Sequence-Specific Inhibition of Small RNA Function
Publication Date
February 24, 2004
Journal
PLOS Biology
Authors
György Hutvágner, Martin J Simard, Craig C Mello & Phillip D Zamore
Volume
2
Issue
4
Pages
e98
DOI
https://dx.plos.org/10.1371/journal.pbio.0020098
Publisher URL
http://journals.plos.org/plosbiology/article?id=10.1371%2Fjournal.pbio.0020098
PubMed
http://www.ncbi.nlm.nih.gov/pubmed/15024405
PubMed Central
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC350664
Europe PMC
http://europepmc.org/abstract/MED/15024405
Web of Science
000221194700013
Scopus
19344378192
Mendeley
http://www.mendeley.com/research/sequencespecific-inhibition-small-rna-function
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CiteULike | Further Information

Mendeley | Further Information

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CrossRef

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/964009"], "description"=>"<div><p>(A) Sequences of the sense and antisense <i>Pp</i>-luc target RNAs (black), the siRNA (red, antisense strand; black, sense strand), and the sense and antisense 2′<i>-O-</i>methyl oligonucleotides (blue) used.</p>\n <p>(B) Sequence-specific depletion of RNAi activity by immobilized 2′-<i>O</i>-methyl oligonucleotides from <i>Drosophila</i> embryo lysate programmed with siRNA. siRNA was incubated with lysate to assemble RISC; then, immobilized 2′-<i>O</i>-methyl oligonucleotide was added. Finally, the beads were removed from the supernatant, and either sense or antisense <sup>32</sup>P-radiolabeled target RNA was added to the supernatant to measure RISC activity for each siRNA strand.</p>\n <p>Symbols and abbreviations: Ø, target RNA before incubation with siRNA-programmed lysate; T, total reaction before depletion; unbound, the supernatant after incubation with the immobilized antisense (AS) or sense (S) 2′-<i>O</i>-methyl oligonucleotides shown in (A). The absence of 5′ cleavage product demonstrates that the sense oligonucleotide depleted RISC containing antisense siRNA, but not sense siRNA, and the antisense oligonucleotide depleted the sense RISC, but not that containing antisense siRNA. Bi, 5′ biotin attached via a six-carbon linker.</p></div>", "links"=>[], "tags"=>["rna", "oligonucleotide", "inhibits", "rnai", "vitro", "embryo"], "article_id"=>633945, "categories"=>["Molecular Biology", "Biotechnology", "Cell Biology", "Genetics"], "users"=>["György Hutvágner", "Martin J Simard", "Craig C Mello", "Phillip D Zamore"], "doi"=>["https://dx.doi.org/10.1371/journal.pbio.0020098.g001"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_A_2_O_Methyl_RNA_Oligonucleotide_Inhibits_RNAi_In_Vitro_in_Drosophila_Embryo_Lysate_/633945", "title"=>"A 2′<i>-O-</i>Methyl RNA Oligonucleotide Inhibits RNAi In Vitro in <i>Drosophila</i> Embryo Lysate", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-22 09:15:24"}
  • {"files"=>["https://ndownloader.figshare.com/files/964092"], "description"=>"<div><p>(A) The immobilized sense 2′-<i>O</i>-methyl oligonucleotide was used to determine the concentration of <sup>32</sup>P-radiolabeled antisense siRNA assembled into RISC in <i>Drosophila</i> embryo. The 2′-<i>O</i>-methyl oligonucleotide and siRNA duplex are shown in <a href=\"http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020098#pbio-0020098-g001\" target=\"_blank\">Figure 1</a>A.</p>\n <p>(B–G) Inhibition of RNAi was measured using free 2′-<i>O</i>-methyl oligonucleotide and 1.3 nM (B), 4.6 nM (C), 9.3 nM (D), 14.5 nM (E), 18 nM (F), and 23.5 nM (G) RISC. The concentration of 2′-<i>O</i>-methyl oligonucleotide required for half-maximal inhibition (IC<sub>50</sub>) was calculated by fitting each dataset to a sigmoidal curve using a Hill coefficient of 1.</p>\n <p>(H) A plot of IC<sub>50</sub> versus RISC concentration suggests that each 2′-<i>O</i>-methyl oligonucleotide binds a single RISC. The data suggest that binding is essentially irreversible.</p></div>", "links"=>[], "tags"=>["oligonucleotides", "irreversible", "inhibitors", "risc"], "article_id"=>634034, "categories"=>["Molecular Biology", "Biotechnology", "Cell Biology", "Genetics"], "users"=>["György Hutvágner", "Martin J Simard", "Craig C Mello", "Phillip D Zamore"], "doi"=>["https://dx.doi.org/10.1371/journal.pbio.0020098.g002"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_2_O_Methyl_Oligonucleotides_Act_as_Stoichiometric_Irreversible_Inhibitors_of_RISC_Function_/634034", "title"=>"2′-<i>O</i>-Methyl Oligonucleotides Act as Stoichiometric, Irreversible Inhibitors of RISC Function", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-22 09:16:04"}
