SHAPE analysis of small RNAs and riboswitches

Methods Enzymol. 2014:549:165-87. doi: 10.1016/B978-0-12-801122-5.00008-8.

Abstract

We describe structural analysis of small RNAs by SHAPE chemical probing. RNAs are treated with 1-methyl-7-nitroisatoic anhydride, a reagent that detects local nucleotide flexibility; and N-methylisatoic anhydride and 1-methyl-6-nitroisatoic anhydride, reagents which together detect higher-order and noncanonical interactions. Chemical adducts are quantified as stops during reverse transcriptase-mediated primer extension. Probing information can be used to infer conformational changes and ligand binding and to develop highly accurate models of RNA secondary structures.

Keywords: Capillary electrophoresis; QuSHAPE; RNA structure; Riboswitch; SHAPE.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Anhydrides / chemistry
  • Base Sequence
  • Electrophoresis, Capillary / methods*
  • Indicators and Reagents
  • Molecular Sequence Data
  • Nucleic Acid Conformation
  • Oxazines / chemistry
  • RNA / chemistry*
  • RNA Folding
  • Riboswitch*
  • ortho-Aminobenzoates / chemistry

Substances

  • 1-methyl-7-nitroisatoic anhydride
  • Anhydrides
  • Indicators and Reagents
  • Oxazines
  • Riboswitch
  • ortho-Aminobenzoates
  • N-methylisatoic anhydride
  • RNA