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Selective 2′-hydroxyl acylation analyzed by protection from exoribonuclease (RNase-detected SHAPE) for direct analysis of covalent adducts and of nucleotide flexibility in RNA

Nature Protocols volume 6, pages 1683–1694 (2011)Cite this article

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Abstract

RNA SHAPE chemistry yields quantitative, single-nucleotide resolution structural information based on the reaction of the 2′-hydroxyl group of conformationally flexible nucleotides with electrophilic SHAPE reagents. However, SHAPE technology has been limited by the requirement that sites of RNA modification be detected by primer extension. Primer extension results in loss of information at both the 5′ and 3′ ends of an RNA and requires multiple experimental steps. Here we describe RNase-detected SHAPE that uses a processive, 3′→5′ exoribonuclease, RNase R, to detect covalent adducts in 5′-end–labeled RNA in a one-tube experiment. RNase R degrades RNA but stops quantitatively three and four nucleotides 3′ of a nucleotide containing a covalent adduct at the ribose 2′-hydroxyl or the pairing face of a nucleobase, respectively. We illustrate this technology by characterizing ligand-induced folding for the aptamer domain of the Escherichia coli thiamine pyrophosphate riboswitch RNA. RNase-detected SHAPE is a facile, two-day approach that can be used to analyze diverse covalent adducts in any RNA molecule, including short RNAs not amenable to analysis by primer extension and RNAs with functionally important structures at their 5′ or 3′ ends.

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Figure 1: RNA SHAPE chemistry.
Figure 2: Model of M. genitalium RNase R and the interactions that mediate covalent adduct detection in RNA.
Figure 3: Representative RNase-detected SHAPE experiment.
Figure 4: Structural transitions in the TPP riboswitch aptamer domain visualized by RNase-detected SHAPE.

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Acknowledgements

We are indebted to A. Malhotra for many productive discussions regarding the mechanism of RNase R enzymes and to Z. Li for the gift of a plasmid encoding M. genitalium RNase R optimized for expression in E. coli. We thank P. Homan for helpful discussions regarding synthesis of 1M7. This work was supported by a grant from the National Science Foundation (MCB-0919666 to K.M.W.); N.A.S. is a Fellow of the UNC Lineberger Comprehensive Cancer Center.

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Authors and Affiliations

  1. Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina, USA

    Kady-Ann Steen, Nathan A Siegfried & Kevin M Weeks

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  1. Kady-Ann Steen
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  2. Nathan A Siegfried
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  3. Kevin M Weeks
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Contributions

K.-A.S. and K.M.W. collaborated on all aspects of the conception and writing of this paper. N.A.S. and K.M.W. wrote the Supporting Protocol on RNase R purification.

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Correspondence to Kady-Ann Steen.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

A representative gel showing purification of the M. genitalium RNase R (75,000 Da) (PDF 215 kb)

Supplementary Data 1

Dataset corresponding to the experiment shown in Figure 3. (XLS 40 kb)

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Steen, KA., Siegfried, N. & Weeks, K. Selective 2′-hydroxyl acylation analyzed by protection from exoribonuclease (RNase-detected SHAPE) for direct analysis of covalent adducts and of nucleotide flexibility in RNA. Nat Protoc 6, 1683–1694 (2011). https://doi.org/10.1038/nprot.2011.373

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