Rapid and specific purification of Argonaute-small RNA complexes from crude cell lysates
UMass Chan Affiliations
Department of Biochemistry and Molecular PharmacologyDocument Type
Journal ArticlePublication Date
2013-02-01Keywords
AnimalsArgonaute Proteins
Base Pairing
Blotting, Northern
Drosophila
Drosophila Proteins
Mass Spectrometry
Mice
MicroRNAs
RNA Interference
RNA, Complementary
RNA, Guide
RNA, Small Interfering
RNA-Induced Silencing Complex
Sensitivity and Specificity
Time Factors
Biochemistry, Biophysics, and Structural Biology
Cells
Genetics and Genomics
Molecular Genetics
Nucleic Acids, Nucleotides, and Nucleosides
Metadata
Show full item recordAbstract
Small 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.Source
RNA. 2013 Feb;19(2):271-9. doi: 10.1261/rna.036921.112. Epub 2012 Dec 18. Link to article on publisher's site
DOI
10.1261/rna.036921.112Permanent Link to this Item
http://hdl.handle.net/20.500.14038/26029PubMed ID
23249751Related Resources
Rights
Copyright © 2013 RNA Society. Freely available online through the RNA Open Access option.ae974a485f413a2113503eed53cd6c53
10.1261/rna.036921.112