Sequence-specific inhibition of small RNA function

Hundreds of microRNAs (miRNAs) and endogenous small interfering RNAs (siRNAs) have been identified from both plants and animals, yet little is known about their biochemical modes of action or biological functions. Here we report that 2'-O-methyl oligonucleotides can act as irreversible, stoichi...

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Bibliographic Details
Published in:PLoS biology 2004-04, Vol.2 (4), p.E98-0465
Main Authors: Hutvágner, György, Simard, Martin J, Mello, Craig C, Zamore, Phillip D
Format: Article
Language:English
Subjects:
Algae
Animals
Animals, Genetically Modified
Biotechnology
Biotinylation
Blotting, Northern
Caenorhabditis
Caenorhabditis elegans
Cell Biology
Drosophila
Eukaryotes
Gene Silencing
Genetic Techniques
Genetics
Genetics/Genomics/Gene Therapy
HeLa Cells
Homo (Human)
Humans
Insects
Medical research
MicroRNAs - chemistry
Molecular Biology/Structural Biology
Molecular Sequence Data
Nematodes
Oligonucleotides - chemistry
Oligonucleotides - pharmacology
Phenotype
Proteins
Ribonucleic acid
RNA
RNA Interference
RNA, Messenger - metabolism
RNA, Small Interfering - chemistry
Studies
Transfection
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Summary:Hundreds of microRNAs (miRNAs) and endogenous small interfering RNAs (siRNAs) have been identified from both plants and animals, yet little is known about their biochemical modes of action or biological functions. Here we report that 2'-O-methyl oligonucleotides can act as irreversible, stoichiometric inhibitors of small RNA function. We show that a 2'-O-methyl oligonucleotide complementary to an siRNA can block mRNA cleavage in Drosophila embryo lysates and HeLa cell S100 extracts and in cultured human HeLa cells. In Caenorhabditis elegans, injection of the 2'-O-methyl oligonucleotide complementary to the miRNA let-7 can induce a let-7 loss-of-function phenocopy. Using an immobilized 2'-O-methyl oligonucleotide, we show that the C. elegans Argonaute proteins ALG-1 and ALG-2, which were previously implicated in let-7 function through genetic studies, are constituents of a let-7-containing protein-RNA complex. Thus, we demonstrate that 2'-O-methyl RNA oligonucleotides can provide an efficient and straightforward way to block small RNA function in vivo and furthermore can be used to identify small RNA-associated proteins that mediate RNA silencing pathways.
ISSN:1545-7885
1544-9173
1545-7885
DOI:10.1371/journal.pbio.0020098