siRNA carrying an (E)-vinylphosphonate moiety at the 5' end of the guide strand augments gene silencing by enhanced binding to human Argonaute-2

Elkayam, Elad; Parmar, Rubina; Brown, Christopher R.; Willoughby, Jennifer L.; Theile, Christopher S.; Manoharan, Muthiah; Joshua-Tor, Leemor

doi:10.1093/nar/gkw1171

siRNA carrying an (E)-vinylphosphonate moiety at the 5' end of the guide strand augments gene silencing by enhanced binding to human Argonaute-2

Journal Article · Mon Nov 28 00:00:00 EST 2016 · Nucleic Acids Research

DOI:https://doi.org/10.1093/nar/gkw1171· OSTI ID:1529625

Elkayam, Elad ^[1]; Parmar, Rubina ^[2]; Brown, Christopher R. ^[2]; Willoughby, Jennifer L. ^[2]; Theile, Christopher S. ^[2]; Manoharan, Muthiah ^[2]; Joshua-Tor, Leemor ^[1]

Keck Structural Biology Lab, Cold Spring Harbor, NY (United States); Howard Hughes Medical Inst., Cold Spring Harbor, NY (United States); Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (United States)
Alnylam Pharmaceuticals, Cambridge, MA (United States)

Efficient gene silencing by RNA interference (RNAi) in vivo requires the recognition and binding of the 5'- phosphate of the guide strand of an siRNA by the Argonaute protein. However, for exogenous siRNAs it is limited by the rapid removal of the 5'- phosphate of the guide strand by metabolic enzymes. Here, we have determined the crystal structure of human Argonaute-2 in complex with the metabolically stable 5'-(E)-vinylphosphonate (5'-E-VP) guide RNA at 2.5-Å resolution. The structure demonstrates how the 5' binding site in the Mid domain of human Argonaute-2 is able to adjust the key residues in the 5'-nucleotide binding pocket to compensate for the change introduced by the modified nucleotide. This observation also explains improved binding affinity of the 5'-E-VP -modified siRNA to human Argonaute-2 in-vitro, as well as the enhanced silencing in the context of the trivalent N-acetylgalactosamine (GalNAc)-conjugated siRNA in mice relative to the un-modified siRNA.

View Accepted Manuscript (DOE)

Research Organization:: Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS)

Sponsoring Organization:: Cold Spring Harbor Laboratory Women in Science Award; USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23); theHoward Hughes Medical Institute

Grant/Contract Number:: AC02-06CH11357

OSTI ID:: 1529625

Journal Information:: Nucleic Acids Research, Journal Name: Nucleic Acids Research Journal Issue: 6 Vol. 45; ISSN 0305-1048

Publisher:: Oxford University PressCopyright Statement

Country of Publication:: United States

Language:: ENGLISH

References (56)

