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

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.

SUBMITTER: Kenyon JC

PROVIDER: S-EPMC3794615 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

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

Kenyon Julia C JC Prestwood Liam J LJ Le Grice Stuart F J SF Lever Andrew M L AM

Nucleic acids research 20130808 18

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 populatio ...[more]

PMID: 23935074

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Project description:Single molecule photoluminescence (PL) spectroscopy of conjugated polymers has shed new light on the complex structure⁻function relationships of these materials. Although extensive work has been carried out using polarization and excitation intensity modulated experiments to elucidate conformation-dependent photophysics, surprisingly little attention has been given to information contained in the PL spectral line shapes. We investigate single molecule PL spectra of the prototypical conjugated polymer poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene] (MEH-PPV) which exists in at least two emissive conformers and can only be observed at dilute levels. Using a model based on the well-known "Missing Mode Effect" (MIME), we show that vibronic progression intervals for MEH-PPV conformers can be explained by relative contributions from particular skeletal vibrational modes. Here, observed progression intervals do not match any ground state Raman active vibrational frequency and instead represent a coalescence of multiple modes in the frequency domain. For example, the higher energy emitting "blue" MEH-PPV form exhibits PL maxima at ~18,200 cm-1 with characteristic MIME progression intervals of ~1200⁻1350 cm-1, whereas the lower energy emitting "red" form peaks at ~17,100 cm-1 with intervals in the range of ~1350⁻1450 cm-1. The main differences in blue and red MEH-PPV chromophores lie in the intra-chain order, or, planarity of monomers within a chromophore segment. We demonstrate that the Raman-active out-of-plane C⁻H wag of the MEH-PPV vinylene group (~966 cm-1) has the greatest influence in determining the observed vibronic progression MIME interval. Namely, larger displacements (intensities)-indicating lower intra-chain order-lower the effective MIME interval. This simple model provides useful insights into the conformational characteristics of the heterogeneous chromophore landscape without requiring costly and time-consuming low temperature or single molecule Raman capabilities.

Dataset Information

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

Publications

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

Similar Datasets

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