Abstract
Here we report a simple and cheap one-step affinity purification protocol for isolating RNAs or proteins that interact with selected functional RNAs. The streptomycin-binding aptamer, termed 'StreptoTag,' is embedded in or fused to either end of any RNA of interest. The resulting hybrid RNA can then be immobilized on a streptomycin affinity matrix. When a complex protein mixture or total cellular lysate is applied to the matrix, subsequent elution with free streptomycin allows efficient recovery of specific ribonucleoprotein or RNA-RNA complexes. The method was successfully used to purify yeast and phage RNA-binding proteins and group II intron, viral and bacterial noncoding RNA (ncRNA)-binding proteins. The selective enrichment of bacterial mRNAs that bind ncRNAs has also been demonstrated. Once the affinity matrix, the RNA construct and the protein extracts have been prepared, the experimental procedure can be performed in 1–2 h.
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Acknowledgements
Work in our laboratory is funded by the Austrian Science fund projects Z-72 and F1703, by the European Community BACRNA FP6-018618 and by the Austrian BMBWK Gen AU programme.
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Windbichler, N., Schroeder, R. Isolation of specific RNA-binding proteins using the streptomycin-binding RNA aptamer. Nat Protoc 1, 637–640 (2006). https://doi.org/10.1038/nprot.2006.95
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DOI: https://doi.org/10.1038/nprot.2006.95
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