.1021\/acs.accounts.8b00650","article-title":"Re-Engineering\nRNA Molecules into Therapeutic Agents","volume":"52","author":"Egli","year":"2019","journal-title":"Acc.\nChem. Res."},{"issue":"36","key":"2026091609114307300_cit13","doi-asserted-by":"publisher","first-page":"6659","DOI":"10.1039\/D5CC00348B","article-title":"Expanding\nthe Binding Space of Argonaute-2:\nIncorporation of Either E or Z Isomers of 6\u2032-Vinylphosphonate\nat the 5\u2032 End of the Antisense Strand Improves RNAi Activity","volume":"61","author":"Datta","year":"2025","journal-title":"Chem. Commun."},{"issue":"55","key":"2026091609114307300_cit14","doi-asserted-by":"publisher","first-page":"6808","DOI":"10.1039\/D1CC02328D","article-title":"Synthesis\nof a Novel Cyclopropyl Phosphonate Nucleotide\nas a Phosphate Mimic","volume":"57","author":"Altenhofer","year":"2021","journal-title":"Chem. Commun."},{"issue":"1","key":"2026091609114307300_cit15","doi-asserted-by":"publisher","first-page":"35","DOI":"10.1089\/oli.2006.0067","article-title":"Tolerance of RNA\nInterference Toward Modifications\nof the 5\u2032 Antisense Phosphate of Small Interfering RNA","volume":"17","author":"Shah","year":"2007","journal-title":"Oligonucleotides"},{"issue":"13","key":"2026091609114307300_cit16","doi-asserted-by":"publisher","first-page":"7581","DOI":"10.1093\/nar\/gkx507","article-title":"5\u2032-Vinylphosphonate\nImproves Tissue Accumulation and Efficacy of Conjugated SiRNAs in\nVivo","volume":"45","author":"Haraszti","year":"2017","journal-title":"Nucleic Acids Res."},{"issue":"47","key":"2026091609114307300_cit17","doi-asserted-by":"publisher","first-page":"10061","DOI":"10.1021\/acs.orglett.4c02903","article-title":"Improved In\nVivo Metabolic Stability\nand Silencing Efficacy of SiRNAs with Phosphorothioate Linkage-Free,\nGalNAc-Conjugated Sense Strands Containing Morpholino-LNA Modifications","volume":"26","author":"Datta","year":"2024","journal-title":"Org. Lett."},{"issue":"6","key":"2026091609114307300_cit18","doi-asserted-by":"publisher","first-page":"904","DOI":"10.1038\/s41587-024-02336-7","article-title":"Enhancing SiRNA Efficacy in Vivo with Extended Nucleic\nAcid Backbones","volume":"43","author":"Yamada","year":"2025","journal-title":"Nat. Biotechnol."},{"issue":"12","key":"2026091609114307300_cit19","doi-asserted-by":"publisher","DOI":"10.1038\/mtna.2015.38","article-title":"Hydrophobically Modified\nSiRNAs Silence\nHuntingtin MRNA in Primary Neurons and Mouse Brain","volume":"4","author":"Alterman","year":"2015","journal-title":"Mol. Ther. Nucleic Acids"},{"key":"2026091609114307300_cit20","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-026-75611-7","article-title":"Single-Dose Administration of Therapeutic\nDivalent\nSiRNA Targeting MECP2 Prevents Lethality in an MECP2 Duplication Mouse\nModel","volume":"17","author":"Hariharan","year":"2026","journal-title":"Nat. Commun."},{"issue":"1","key":"2026091609114307300_cit21","doi-asserted-by":"publisher","first-page":"71","DOI":"10.1016\/j.bbagrm.2015.08.007","article-title":"RISC Assembly: Coordination between\nSmall RNAs and\nArgonaute Proteins","volume":"1859","author":"Kobayashi","year":"2016","journal-title":"Biochim. Biophys. Acta,\nGene Regul. Mech."},{"key":"2026091609114307300_cit22","doi-asserted-by":"publisher","first-page":"135","DOI":"10.1016\/j.omtn.2022.06.009","article-title":"Chemical Optimization\nof SiRNA for Safe and Efficient Silencing of Placental SFLT1","volume":"29","author":"Davis","year":"2022","journal-title":"Mol. Ther. Nucleic Acids"},{"issue":"3","key":"2026091609114307300_cit23","doi-asserted-by":"publisher","first-page":"1221","DOI":"10.1093\/nar\/gkab544","article-title":"Chirality\nMatters: Stereo-Defined Phosphorothioate Linkages at the Termini of\nSmall Interfering RNAs Improve Pharmacology in Vivo","volume":"50","author":"Jahns","year":"2022","journal-title":"Nucleic Acids Res."},{"issue":"6","key":"2026091609114307300_cit24","doi-asserted-by":"publisher","first-page":"664","DOI":"10.1134\/S1068162017060024","article-title":"New Oligodeoxynucleotide\nDerivatives Containing N-(Methanesulfonyl)-Phosphoramidate (Mesyl\nPhosphoramidate) Internucleotide Group","volume":"43","author":"Chelobanov","year":"2017","journal-title":"Russ.\nJ. Bioorg. Chem."