Mapping RNA Structure In Vitro with SHAPE Chemistry and Next-Generation Sequencing (SHAPE-Seq)

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1490)

Abstract

Mapping RNA structure with selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry has proven to be a versatile method for characterizing RNA structure in a variety of contexts. SHAPE reagents covalently modify RNAs in a structure-dependent manner to create adducts at the 2′-OH group of the ribose backbone at nucleotides that are structurally flexible. The positions of these adducts are detected using reverse transcriptase (RT) primer extension, which stops one nucleotide before the modification, to create a pool of cDNAs whose lengths reflect the location of SHAPE modification. Quantification of the cDNA pools is used to estimate the “reactivity” of each nucleotide in an RNA molecule to the SHAPE reagent. High reactivities indicate nucleotides that are structurally flexible, while low reactivities indicate nucleotides that are inflexible. These SHAPE reactivities can then be used to infer RNA structures by restraining RNA structure prediction algorithms. Here, we provide a state-of-the-art protocol describing how to perform in vitro RNA structure probing with SHAPE chemistry using next-generation sequencing to quantify cDNA pools and estimate reactivities (SHAPE-Seq). The use of next-generation sequencing allows for higher throughput, more consistent data analysis, and multiplexing capabilities. The technique described herein, SHAPE-Seq v2.0, uses a universal reverse transcription priming site that is ligated to the RNA after SHAPE modification. The introduced priming site allows for the structural analysis of an RNA independent of its sequence.

Key words

SHAPE SHAPE-Seq RNA RNA structure probing RNA structure mapping Next-generation sequencing RNA structure RNA folding 

Notes

Acknowledgements

We thank Alex Settle for assistance on experimental procedures, Peter Schweitzer and the Cornell Life Sciences Core facility for sequencing support, and David Loughrey and James Chappell for helpful comments in reviewing this manuscript. This work was supported by the National Science Foundation Graduate Research Fellowship Program (grant number DGE-1144153 to K.E.W.); the Cornell University Center for Life Sciences Enterprises, a New York State Center for Advanced Technology supported by New York State and industrial partners (grant number C110124 to J.B.L.); and a New Innovator Award through the National Institute of General Medical Sciences of the National Institutes of Health (grant number DP2GM110838 to J.B.L.). K.E.W. is a Fleming Scholar in the Robert F. Smith School of Chemical and Biomolecular Engineering at Cornell University. J.B.L. is an Alfred P. Sloan Research Fellow.

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Robert F. Smith School of Chemical and Biomolecular EngineeringCornell UniversityIthacaUSA

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