Abstract
RNA affinity tags (aptamers) have emerged as useful tools for the isolation of RNAs and ribonucleoprotein complexes from cell extracts. The streptavidin binding RNA aptamer binds with high affinity and is quickly and cleanly eluted with biotin under mild conditions that retain intact complexes. We describe the use of the streptavidin binding aptamer as a tool for purification and discuss strategies towards the design and production of tagged RNAs with a focus on structured target RNAs. The aptamer site can be further exploited as a unique region for the hybridization of oligonucleotide probes and localization by fluorescent in situ hybridization (FISH). The aptamer insertion will allow the localization of a population of RNA species (such as mutants) to be viewed specifically, while in the presence of the wild type RNA. We describe the production of labeled oligonucleotide probes and the preparation of yeast cells for the localization of RNAs by FISH.
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Acknowledgments
This work was supported by a National Institute of Health grant (R01GM082875) to DRE.
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Walker, S.C., Good, P.D., Gipson, T.A., Engelke, D.R. (2011). The Dual Use of RNA Aptamer Sequences for Affinity Purification and Localization Studies of RNAs and RNA–Protein Complexes. In: Gerst, J. (eds) RNA Detection and Visualization. Methods in Molecular Biology, vol 714. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-005-8_26
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DOI: https://doi.org/10.1007/978-1-61779-005-8_26
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