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Nucleic Acids Res. 2013 Oct;41(18):e174. doi: 10.1093/nar/gkt690. Epub 2013 Aug 8.

In-gel probing of individual RNA conformers within a mixed population reveals a dimerization structural switch in the HIV-1 leader.

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  • 1Department of Medicine, University of Cambridge, Addenbrooke's Hospital, Hills Road, Cambridge, Cambridgeshire, CB2 0QQ, UK and HIV-Drug Resistance Program, Centre for Cancer Research, National Cancer Institute, P.O. Box B, Building 535, Frederick, MD 21702-1201, USA.

Abstract

Definitive secondary structural mapping of RNAs in vitro can be complicated by the presence of more than one structural conformer or multimerization of some of the molecules. Until now, probing a single structure of conformationally flexible RNA molecules has typically relied on introducing stabilizing mutations or adjusting buffer conditions or RNA concentration. Here, we present an in-gel SHAPE (selective 2'OH acylation analysed by primer extension) approach, where a mixed structural population of RNA molecules is separated by non-denaturing gel electrophoresis and the conformers are individually probed within the gel matrix. Validation of the technique using a well-characterized RNA stem-loop structure, the HIV-1 trans-activation response element, showed that authentic structure was maintained and that the method was accurate and highly reproducible. To further demonstrate the utility of in-gel SHAPE, we separated and examined monomeric and dimeric species of the HIV-1 packaging signal RNA. Extensive differences in acylation sensitivity were seen between monomer and dimer. The results support a recently proposed structural switch model of RNA genomic dimerization and packaging, and demonstrate the discriminatory power of in-gel SHAPE.

PMID:
23935074
[PubMed - indexed for MEDLINE]
PMCID:
PMC3794615
Free PMC Article
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