Native Purification and Analysis of Long RNAs

Methods Enzymol. 2015;558:3-37. doi: 10.1016/bs.mie.2015.01.008. Epub 2015 Feb 27.

Abstract

The purification and analysis of long noncoding RNAs (lncRNAs) in vitro is a challenge, particularly if one wants to preserve elements of functional structure. Here, we describe a method for purifying lncRNAs that preserves the cotranscriptionally derived structure. The protocol avoids the misfolding that can occur during denaturation-renaturation protocols, thus facilitating the folding of long RNAs to a native-like state. This method is simple and does not require addition of tags to the RNA or the use of affinity columns. LncRNAs purified using this type of native purification protocol are amenable to biochemical and biophysical analysis. Here, we describe how to study lncRNA global compaction in the presence of divalent ions at equilibrium using sedimentation velocity analytical ultracentrifugation and analytical size-exclusion chromatography as well as how to use these uniform RNA species to determine robust lncRNA secondary structure maps by chemical probing techniques like selective 2'-hydroxyl acylation analyzed by primer extension and dimethyl sulfate probing.

Keywords: Analytical ultracentrifugation; Chemical probing; DMS; Long noncoding RNA; Native purification; RNA folding; SHAPE; Secondary structure; Size-exclusion chromatography.

Publication types

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

MeSH terms

  • Acylation
  • Cations, Divalent
  • Chromatography, Gel / methods
  • Chromatography, High Pressure Liquid / methods
  • Genetic Vectors / chemistry*
  • Genetic Vectors / metabolism
  • Humans
  • Hydroxyl Radical / chemistry
  • In Vitro Techniques / instrumentation
  • In Vitro Techniques / methods*
  • Magnesium / chemistry
  • Molecular Probes / chemistry
  • Nucleic Acid Conformation
  • Plasmids / chemistry*
  • Plasmids / metabolism
  • RNA Folding
  • RNA, Long Noncoding / chemistry
  • RNA, Long Noncoding / isolation & purification*
  • RNA, Long Noncoding / metabolism
  • Sequence Analysis, RNA
  • Sulfuric Acid Esters / chemistry
  • Transcription, Genetic*
  • Ultracentrifugation / methods

Substances

  • Cations, Divalent
  • Molecular Probes
  • RNA, Long Noncoding
  • Sulfuric Acid Esters
  • Hydroxyl Radical
  • Magnesium
  • dimethyl sulfate