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WIREs RNA
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RNA structural analysis by evolving SHAPE chemistry

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RNA is central to the flow of biological information. From transcription to splicing, RNA localization, translation, and decay, RNA is intimately involved in regulating every step of the gene expression program, and is thus essential for health and understanding disease. RNA has the unique ability to base‐pair with itself and other nucleic acids to form complex structures. Hence the information content in RNA is not simply its linear sequence of bases, but is also encoded in complex folding of RNA molecules. A general chemical functionality that all RNAs have is a 2′‐hydroxyl group in the ribose ring, and the reactivity of the 2′‐hydroxyl in RNA is gated by local nucleotide flexibility. In other words, the 2′‐hydroxyl is reactive at single‐stranded and conformationally flexible positions but is unreactive at nucleotides constrained by base‐pairing. Recent efforts have been focused on developing reagents that modify RNA as a function of RNA 2′ hydroxyl group reactivity. Such RNA structure probing techniques can be read out by primer extension in experiments termed RNA SHAPE (selective 2′‐ hydroxyl acylation and primer extension). Herein, we describe the efforts devoted to the design and utilization of SHAPE probes for characterizing RNA structure. We also describe current technological advances that are being applied to utilize SHAPE chemistry with deep sequencing to probe many RNAs in parallel. The merging of chemistry with genomics is sure to open the door to genome‐wide exploration of RNA structure and function. WIREs RNA 2014, 5:867–881. doi: 10.1002/wrna.1253

Mechanisms of SHAPE chemistry and electrophiles used for acylation. (a) Reaction pathway of RNA 2′‐O‐acylation. LG is short for leaving group. (b) Examples of SHAPE electrophiles developed for RNA structure probing. Blue spheres mark the site of 2′‐OH attack.
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Methods to merge RNA structure probing with deep sequencing. (A) Experimental outline of parallel analysis of RNA structure (PARS). (B) Experimental outline of SHAPE‐seq.
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Synthesis and utilization of the first in vivo SHAPE reagents. (A) Synthesis of FAI and NAI as 1.0 M stock solution of 1:1 compound:imidazole in DMSO. Note that CO2 is also produced during the reaction but it evolves from solution. (B) SHAPE modification patterns with differences between in vivo and in vitro calculated. (C) Differential SHAPE modifications mapped onto the crystal structure of 5S rRNA. (D) Zoomed in view of residue A49. (E) Zoomed in view of reside U84.
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SHAPE probing on the SAM‐I riboswitch. (A) Denaturing gel electrophoresis diagram for SHAPE probing, as a function of increasing magnesium (−/+) S‐adenosyl methionine (SAM). (B) Ligand‐dependent changes in apparent melting temperatures mapped on the secondary structure of the SAM‐I riboswitch. (C) Additional nucleotide analog interference mapping of guanine (inosine, yellow) and adenine (2‐amino purine, red; di‐amino purine, cyan) identifies specific nucleotide positions as important for folding and/or binding. (Reprinted with permission from Ref Copyright 2010 Cell Press)
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RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems
RNA Structure and Dynamics > RNA Structure, Dynamics and Chemistry
RNA Methods

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