5′-( E )-Vinylphosphonate: A Stable Phosphate Mimic Can Improve the RNAi Activity of siRNA-GalNAc Conjugates Parmar, Rubina; Willoughby, Jennifer L. S.; Liu, Jingxuan ChemBioChem, Vol. 17, Issue 11 https://doi.org/10.1002/cbic.201600130	journal	April 2016
Evidence for the conformation about the C(5′)-O(5′) bond of AMP complexed to AMP kinase: Substrate properties of a vinyl phosphonate analog of AMP Hampton, Alexander; Kappler, Francis; Perini, Florian Bioorganic Chemistry, Vol. 5, Issue 1 https://doi.org/10.1016/0045-2068(76)90014-6	journal	March 1976
Stabilizing effects of the RNA 2′-substituent: crystal structure of an oligodeoxynucleotide duplex containing 2′-O-methylated adenosines Lubini, Paolo; Zürcher, Werner; Egli, Martin Chemistry & Biology, Vol. 1, Issue 1 https://doi.org/10.1016/1074-5521(94)90039-6	journal	September 1994
RNA interference and chemically modified small interfering RNAs Manoharan, Muthiah Current Opinion in Chemical Biology, Vol. 8, Issue 6 https://doi.org/10.1016/j.cbpa.2004.10.007	journal	December 2004
The Structure of Human Argonaute-2 in Complex with miR-20a Elkayam, Elad; Kuhn, Claus-D.; Tocilj, Ante Cell, Vol. 150, Issue 1 https://doi.org/10.1016/j.cell.2012.06.021	journal	July 2012
Singles Engage the RNA Interference Pathway Davidson, Beverly L.; Monteys, Alex Mas Cell, Vol. 150, Issue 5 https://doi.org/10.1016/j.cell.2012.08.008	journal	August 2012
The Making of a Slicer: Activation of Human Argonaute-1 Faehnle, Christopher R.; Elkayam, Elad; Haase, Astrid D. Cell Reports, Vol. 3, Issue 6 https://doi.org/10.1016/j.celrep.2013.05.033	journal	June 2013
Eukaryote-Specific Insertion Elements Control Human ARGONAUTE Slicer Activity Nakanishi, Kotaro; Ascano, Manuel; Gogakos, Tasos Cell Reports, Vol. 3, Issue 6 https://doi.org/10.1016/j.celrep.2013.06.010	journal	June 2013
Chemically modified siRNA: tools and applications Watts, J.; Deleavey, G.; Damha, M. Drug Discovery Today, Vol. 13, Issue 19-20 https://doi.org/10.1016/j.drudis.2008.05.007	journal	October 2008
Allele-Selective Inhibition of Mutant Atrophin-1 Expression by Duplex and Single-Stranded RNAs Hu, Jiaxin; Liu, Jing; Narayanannair, K. Jayaprakash Biochemistry, Vol. 53, Issue 28 https://doi.org/10.1021/bi500610r	journal	July 2014
Lipid Nanoparticles Improve Activity of Single-Stranded siRNA and Gapmer Antisense Oligonucleotides in Animals Prakash, Thazha P.; Lima, Walt F.; Murray, Heather M. ACS Chemical Biology, Vol. 8, Issue 7 https://doi.org/10.1021/cb4001316	journal	May 2013
Fully 2‘-Modified Oligonucleotide Duplexes with Improved in Vitro Potency and Stability Compared to Unmodified Small Interfering RNA Allerson, Charles R.; Sioufi, Namir; Jarres, Russell Journal of Medicinal Chemistry, Vol. 48, Issue 4 https://doi.org/10.1021/jm049167j	journal	February 2005
Argonaute MID domain takes centre stage Faehnle, Christopher R.; Joshua‐Tor, Leemor EMBO reports, Vol. 11, Issue 8 https://doi.org/10.1038/embor.2010.110	journal	July 2010
Structural analysis of 5′-mRNA–cap interactions with the human AGO2 MID domain Frank, Filipp; Fabian, Marc R.; Stepinski, Janusz EMBO reports, Vol. 12, Issue 5 https://doi.org/10.1038/embor.2011.48	journal	April 2011
Preclinical Development of a Subcutaneous ALAS1 RNAi Therapeutic for Treatment of Hepatic Porphyrias Using Circulating RNA Quantification Chan, Amy; Liebow, Abigail; Yasuda, Makiko Molecular Therapy - Nucleic Acids, Vol. 4 https://doi.org/10.1038/mtna.2015.36	journal	January 2015
Defining fundamental steps in the assembly of the Drosophila RNAi enzyme complex Iwasaki, Shintaro; Sasaki, Hiroshi M.; Sakaguchi, Yuriko Nature, Vol. 521, Issue 7553 https://doi.org/10.1038/nature14254	journal	March 2015
RNAi therapeutics: a potential new class of pharmaceutical drugs Bumcrot, David; Manoharan, Muthiah; Koteliansky, Victor Nature Chemical Biology, Vol. 2, Issue 12 https://doi.org/10.1038/nchembio839	journal	December 2006
An RNAi therapeutic targeting antithrombin to rebalance the coagulation system and promote hemostasis in hemophilia Sehgal, Alfica; Barros, Scott; Ivanciu, Lacramioara Nature Medicine, Vol. 21, Issue 5 https://doi.org/10.1038/nm.3847	journal	April 2015
