Affordable Access

deepdyve-link deepdyve-link
Publisher Website

Use of aptamer tagging to identify in vivo protein binding partners of small regulatory RNAs.

  • Corcoran, Colin P1
  • Rieder, Renate
  • Podkaminski, Dimitri
  • Hofmann, Benjamin
  • Vogel, Jörg
  • 1 Institute for Molecular Infection Biology, University of Würzburg, Würzburg, Germany. , (Germany)
Published Article
Methods in molecular biology (Clifton, N.J.)
Publication Date
Jan 01, 2012
DOI: 10.1007/978-1-61779-949-5_11
PMID: 22736004


Small regulatory RNAs (sRNAs) are short, generally noncoding RNAs that act posttranscriptionally to control target gene expression. Over the past 10 years there has been a rapid expansion in the discovery and characterization of sRNAs in a diverse range of bacteria. Paradigm shifts in our understanding of the breadth of posttranscriptional control by sRNAs were achieved in a number of pioneering studies that involved immunoprecipitation of a known RNA chaperone, the near-ubiquitous Hfq, followed by sequencing to identify novel putative regulators and targets. To perform the converse experiment, we previously developed a method which uses an aptamer-tagged sRNA to allow purification of in vivo assembled RNA-protein complexes and subsequent identification of bound proteins. We successfully implemented this protocol using the Hfq-associated sRNA, InvR, tagged with a tandem repeat of the commonly used MS2-aptamer. Incorporation of the aptamer had no effect on sRNA stability or activity. InvR-MS2 could be effectively purified along with associated proteins, such as Hfq, using maltose binding protein fused to the MS2 coat protein (MBP-MS2) immobilized on an amylose column. Mass-spectroscopy was also used to identify previously uncharacterized protein partners. These results have been described previously (Said et al., Nucleic Acids Res 37:e133, 2009) and thus the figures presented here are intended solely as an illustrative guide to complement this detailed step-by-step protocol.

Report this publication


Seen <100 times