title: The dual use of RNA aptamer sequences for affinity purification and localization studies of RNAs and RNA-protein complexes.
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.
authors: Department of Biological Chemistry, University of Michigan, Ann Arbor, MI, USA. scwalker.mail@gmail.com
authors: Scott C
authors: SC
authors: Walker
authors: Paul D
authors: PD
authors: Good
authors: Theresa A
authors: TA
authors: Gipson
authors: David R
authors: DR
authors: Engelke
mesh_terms: D052157:Aptamers, Nucleotide / Q000378:metabolism*; D001483:Base Sequence; D017404:In Situ Hybridization, Fluorescence; D046911:Macromolecular Substances / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000235:genetics; D010539:Permeability; D016133:Polymerase Chain Reaction; D021381:Protein Transport; D012313:RNA / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000302:isolation & purification* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000378:metabolism; D019809:Streptavidin / Q000378:metabolism
chemical_list: D052157:Aptamers, Nucleotide; D046911:Macromolecular Substances; D015345:Oligonucleotide Probes; D012261:Ribonucleoproteins; D012313:RNA; D019809:Streptavidin