Leaner, meaner siRNAs Nawy, Tal Nature Methods, Vol. 9, Issue 11 https://doi.org/10.1038/nmeth.2240	journal	November 2012
Purified Argonaute2 and an siRNA form recombinant human RISC Rivas, Fabiola V.; Tolia, Niraj H.; Song, Ji-Joon Nature Structural & Molecular Biology, Vol. 12, Issue 4 https://doi.org/10.1038/nsmb918	journal	March 2005
Crystal structure of the MID-PIWI lobe of a eukaryotic Argonaute protein Boland, A.; Huntzinger, E.; Schmidt, S. Proceedings of the National Academy of Sciences, Vol. 108, Issue 26 https://doi.org/10.1073/pnas.1103946108	journal	June 2011
Binding and Cleavage Specificities of Human Argonaute2 Lima, Walt F.; Wu, Hongjiang; Nichols, Josh G. Journal of Biological Chemistry, Vol. 284, Issue 38 https://doi.org/10.1074/jbc.m109.010835	journal	July 2009
Sequence, Chemical, and Structural Variation of Small Interfering RNAs and Short Hairpin RNAs and the Effect on Mammalian Gene Silencing Harborth, Jens; Elbashir, Sayda M.; Vandenburgh, Kim Antisense and Nucleic Acid Drug Development, Vol. 13, Issue 2 https://doi.org/10.1089/108729003321629638	journal	April 2003
Identification of metabolically stable 5′-phosphate analogs that support single-stranded siRNA activity Prakash, Thazha P.; Lima, Walt F.; Murray, Heather M. Nucleic Acids Research, Vol. 43, Issue 6 https://doi.org/10.1093/nar/gkv162	journal	March 2015
Real-time quantification of microRNAs by stem-loop RT-PCR Chen, C. Nucleic Acids Research, Vol. 33, Issue 20 https://doi.org/10.1093/nar/gni178	journal	November 2005
Solving structures of protein complexes by molecular replacement with Phaser McCoy, Airlie J. Acta Crystallographica Section D Biological Crystallography, Vol. 63, Issue 1 https://doi.org/10.1107/s0907444906045975	journal	December 2006
Features and development of Coot Emsley, P.; Lohkamp, B.; Scott, W. G. Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 4 https://doi.org/10.1107/s0907444910007493	journal	March 2010
Data processing and analysis with the autoPROC toolbox Vonrhein, Clemens; Flensburg, Claus; Keller, Peter Acta Crystallographica Section D Biological Crystallography, Vol. 67, Issue 4 https://doi.org/10.1107/s0907444911007773	journal	March 2011
R2D2, a Bridge Between the Initiation and Effector Steps of the Drosophila RNAi Pathway Liu, Q. Science, Vol. 301, Issue 5641 https://doi.org/10.1126/science.1088710	journal	September 2003
The Crystal Structure of Human Argonaute2 Schirle, N. T.; MacRae, I. J. Science, Vol. 336, Issue 6084 https://doi.org/10.1126/science.1221551	journal	April 2012
Argonaute2 expression is post-transcriptionally coupled to microRNA abundance Martinez, N. J.; Gregory, R. I. RNA, Vol. 19, Issue 5 https://doi.org/10.1261/rna.036434.112	journal	March 2013
Quantitative evaluation of siRNA delivery in vivo Pei, Y.; Hancock, P. J.; Zhang, H. RNA, Vol. 16, Issue 12 https://doi.org/10.1261/rna.2255810	journal	October 2010
PHENIX: a comprehensive Python-based system for macromolecular structure solution. Adams, Paul D.; Afonine, Pavel V.; Bunkóczi, Gábor Apollo - University of Cambridge Repository https://doi.org/10.17863/cam.45787	text	January 2010
Unique Gene-Silencing and Structural Properties of 2′-Fluoro-Modified siRNAs Manoharan, Muthiah; Akinc, Akin; Pandey, Rajendra K. Angewandte Chemie International Edition, Vol. 50, Issue 10 https://doi.org/10.1002/anie.201006519	journal	January 2011
Uptake and degradation of cytoplasmic RNA by hepatic lysosomes. Quantitative relationship to RNA turnover Heydrick, S. J.; Lardeux, B. R.; Mortimore, G. E. Journal of Biological Chemistry, Vol. 266, Issue 14 https://doi.org/10.1016/S0021-9258(18)31516-3	journal	May 1991
A Dicer-2-Dependent 80S Complex Cleaves Targeted mRNAs during RNAi in Drosophila Pham, John W.; Pellino, Janice L.; Lee, Young Sik Cell, Vol. 117, Issue 1 https://doi.org/10.1016/S0092-8674(04)00258-2	journal	April 2004
Passenger-Strand Cleavage Facilitates Assembly of siRNA into Ago2-Containing RNAi Enzyme Complexes Matranga, Christian; Tomari, Yukihide; Shin, Chanseok Cell, Vol. 123, Issue 4 https://doi.org/10.1016/j.cell.2005.08.044	journal	November 2005
The Structure of Human Argonaute-2 in Complex with miR-20a Elkayam, Elad; Kuhn, Claus-D.; Tocilj, Ante Cell, Vol. 150, Issue 1 https://doi.org/10.1016/j.cell.2012.05.017	journal	July 2012