  • {"files"=>["https://ndownloader.figshare.com/files/964181"], "description"=>"<div><p>(A) Inhibition of sense target cleavage by an antisense 2′<i>-O-</i>methyl oligonucleotide requires an approximately 40-fold higher concentration than by a sense oligonucleotide. The antisense oligonucleotide can pair completely with the sense target RNA, but not with the antisense siRNA-programmed RISC. The IC<sub>50</sub> value and the RISC concentration are indicated. Also shown are the sequences of the sense <i>Pp</i>-luc RNA target (black), the siRNA (red, antisense strand; black, sense strand), and the 2′<i>-O-</i>methyl oligonucleotide (blue).</p>\n <p>(B) The same antisense 2′-<i>O</i>-methyl oligonucleotide is an effective competitor of antisense target cleavage. In this experiment, inhibition occurs via binding of the antisense oligonucleotide to the sense siRNA-programmed RISC, not the target RNA. The IC<sub>50</sub> value and the RISC concentration are indicated. Also shown are the sequences of the <i>Pp</i>-luc antisense RNA target (black), the siRNA (red, antisense strand; black, sense strand), and the 2′<i>-O-</i>methyl oligonucleotide (blue). The G:U wobble in the siRNA duplex in (B) acts to direct the sense strand into RISC and improving its efficacy in target cleavage.</p></div>", "links"=>[], "tags"=>["antisense"], "article_id"=>634121, "categories"=>["Molecular Biology", "Biotechnology", "Cell Biology", "Genetics"], "users"=>["György Hutvágner", "Martin J Simard", "Craig C Mello", "Phillip D Zamore"], "doi"=>["https://dx.doi.org/10.1371/journal.pbio.0020098.g003"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_RISC_Does_Not_Act_through_an_Antisense_Mechanism_/634121", "title"=>"RISC Does Not Act through an Antisense Mechanism", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-22 09:16:42"}
  • {"files"=>["https://ndownloader.figshare.com/files/964273"], "description"=>"<div><p>(A–D) HeLa cells were transfected with 1 nM (A), 5 nM (B), 10 nM (C), or 25 nM (D) siRNA-targeting <i>Pp-</i>luc mRNA. The next day the cells were cotransfected with <i>Rr-</i>luc<i>-</i>and <i>Pp</i>-luc-expressing plasmids together with various amounts of a 31-nt 2′<i>-O-</i>methyl oligonucleotide complementary to the antisense strand of the siRNA. The half-maximal concentration of 2′-<i>O</i>-methyl oligonucleotide required to inhibit (IC<sub>50</sub>) was determined by fitting the data to a sigmoidal curve using a Hill coefficient of 1.</p>\n <p>(E) IC<sub>50</sub> plotted as a function of the concentration of transfected siRNA.</p></div>", "links"=>[], "tags"=>["oligonucleotide", "potent", "inhibitor", "rnai", "cultured", "hela"], "article_id"=>634207, "categories"=>["Molecular Biology", "Biotechnology", "Cell Biology", "Genetics"], "users"=>["György Hutvágner", "Martin J Simard", "Craig C Mello", "Phillip D Zamore"], "doi"=>["https://dx.doi.org/10.1371/journal.pbio.0020098.g004"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_A_2_O_Methyl_Oligonucleotide_Is_a_Potent_Inhibitor_of_RNAi_in_Human_Cultured_HeLa_Cells_/634207", "title"=>"A 2′-<i>O</i>-Methyl Oligonucleotide Is a Potent Inhibitor of RNAi in Human Cultured HeLa Cells", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-22 09:17:20"}
  • {"files"=>["https://ndownloader.figshare.com/files/964352"], "description"=>"<div><p>(A) Sequence of the <i>let-7</i>-complementary site in the target RNA (black), of the siRNA (red, antisense strand; black, sense strand), and of the <i>let-7</i>-complementary 2′<i>-O-</i>methyl oligonucleotide (blue).</p>\n <p>(B) Schematic representation of the target RNA, which contained both <i>Pp</i>-luc and antisense <i>let-7</i> sequences.</p>\n <p>(C) <i>Drosophila</i> embryo lysate (left) was programmed with <i>let-7</i> siRNA; then, the target RNA and the 2′<i>-O-</i>methyl oligonucleotide were added together. Target RNA and 2′-<i>O</i>-methyl oligonucleotide (right) were added to HeLa S100 extract, which contains endogenous human <i>let-7</i>-programmed RISC.</p>\n <p>(D) An RNA target containing both <i>Pp</i>-luc and antisense <i>let-7</i> sequence can be simultaneously targeted by <i>Pp</i>-luc siRNA and endogenous <i>let-7</i> in HeLa S100 lysate. The <i>let-7</i>-complementary 2′-<i>O</i>-methyl oligonucleotide blocks <i>let-7</i>-programmed, but not <i>Pp</i>-luc siRNA-programmed, RISC function. The bottom panel shows the same samples analyzed separately to better resolve the <i>let-7</i> 5′ cleavage product.