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

Lucks, Julius B. and Mortimer, Stefanie A. and Trapnell, Cole and Luo, Shujun and Aviran, Sharon and Schroth, Gary P. and Pachter, Lior and Doudna, Jennifer A. and Arkin, Adam P. (2011) Multiplexed RNA structure characterization with selective 2'-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq). Proceedings of the National Academy of Sciences of the United States of America, 108 (27). pp. 11063-11068. ISSN 0027-8424. PMCID PMC3131332. https://resolver.caltech.edu/CaltechAUTHORS:20170306-104204159

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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.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://dx.doi.org/10.1073/pnas.1106501108DOIArticle
http://www.pnas.org/content/108/27/11063.abstractPublisherArticle
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3131332/PubMed CentralArticle
http://www.pnas.org/content/108/27/11063/suppl/DCSupplementalPublisherSupporting Information
ORCID:
AuthorORCID
Pachter, Lior0000-0002-9164-6231
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.
Funders:
Funding AgencyGrant Number
Howard Hughes Medical Institute (HHMI)UNSPECIFIED
Leukemia and Lymphoma SocietyUNSPECIFIED
NSF04-570/0540879
Miller Institute for Basic Research in ScienceUNSPECIFIED
Subject Keywords:chemical probing; RNA sequencing; RNA folding genomics
Issue or Number:27
PubMed Central ID:PMC3131332
Record Number:CaltechAUTHORS:20170306-104204159
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20170306-104204159
Official Citation:Julius B. Lucks, Stefanie A. Mortimer, Cole Trapnell, Shujun Luo, Sharon Aviran, Gary P. Schroth, Lior Pachter, Jennifer A. Doudna, and Adam P. Arkin Multiplexed RNA structure characterization with selective 2′-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq) PNAS 2011 108 (27) 11063-11068; published ahead of print June 3, 2011, doi:10.1073/pnas.1106501108
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:74778
Collection:CaltechAUTHORS
Deposited By: George Porter
Deposited On:06 Mar 2017 18:52
Last Modified:24 Feb 2020 10:30

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