A mutate-and-map strategy accurately infers the base pairs of a 35-nucleotide model RNA

RNA. 2011 Mar;17(3):522-34. doi: 10.1261/rna.2516311. Epub 2011 Jan 14.

Abstract

We present a rapid experimental strategy for inferring base pairs in structured RNAs via an information-rich extension of classic chemical mapping approaches. The mutate-and-map method, previously applied to a DNA/RNA helix, systematically searches for single mutations that enhance the chemical accessibility of base-pairing partners distant in sequence. To test this strategy for structured RNAs, we have carried out mutate-and-map measurements for a 35-nt hairpin, called the MedLoop RNA, embedded within an 80-nt sequence. We demonstrate the synthesis of all 105 single mutants of the MedLoop RNA sequence and present high-throughput DMS, CMCT, and SHAPE modification measurements for this library at single-nucleotide resolution. The resulting two-dimensional data reveal visually clear, punctate features corresponding to RNA base pair interactions as well as more complex features; these signals can be qualitatively rationalized by comparison to secondary structure predictions. Finally, we present an automated, sequence-blind analysis that permits the confident identification of nine of the 10 MedLoop RNA base pairs at single-nucleotide resolution, while discriminating against all 1460 false-positive base pairs. These results establish the accuracy and information content of the mutate-and-map strategy and support its feasibility for rapidly characterizing the base-pairing patterns of larger and more complex RNA systems.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Pairing*
  • Base Sequence
  • DNA / chemistry*
  • DNA / genetics
  • Molecular Sequence Data
  • Mutagenesis
  • Mutation / genetics*
  • Nucleic Acid Conformation*
  • RNA / chemistry*
  • RNA / genetics

Substances

  • RNA
  • DNA