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.
Highlights
A variety of biochemical techniques exist to examine the secondary structure of RNA molecules via nuclease mapping [1] or chemical modification [2]
Acylation sensitivity of each nucleotide is correlated with the nuclear magnetic resonance (NMR) disorder parameter S2, suggesting that modification is sensitive to tertiary structure and may have a future role as a three-dimensional probing tool [4]
Using in-gel probing of the HIV-1 packaging signal monomer and dimer, we show significant differences across much of the structure, but consistent low acylation sensitivity at the DIS
Summary
A variety of biochemical techniques exist to examine the secondary structure of RNA molecules via nuclease mapping [1] or chemical modification [2]. A group of compounds have been reported that acylate the ribose 20OH where the backbone is flexible, which preferentially occurs within single-stranded regions [3]. This technique, designated SHAPE (selective 20OH acylation analysed by primer extension), provides faster and more extensive structural mapping, as the acylating agent modifies each nucleotide irrespective of its nucleobase. Acylation sensitivity of each nucleotide is correlated with the nuclear magnetic resonance (NMR) disorder parameter S2, suggesting that modification is sensitive to tertiary structure and may have a future role as a three-dimensional probing tool [4]. Sample analysis via automated capillary electrophoresis has accelerated structural mapping, making high-throughput SHAPE vastly more powerful than previous methods [5]
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