Department of Biochemistry and Molecular Pharmacology; Program in Molecular Medicine
Animals; Animals, Genetically Modified; Biotinylation; Blotting, Northern; Caenorhabditis elegans; Drosophila; Gene Silencing; Genetic Techniques; HeLa Cells; Humans; MicroRNAs; Molecular Sequence Data; Oligonucleotides; Phenotype; RNA Interference; RNA, Messenger; RNA, Small Interfering; Transfection
Biochemistry, Biophysics, and Structural Biology | Life Sciences | Medicine and Health Sciences
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.
Hutvagner, Gyorgy; Simard, Martin J.; Mello, Craig C.; and Zamore, Phillip D., "Sequence-specific inhibition of small RNA function" (2004). Open Access Articles. 2371.