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Determination of the Secondary Structure of an RNA fragment in Solution: Selective 2`- Hydroxyl Acylation Analyzed by Primer Extension Assay (SHAPE)
bio-protocol
  • Manuel Miras, Consejo Superior de Investigaciones Científicas (CSIC)
  • Raquel N. Sempere, Consejo Superior de Investigaciones Científicas (CSIC)
  • Jelena Jeremic Kraft, Iowa State University
  • W. Allen Miller, Iowa State University
  • Miguel A. Aranda, Consejo Superior de Investigaciones Científicas (CSIC)
  • Veronica Truniger, Consejo Superior de Investigaciones Científicas (CSIC)
Document Type
Article
Publication Version
Published Version
Publication Date
1-1-2015
Abstract

This protocol describes the methodology for the determination of the secondary structure of an RNA fragment in solution using Selective 2´-Hydroxyl Acylation analyzed by Primer Extension, abbreviation SHAPE. It consists in the very fast chemical modification of flexible and therefore possibly single-stranded nucleotides in a sequence-independent manner using benzoyl cyanide (BzCN), forming 2´-O-adducts. The modifications in the RNA are then analyzed by primer extension. Reverse transcriptase is blocked by the 2´-O-adducts formed. The advantage of the method is, first, that not each RNA molecule studied but the primer used in the extension reaction is labelled and, second, that the resulting cDNA analyzed in sequencing gels is much more stable than the modified RNA

Comments

This article is from Bio-protocol 5 (2015): e1386. Posted with permission.

Rights
This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Copyright Owner
Bio-protocol LLC
Language
en
File Format
application/pdf
Citation Information
Manuel Miras, Raquel N. Sempere, Jelena Jeremic Kraft, W. Allen Miller, et al.. "Determination of the Secondary Structure of an RNA fragment in Solution: Selective 2`- Hydroxyl Acylation Analyzed by Primer Extension Assay (SHAPE)" bio-protocol Vol. 5 Iss. 2 (2015) p. e1386-1 - e1386-9
Available at: http://works.bepress.com/wallen_miller/9/