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Time-resolved RNA SHAPE chemistry: quantitative RNA structure analysis in one-second snapshots and at single-nucleotide resolution

Nature Protocols volume 4, pages 1413–1421 (2009)Cite this article

Abstract

RNA selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry exploits the discovery that conformationally dynamic nucleotides preferentially adopt configurations that facilitate reaction between the 2′-OH group and a hydroxyl-selective electrophile, such as benzoyl cyanide (BzCN), to form a 2′-O-adduct. BzCN is ideally suited for quantitative, time-resolved analysis of RNA folding and ribonucleoprotein (RNP) assembly mechanisms because this reagent both reacts with flexible RNA nucleotides and also undergoes auto-inactivating hydrolysis with a half-life of 0.25 s at 37 °C. RNA folding is initiated by addition of Mg2+ or protein, or other change in solution conditions, and nucleotide resolution structural images are obtained by adding aliquots of the evolving reaction to BzCN and then 'waiting' for 1 second. Sites of the 2′-O-adduct formation are subsequently scored as stops to primer extension using reverse transcriptase. This time-resolved SHAPE protocol makes it possible to obtain 1-second structural snapshots in time-resolved kinetic studies for RNAs of arbitrary length and complexity in a straightforward and concise experiment.

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Figure 1: The mechanism of RNA SHAPE chemistry with BzCN.
Figure 2: Analysis of time–progress curves.
Figure 3: Electropherogram analysis using ShapeFinder24.
Figure 4: Representative time-resolved SHAPE analysis of the RNase P specificity domain RNA.
Figure 5: Mechanism for folding of the RNase P specificity domain determined by time-resolved SHAPE.

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Acknowledgements

This work was supported by a grant from the National Science Foundation (MCB-0416941 to K.M.W.).

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

    Stefanie A Mortimer & Kevin M Weeks

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  1. Stefanie A Mortimer
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S.A.M. and K.M.W. collaborated on all aspects of the conception, design, presentation and writing of this paper.

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Correspondence to Kevin M Weeks.

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Mortimer, S., Weeks, K. Time-resolved RNA SHAPE chemistry: quantitative RNA structure analysis in one-second snapshots and at single-nucleotide resolution. Nat Protoc 4, 1413–1421 (2009). https://doi.org/10.1038/nprot.2009.126

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