Mapping RNA structure in vitro with SHAPE chemistry and next-generation sequencing (SHAPE-Seq)

Kyle E. Watters, Julius B. Lucks*

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingChapter

9 Scopus citations

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 nextgeneration 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.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages135-162
Number of pages28
DOIs
StatePublished - Sep 1 2016

Publication series

NameMethods in Molecular Biology
Volume1490
ISSN (Print)1064-3745

Keywords

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

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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