Journal
NUCLEIC ACIDS RESEARCH
Volume 41, Issue 18, Pages -Publisher
OXFORD UNIV PRESS
DOI: 10.1093/nar/gkt690
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Funding
- Biomedical Research Centre and the Medical Research Council [G0800142]
- Intramural Research Program of the National Institutes of Health, National Cancer Institute
- Medical Research Council [G0800142]
- MRC [G0800142] Funding Source: UKRI
- Medical Research Council [G0800142] Funding Source: researchfish
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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.
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