Rapid and specific purification of Argonaute-small RNA complexes from crude cell lysates
UMass Chan AffiliationsDepartment of Biochemistry and Molecular Pharmacology
Document TypeJournal Article
RNA, Small Interfering
RNA-Induced Silencing Complex
Sensitivity and Specificity
Biochemistry, Biophysics, and Structural Biology
Genetics and Genomics
Nucleic Acids, Nucleotides, and Nucleosides
MetadataShow full item record
AbstractSmall interfering RNAs (siRNAs) direct Argonaute proteins, the core components of the RNA-induced silencing complex (RISC), to cleave complementary target RNAs. Here, we describe a method to purify active RISC containing a single, unique small RNA guide sequence. We begin by capturing RISC using a complementary 2'-O-methyl oligonucleotide tethered to beads. Unlike other methods that capture RISC but do not allow its recovery, our strategy purifies active, soluble RISC in good yield. The method takes advantage of the finding that RISC partially paired to a target through its siRNA guide dissociates more than 300 times faster than a fully paired siRNA in RISC. We use this strategy to purify fly Ago1- and Ago2-RISC, as well as mouse AGO2-RISC. The method can discriminate among RISCs programmed with different guide strands, making it possible to deplete and recover specific RISC populations. Endogenous microRNA:Argonaute complexes can also be purified from cell lysates. Our method scales readily and takes less than a day to complete.
RNA. 2013 Feb;19(2):271-9. doi: 10.1261/rna.036921.112. Epub 2012 Dec 18. Link to article on publisher's site
Permanent Link to this Itemhttp://hdl.handle.net/20.500.14038/26029
RightsCopyright © 2013 RNA Society. Freely available online through the RNA Open Access option.