Purification of RNA and RNA-protein complexes by an R17 coat protein affinity method

Vivian J. Bardwell, Marvin Wickens

Research output: Contribution to journalArticlepeer-review

61 Scopus citations

Abstract

We describe an affinity chromatography method to isolate specific RNAs and RNA-protein complexes formed in vivo or in vitro. It exploits the highly selective binding of the coat protein of bacteriophage R17 to a short hairpin in its genomic RNA. RNA containing that hairpin binds to coat protein that has been covalently bound to a solid support. Bound RNA-protein complexes can be eluted with excess R17 recognition sites. Using purified RNA, we demonstrate that binding to immobilized coat protein is highly specific and enables one to separate an RNA of interest from a large excess of other RNAs in a single step. Surprisingly, binding of an RNA containing non-R17 sequences to the support requires two recognition sites in tandem; a single site is insufficient. We determine optimal conditions for purification of specific RNAs by comparing specific binding (retention of RNAs with recognition sites) to non-specific binding (retention of RNAs without recognition sites) over a range of experimental conditions. These results suggest that binding of immobilized coat protein to RNAs containing two sites is cooperative. We illustrate the potential utility of the approach in purifying RNA-proteln complexes by demonstrating that a U1 snRNP formed in vivo on an RNA containing tandem recognition sites is selectively retained by the coat protein support.

Original languageEnglish (US)
Pages (from-to)6587-6594
Number of pages8
JournalNucleic acids research
Volume18
Issue number22
DOIs
StatePublished - Nov 25 1990
Externally publishedYes

Bibliographical note

Funding Information:
DNA oligonucleotide synthesis was performed by the University of Wisconsin Protein Sequence-DNA Synthesis Facility. V.B. was supported by a Natural Sciences and Engineering Research Council of Canada postgraduate fellowship. This work is supported by the Public Health Service research grant GM31892 and Research Career Development Award GM00521 from the National Institutes of Health to M.W.

Copyright:
Copyright 2010 Elsevier B.V., All rights reserved.

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