Welcome to the new version of CaltechAUTHORS. Login is currently restricted to library staff. If you notice any issues, please email coda@library.caltech.edu
Published July 5, 2011 | Supplemental Material + Published
Journal Article Open

Multiplexed RNA structure characterization with selective 2'-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq)

Abstract

New regulatory roles continue to emerge for both natural and engineered noncoding RNAs, many of which have specific secondary and tertiary structures essential to their function. Thus there is a growing need to develop technologies that enable rapid characterization of structural features within complex RNA populations. We have developed a high-throughput technique, SHAPE-Seq, that can simultaneously measure quantitative, single nucleotide-resolution secondary and tertiary structural information for hundreds of RNA molecules of arbitrary sequence. SHAPE-Seq combines selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry with multiplexed paired-end deep sequencing of primer extension products. This generates millions of sequencing reads, which are then analyzed using a fully automated data analysis pipeline, based on a rigorous maximum likelihood model of the SHAPE-Seq experiment. We demonstrate the ability of SHAPE-Seq to accurately infer secondary and tertiary structural information, detect subtle conformational changes due to single nucleotide point mutations, and simultaneously measure the structures of a complex pool of different RNA molecules. SHAPE-Seq thus represents a powerful step toward making the study of RNA secondary and tertiary structures high throughput and accessible to a wide array of scientific pursuits, from fundamental biological investigations to engineering RNA for synthetic biological systems.

Additional Information

© 2011 National Academy of Sciences. Freely available online through the PNAS open access option. Contributed by Jennifer A. Doudna, May 1, 2011 (sent for review February 9, 2011) The authors thank Michael Eisen, Jacqueline Villalta, Oh Kyu Yoon, Leath Tonkin, Devin Scannell, Jennifer Kuehl, and Keith Keller for advice and assistance. We thank Rhiju Das for insightful reading of the manuscript. We also thank Phil Homan (University of North Carolina, Chapel Hill, NC) and Kevin Weeks (University of North Carolina, Chapel Hill, NC) for the generous gift of 1M7. J.A.D. is a Howard Hughes Medical Institute (HHMI) Investigator, and this work was supported in part by the HHMI. S.A.M. is a fellow of the Leukemia and Lymphoma Society. A.P.A., J.B.L., and S.A. acknowledge support from the Synthetic Biology Engineering Research Center under National Science Foundation Grant 04-570/0540879. J.B.L. and L.P. thank the Miller Institute for financial support, and a stimulating environment in which this work was conceived. Author contributions: J.B.L., S.A.M., C.T., S.L., S.A., G.P.S., L.P., J.A.D., and A.P.A. designed research; J.B.L., S.A.M., C.T., S.L., and S.A. performed research; J.B.L., S.A.M., C.T., S.L., and S.A. contributed new reagents/analytic tools; J.B.L., S.A.M., C.T., S.L., S.A., and L.P. analyzed data; and J.B.L., S.A.M., C.T., S.L., S.A., G.P.S., L.P., J.A.D., and A.P.A. wrote the paper. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1106501108/-/DCSupplemental. The authors declare no conflict of interest.

Attached Files

Published - 11063.full.pdf

Supplemental Material - pnas.1106501108_SI.pdf

Files

pnas.1106501108_SI.pdf
Files (2.5 MB)
Name Size Download all
md5:0ab98c10a20afc2ece0ed37d3525e96c
1.4 MB Preview Download
md5:c64d402d83c6bf3b5cc09e2e5431e1e7
1.1 MB Preview Download

Additional details

Created:
August 22, 2023
Modified:
October 24, 2023