},{"issue":"7","key":"2026091609114307300_cit25","doi-asserted-by":"publisher","first-page":"6870","DOI":"10.1021\/acs.jmedchem.4c02528","article-title":"Metabolic\nStability\nand Targeted Delivery of Oligonucleotides: Advancing RNA Therapeutics\nBeyond The Liver","volume":"68","author":"Anand","year":"2025","journal-title":"J. Med. Chem."},{"issue":"3","key":"2026091609114307300_cit26","doi-asserted-by":"publisher","DOI":"10.1002\/prp2.70096","article-title":"In Vitro Structure\u2013Activity\nRelationship Stability Study of Antisense Oligonucleotide Therapeutics\nUsing Biological Matrices and Nucleases","volume":"13","author":"Lovric\u0301","year":"2025","journal-title":"Pharmacol.\nRes. Perspect."},{"issue":"8","key":"2026091609114307300_cit27","doi-asserted-by":"publisher","first-page":"330","DOI":"10.1101\/PDB.TOP100768","article-title":"Alkaline Phosphatase","volume":"2020","author":"Green","year":"2020","journal-title":"Cold Spring\nHarb. Protoc."},{"issue":"4","key":"2026091609114307300_cit28","doi-asserted-by":"publisher","first-page":"1293","DOI":"10.1021\/ja056528r","article-title":"Alkaline Phosphatase Mono- and Diesterase Reactions:\nComparative Transition State Analysis","volume":"128","author":"Zalatan","year":"2006","journal-title":"J. Am.\nChem. Soc."},{"issue":"5","key":"2026091609114307300_cit29","doi-asserted-by":"publisher","first-page":"600","DOI":"10.1016\/j.molcel.2011.02.004","article-title":"Coupled 5\u2032 Nucleotide Recognition\nand Processivity in Xrn1-Mediated MRNA Decay","volume":"41","author":"Jinek","year":"2011","journal-title":"Mol. Cell"},{"issue":"38","key":"2026091609114307300_cit30","doi-asserted-by":"publisher","first-page":"11841","DOI":"10.1073\/pnas.1506116112","article-title":"Biochemical Isolation of Argonaute\nProtein Complexes\nby Ago-APP","volume":"112","author":"Hauptmann","year":"2015","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"2026091609114307300_cit31","doi-asserted-by":"publisher","first-page":"1777","DOI":"10.1101\/gr.251421.119","article-title":"Global Analyses of the Dynamics of Mammalian MicroRNA\nMetabolism","volume":"29","author":"Kingston","year":"2019","journal-title":"Genome Res."},{"key":"2026091609114307300_cit32","first-page":"224","article-title":"[29] Alkaline\nHydrolysis of RNA","volume-title":"Methods Enzymol.","author":"Bock","year":"1967"},{"issue":"4","key":"2026091609114307300_cit33","doi-asserted-by":"publisher","first-page":"199","DOI":"10.1089\/nat.2023.0067","article-title":"Therapeutic\nSiRNA Loaded to RISC as Single and Double Strands Requires an Appropriate\nQuantitative Assay for RISC PK Assessment","volume":"34","author":"Xu","year":"2024","journal-title":"Nucleic\nAcid Ther."},{"issue":"2","key":"2026091609114307300_cit34","doi-asserted-by":"publisher","DOI":"10.1016\/j.omtn.2025.102516","article-title":"Mechanistic Intracellular PK\/PD Modeling\nto Inform Development Strategies for Small Interfering RNA Therapeutics","volume":"36","author":"Chen","year":"2025","journal-title":"Mol. Ther. Nucleic Acids"},{"issue":"6","key":"2026091609114307300_cit35","doi-asserted-by":"publisher","DOI":"10.15252\/embr.202255806","article-title":"A Tiny\nLoop in the Argonaute PIWI\nDomain Tunes Small RNA Seed Strength","volume":"24","author":"Xiao","year":"2023","journal-title":"EMBO Rep."}],"container-title":["Journal of the American\nChemical Society"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/jacs.6c03647","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/jacsat\/article-pdf\/148\/36\/38608\/68287608\/jacs.6c03647.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/jacsat\/article-pdf\/148\/36\/38608\/68287608\/jacs.6c03647.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,16]],"date-time":"2026-09-16T13:36:08Z","timestamp":1789565768000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/jacsat\/article\/148\/36\/38608\/5413545\/Programmable-Recognition-of-Organyl-5-Phosphates"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,3]]},"references-count":35,"journal-issue":{"issue":"36","published-online":{"date-parts":[[2026,9,3]]},"published-print":{"date-parts":[[2026,9,16]]}},"URL":"https:\/\/doi.org\/10.1021\/jacs.6c03647","relation":{},"ISSN":["0002-7863","1520-5126"],"issn-type":[{"value":"0002-7863","type":"print"},{"value":"1520-5126","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,3]]}}}