Single-Stranded RNAs Use RNAi to Potently and Allele-Selectively Inhibit Mutant Huntingtin Expression Yu, Dongbo; Pendergraff, Hannah; Liu, Jing Cell, Vol. 150, Issue 5 https://doi.org/10.1016/j.cell.2012.08.002	journal	August 2012
Single-Stranded siRNAs Activate RNAi in Animals Lima, Walt F.; Prakash, Thazha P.; Murray, Heather M. Cell, Vol. 150, Issue 5 https://doi.org/10.1016/j.cell.2012.08.014	journal	August 2012
Argonaute Divides Its RNA Guide into Domains with Distinct Functions and RNA-Binding Properties Wee, Liang Meng; Flores-Jasso, C. Fabián; Salomon, William E. Cell, Vol. 151, Issue 5 https://doi.org/10.1016/j.cell.2012.10.036	journal	November 2012
Hsc70/Hsp90 Chaperone Machinery Mediates ATP-Dependent RISC Loading of Small RNA Duplexes Iwasaki, Shintaro; Kobayashi, Maki; Yoda, Mayuko Molecular Cell, Vol. 39, Issue 2 https://doi.org/10.1016/j.molcel.2010.05.015	journal	July 2010
Multivalent N -Acetylgalactosamine-Conjugated siRNA Localizes in Hepatocytes and Elicits Robust RNAi-Mediated Gene Silencing Nair, Jayaprakash K.; Willoughby, Jennifer L. S.; Chan, Amy Journal of the American Chemical Society, Vol. 136, Issue 49 https://doi.org/10.1021/ja505986a	journal	November 2014
Structural Analysis of Human Argonaute-2 Bound to a Modified siRNA Guide Schirle, Nicole T.; Kinberger, Garth A.; Murray, Heather F. Journal of the American Chemical Society, Vol. 138, Issue 28 https://doi.org/10.1021/jacs.6b04454	journal	July 2016
Positional Effect of Chemical Modifications on Short Interference RNA Activity in Mammalian Cells Prakash, Thazha P.; Allerson, Charles R.; Dande, Prasad Journal of Medicinal Chemistry, Vol. 48, Issue 13 https://doi.org/10.1021/jm050044o	journal	June 2005
Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans Fire, Andrew; Xu, SiQun; Montgomery, Mary K. Nature, Vol. 391, Issue 6669 https://doi.org/10.1038/35888	journal	February 1998
Crystal structure and improved antisense properties of 2'-O-(2-methoxyethyl)-RNA Manoharan, Muthiah; Egli, Martin; Teplova, Marianna Nature Structural Biology, Vol. 6, Issue 6, p. 535-539 https://doi.org/10.1038/9304	journal	June 1999
Crystal structure and ligand binding of the MID domain of a eukaryotic Argonaute protein Boland, Andreas; Tritschler, Felix; Heimstädt, Susanne EMBO reports, Vol. 11, Issue 7 https://doi.org/10.1038/embor.2010.81	journal	June 2010
Structural basis for 5′-nucleotide base-specific recognition of guide RNA by human AGO2 Frank, Filipp; Sonenberg, Nahum; Nagar, Bhushan Nature, Vol. 465, Issue 7299 https://doi.org/10.1038/nature09039	journal	May 2010
Long-lived microRNA-Argonaute complexes in quiescent cells can be activated to regulate mitogenic responses Olejniczak, S. H.; La Rocca, G.; Gruber, J. J. Proceedings of the National Academy of Sciences, Vol. 110, Issue 1 https://doi.org/10.1073/pnas.1219958110	journal	December 2012
Binding and Cleavage Specificities of Human Argonaute2 Lima, Walt F.; Wu, Hongjiang; Nichols, Josh G. Journal of Biological Chemistry, Vol. 284, Issue 38 https://doi.org/10.1074/jbc.M109.010835	journal	July 2009
Solving structures of protein complexes by molecular replacement with Phaser McCoy, Airlie J. Acta Crystallographica Section D Biological Crystallography, Vol. 63, Issue 1 https://doi.org/10.1107/S0907444906045975	journal	December 2006
PHENIX: a comprehensive Python-based system for macromolecular structure solution Adams, Paul D.; Afonine, Pavel V.; Bunkóczi, Gábor Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 2, p. 213-221 https://doi.org/10.1107/S0907444909052925	journal	January 2010
Features and development of Coot Emsley, P.; Lohkamp, B.; Scott, W. G. Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 4 https://doi.org/10.1107/S0907444910007493	journal	March 2010
Data processing and analysis with the autoPROC toolbox Vonrhein, Clemens; Flensburg, Claus; Keller, Peter Acta Crystallographica Section D Biological Crystallography, Vol. 67, Issue 4 https://doi.org/10.1107/S0907444911007773	journal	March 2011
Structural basis for microRNA targeting Schirle, N. T.; Sheu-Gruttadauria, J.; MacRae, I. J. Science, Vol. 346, Issue 6209 https://doi.org/10.1126/science.1258040	journal	October 2014