</p>\n <p>(E) <i>Drosophila</i> embryo lysate was programmed with <i>let-7</i> siRNA and then incubated with biotinylated 2′-<i>O</i>-methyl oligonucleotide tethered to paramagnetic streptavidin beads. The beads were removed and the supernatant tested for RNAi activity.</p>\n <p>Symbols and abbreviations: Ø, target RNA before incubation with siRNA-programmed lysate; T, total reaction before depletion; unbound, the supernatant after incubation with the paramagnetic beads. “Mock” indicates that no oligonucleotide was used on the beads; “<i>let-7</i>” indicates that the beads contained the <i>let-7</i>-complementary oligonucleotide shown in (A).</p></div>", "links"=>[], "tags"=>["complementary", "oligonucleotide", "blocks", "endogenous", "risc"], "article_id"=>634285, "categories"=>["Molecular Biology", "Biotechnology", "Cell Biology", "Genetics"], "users"=>["György Hutvágner", "Martin J Simard", "Craig C Mello", "Phillip D Zamore"], "doi"=>["https://dx.doi.org/10.1371/journal.pbio.0020098.g005"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_A_Complementary_2_O_Methyl_Oligonucleotide_Blocks_Endogenous_let_7_Containing_RISC_Function_/634285", "title"=>"A Complementary 2′<i>-O-</i>Methyl Oligonucleotide Blocks Endogenous <i>let-7</i>-Containing RISC Function", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-22 09:17:56"}
  • {"files"=>["https://ndownloader.figshare.com/files/964440"], "description"=>"<div><p>(A) Wild-type and <i>lin-41(ma104)</i> L2-stage C. elegans larvae were injected with either a 2′<i>-O-</i>methyl oligonucleotide complementary to <i>let-7</i> miRNA (<a href=\"http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020098#pbio-0020098-g005\" target=\"_blank\">Figure 5</a>A) or an unrelated <i>Pp</i>-luc 2′-<i>O</i>-methyl oligonucleotide. Absence of alae and presence of bursting vulvae were scored when the injected animals reached adulthood.</p>\n <p>(B) Isolation of <i>let-7</i>-associated proteins with a tethered 2′-<i>O</i>-methyl oligonucleotide. Northern blot analysis of <i>let-7</i> miRNA remaining in the supernatant of the worm lysate after incubation with the <i>let-7</i>-complementary (<i>let-7</i>) or <i>Pp</i>-luc (unrelated) oligonucleotide. Input represents the equivalent of 50% of the total extract incubated with tethered oligonucleotide.</p>\n <p>(C) Western blot analysis of the GFP-tagged ALG-1 and ALG-2 proteins associated with <i>let-7</i>. The upper band corresponds to GFP-tagged ALG-1 and the lower to GFP-tagged ALG-2. Extracts from a transgenic strain expressing the tagged proteins was incubated with the indicated tethered 2′-<i>O</i>-methyl oligonucleotide; then, the beads were washed and bound proteins were fractionated on an 8% SDS-polyacrylamide gel. Western blots were probed using anti-GFP monoclonal or anti-RDE-4 polyclonal antibody. The RDE-4-specific band is marked with an asterisk (<a href=\"http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020098#pbio-0020098-Tabara2\" target=\"_blank\">Tabara et al. 2002</a>).</p>\n <p>(D and E) Analysis of <i>let-7</i> miRNA in ALG-1/ALG-2 complexes (D). Extracts prepared from mixed-stage wild-type worms (N2) or from GFP::ALG-1/ALG-2 transgenic worms were immunoprecipitated using anti-GFP monoclonal antibodies. The unbound and immunoprecipitated RNAs were analyzed by Northern blot hybridization for <i>let-7</i> (D), and 5% of the immunoprecipitated protein was analyzed by Western blotting for GFP to confirm recovery of the GFP-tagged ALG-1/ALG-2 proteins (E).</p></div>", "links"=>[], "tags"=>["oligonucleotide", "complementary", "mirna", "phenocopy"], "article_id"=>634378, "categories"=>["Molecular Biology", "Biotechnology", "Cell Biology", "Genetics"], "users"=>["György Hutvágner", "Martin J Simard", "Craig C Mello", "Phillip D Zamore"], "doi"=>["https://dx.doi.org/10.1371/journal.pbio.0020098.g006"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Injection_of_a_2_O_Methyl_Oligonucleotide_Complementary_to_let_7_miRNA_Can_Phenocopy_the_Loss_of_let_7_Function_in_C_elegans_/634378", "title"=>"Injection of a 2′-<i>O</i>-Methyl Oligonucleotide Complementary to <i>let-7</i> miRNA Can Phenocopy the Loss of <i>let-7</i> Function in C. elegans", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-22 09:18:30"}

PMC Usage Stats | Further Information

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Relative Metric

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