Summary
Isolation of ribonucleoprotein particles from living cells and cell lysates has allowed the identification of both simple bimolecular interactions and the members of large, extended complexes. A number of different strategies have been devised to isolate these complexes by using affinity purification methods that are specific for the RNA rather than the protein components of these complexes. We describe the use of two such RNA affinity tags: small RNAs that bind with high affinity and specificity to either Sephadex beads or streptavidin affinity resins and can be eluted under mild, native conditions that retain intact complexes. The tags can be inserted into appropriate locations in genes encoding the RNA components, and ribonucleoproteins can be assembled either in vivo or in vitro before affinity isolation. Strategies toward the design and production of these tagged RNA sequences are discussed, and the purification procedure is outlined.
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Acknowledgments
This work was supported by a National Institute of Health grant (R01 6M34869) to D.R.E. We thank both Shaohua Xiao and Rebecca Haeusler for their helpful comments and review of the text.
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Walker, S.C., Scott, F.H., Srisawat, C., Engelke, D.R. (2008). RNA Affinity Tags for the Rapid Purification and Investigation of RNAs and RNA–Protein Complexes. In: Lin, RJ. (eds) RNA-Protein Interaction Protocols. Methods in Molecular Biology, vol 488. Humana Press. https://doi.org/10.1007/978-1-60327-475-3_3
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DOI: https://doi.org/10.1007/978-1-60327-475-3_3
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