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Characterizing RNA structures in vitro and in vivo with selective 2’-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq)

Kyle E. Watters, Angela M Yu, Eric J. Strobel, Alex H. Settle, View ORCID ProfileJulius B. Lucks
doi: https://doi.org/10.1101/034470
Kyle E. Watters
1School of Chemical and Biomolecular Engineering, Cornell University, Ithaca NY 14853
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Angela M Yu
1School of Chemical and Biomolecular Engineering, Cornell University, Ithaca NY 14853
2Tri-Institutional Program in Computational Biology and Medicine, Cornell University, Ithaca, New York, Weill Cornell Medical College, New York, New York, Memorial Sloan-Kettering Cancer Center, New York, New York
3Computational Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10065
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Eric J. Strobel
1School of Chemical and Biomolecular Engineering, Cornell University, Ithaca NY 14853
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Alex H. Settle
1School of Chemical and Biomolecular Engineering, Cornell University, Ithaca NY 14853
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Julius B. Lucks
1School of Chemical and Biomolecular Engineering, Cornell University, Ithaca NY 14853
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  • ORCID record for Julius B. Lucks
  • For correspondence: jblucks@cornell.edu
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Abstract

RNA molecules adopt a wide variety of structures that perform many cellular functions, including catalysis, small molecule sensing, and cellular defense, among others. Our ability to characterize, predict, and design RNA structures are key factors for understanding and controlling the biological roles of RNAs. Fortunately, there has been rapid progress in this area, especially with respect to experimental methods that can characterize RNA structures in a high throughput fashion using chemical probing and next-generation sequencing. Here, we describe one such method, selective 2’-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq), which measures nucleotide resolution flexibility information for RNAs in vitro and in vivo. We outline the process of designing and performing a SHAPE-Seq experiment and describe methods for using experimental SHAPE-Seq data to restrain computational folding algorithms to generate more accurate predictions of RNA secondary structure. We also provide a number of examples of SHAPE-Seq reactivity spectra obtained in vitro and in vivo and discuss important considerations for performing SHAPE-Seq experiments, both in terms of collecting and analyzing data. Finally we discuss improvements and extensions of these experimental and computational techniques that promise to deepen our knowledge of RNA folding and function.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted December 15, 2015.
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Characterizing RNA structures in vitro and in vivo with selective 2’-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq)
Kyle E. Watters, Angela M Yu, Eric J. Strobel, Alex H. Settle, Julius B. Lucks
bioRxiv 034470; doi: https://doi.org/10.1101/034470
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Characterizing RNA structures in vitro and in vivo with selective 2’-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq)
Kyle E. Watters, Angela M Yu, Eric J. Strobel, Alex H. Settle, Julius B. Lucks
bioRxiv 034470; doi: https://doi.org/10.1101/034470

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