Cited By (9)

Chimeric siRNAs with chemically modified pentofuranose and hexopyranose nucleotides: altritol-nucleotide (ANA) containing GalNAc–siRNA conjugates: in vitro and in vivo RNAi activity and resistance to 5′-exonuclease Kumar, Pawan; Degaonkar, Rohan; Guenther, Dale C. Nucleic Acids Research, Vol. 48, Issue 8 https://doi.org/10.1093/nar/gkaa125	journal	March 2020
5΄-Vinylphosphonate improves tissue accumulation and efficacy of conjugated siRNAs in vivo Haraszti, Reka A.; Roux, Loic; Coles, Andrew H. Nucleic Acids Research, Vol. 45, Issue 13 https://doi.org/10.1093/nar/gkx507	journal	June 2017
Impact of enhanced metabolic stability on pharmacokinetics and pharmacodynamics of GalNAc–siRNA conjugates Nair, Jayaprakash K.; Attarwala, Husain; Sehgal, Alfica Nucleic Acids Research, Vol. 45, Issue 19 https://doi.org/10.1093/nar/gkx818	journal	September 2017
Current Development of siRNA Bioconjugates: From Research to the Clinic Chernikov, Ivan V.; Vlassov, Valentin V.; Chernolovskaya, Elena L. Frontiers in Pharmacology, Vol. 10 https://doi.org/10.3389/fphar.2019.00444	journal	April 2019
The Medicinal Chemistry of Antisense Oligonucleotides Watts, Jonathan K. Oligonucleotide‐Based Drugs and Therapeutics: Preclinical and Clinical Considerations for Development https://doi.org/10.1002/9781119070153.ch2	book	June 2018
Squaramide‐Based 5’‐Phosphate Replacements Bind to the DNA Repair Exonuclease SNM1A Dürr, Eva‐Maria; Doherty, William; Lee, Sook Y. ChemistrySelect, Vol. 3, Issue 45 https://doi.org/10.1002/slct.201803375	journal	December 2018
Reversal of siRNA-mediated gene silencing in vivo Zlatev, Ivan; Castoreno, Adam; Brown, Christopher R. Nature Biotechnology, Vol. 36, Issue 6 https://doi.org/10.1038/nbt.4136	journal	May 2018
Selection of GalNAc-conjugated siRNAs with limited off-target-driven rat hepatotoxicity Janas, Maja M.; Schlegel, Mark K.; Harbison, Carole E. Nature Communications, Vol. 9, Issue 1 https://doi.org/10.1038/s41467-018-02989-4	journal	February 2018
5′-Morpholino modification of the sense strand of an siRNA makes it a more effective passenger Kumar, Pawan; Parmar, Rubina G.; Brown, Christopher R. Chemical Communications, Vol. 55, Issue 35 https://doi.org/10.1039/c9cc00977a	journal	January 2019

Similar Records

A Potential Protein-RNA Recognition Event Along the RISC-Loading Pathway from the Structure of A. aeolicus Argonaute with Externally Bound siRNA

Journal Article · Sat Dec 31 23:00:00 EST 2005 · Structure · OSTI ID:930084

Structure of the guide-strand-containing argonaute silencing complex

Journal Article · Wed Jan 14 23:00:00 EST 2009 · Nature · OSTI ID:1007015

Overcoming GNA/RNA base-pairing limitations using isonucleotides improves the pharmacodynamic activity of ESC+ GalNAc-siRNAs

Journal Article · Wed Oct 13 20:00:00 EDT 2021 · Nucleic Acids Research · OSTI ID:1826226

Related Subjects

59 BASIC BIOLOGICAL SCIENCES

siRNA carrying an (E)-vinylphosphonate moiety at the 5' end of the guide strand augments gene silencing by enhanced binding to human Argonaute-2

Citation Formats

References (56)

Cited By (9)

Similar Records

Related Subjects